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. 2023 Mar 16;11(2):e04709-22. doi: 10.1128/spectrum.04709-22

Clarifying Microbial Nitrous Oxide Reduction under Aerobic Conditions: Tolerant, Intolerant, and Sensitive

Zhiyue Wang a,b, Nisha Vishwanathan c, Sophie Kowaliczko c, Satoshi Ishii c,d,✉
Editor: Victor Gonzaleze
Reviewed by: Maria Delgadof
PMCID: PMC10100939  PMID: 36926990

ABSTRACT

One of the major challenges for the bioremediation application of microbial nitrous oxide (N2O) reduction is its oxygen sensitivity. While a few strains were reported capable of reducing N2O under aerobic conditions, the N2O reduction kinetics of phylogenetically diverse N2O reducers are not well understood. Here, we analyzed and compared the kinetics of clade I and clade II N2O-reducing bacteria in the presence or absence of oxygen (O2) by using a whole-cell assay with N2O and O2 microsensors. Among the seven strains tested, N2O reduction of Stutzerimonas stutzeri TR2 and ZoBell was not inhibited by oxygen (i.e., oxygen tolerant). Paracoccus denitrificans, Azospirillum brasilense, and Gemmatimonas aurantiaca reduced N2O in the presence of O2 but slower than in the absence of O2 (i.e., oxygen sensitive). N2O reduction of Pseudomonas aeruginosa and Dechloromonas aromatica did not occur when O2 was present (i.e., oxygen intolerant). Amino acid sequences and predicted structures of NosZ were highly similar among these strains, whereas oxygen-tolerant N2O reducers had higher oxygen consumption rates. The results suggest that the mechanism of O2 tolerance is not directly related to NosZ structure but is rather related to the scavenging of O2 in the cells and/or accessory proteins encoded by the nos cluster.

IMPORTANCE Some bacteria can reduce N2O in the presence of O2, whereas others cannot. It is unclear whether this trait of aerobic N2O reduction is related to the phylogeny and structure of N2O reductase. The understanding of aerobic N2O reduction is critical for guiding emission control, due to the common concurrence of N2O and O2 in natural and engineered systems. This study provided the N2O reduction kinetics of various bacteria under aerobic and anaerobic conditions and classified the bacteria into oxygen-tolerant, -sensitive, and -intolerant N2O reducers. Oxygen-tolerant N2O reducers rapidly consumed O2, which could help maintain the low O2 concentration in the cells and keep their N2O reductase active. These findings are important and useful when selecting N2O reducers for bioremediation applications.

KEYWORDS: nitrous oxide reduction, oxygen sensitivity, microsensor, kinetics, enzyme kinetics, microsensors

INTRODUCTION

Nitrous oxide (N2O) is a potent greenhouse gas and a stratospheric ozone layer destructor (1). The use of microbial N2O reduction has a potential to mitigate N2O emissions (2, 3). This reaction is catalyzed by nitrous oxide reductase (N2OR) encoded by the nos cluster (4). N2OR is the only known enzyme so far capable of biologically reducing N2O to N2 and is carried by both denitrifying and nondenitrifying microorganisms (5).

N2OR is generally believed to be sensitive to oxygen (O2), which may limit the bioremediation application of N2OR in a standard aerobic environment. Exposure to oxygen may change the configuration of the copper-based catalytic sites and inactivate N2OR (6). Such inactivation could potentially protect the enzyme from irreversible damage and the production of reactive oxygen radicals upon transient exposure to oxygen (7). This process could also contribute to the sensitivity of N2OR to oxygen at the enzyme level. In addition to the effect on the enzyme itself, O2 can also influence the transcription of the nos cluster. The O2-sensing transcription regulators, such as FNR and NNR, as well as small RNA, can suppress the transcription of nos (8, 9).

While the impact of O2 on microbial N2O reduction has been well documented, some denitrifying bacterial strains have been reported to reduce N2O in the presence of O2 (i.e., aerobic N2O reduction) (10, 11). However, the ecophysiology of aerobic N2O reduction remains largely unclear. Questions that remained unanswered include whether the O2 sensitivity of N2OR is related to their structure and how widely aerobic N2O reducers occur in the N2OR phylogeny.

There are two distinct clades (clade I and II) for nosZ, which is the key functional gene of N2OR (12). Genomic differences between the two clades are associated with nos cluster organization, the translocation pathway, and co-occurrence with other denitrifying genes (13). Several studies have reported the physiological differences between the two clades. Yoon et al. (14) report that clade II bacteria (Dechloromonas aromatica and Anaeromyxobacter dehalogenans) showed high affinities to N2O but lower maximum reduction rates than those of clade I bacteria (Stutzerimonas stutzeri, formerly known as Pseudomonas stutzeri [15], and Shewanella loihica). In contrast, Suenaga et al. (3) found that the N2O reduction biokinetics could not be used to distinguish the clade I bacteria (S. stutzeri and Paracoccus denitrificans) and clade II bacteria studied (Azospira spp.). Nevertheless, it is still unclear how clade I and II N2O reducers behave in the presence of O2.

Therefore, the objectives of this study were to (i) characterize the oxygen sensitivity of various N2O reducing bacteria, (ii) classify N2OR based on their oxygen sensitivity, and (iii) examine the relationships between N2OR oxygen sensitivity, nosZ phylogeny (clade I versus clade II), and the predicted N2OR structures.

RESULTS

Michaelis-Menten kinetics of aerobic and anaerobic N2O reduction.

By fitting the N2O reduction results normalized by the optical density (OD) at 600 nm wavelength to the Michaelis-Menten model, we obtained the maximum rate (Vmax) and Michaelis constant (Km) values for various N2O-reducing strains under aerobic and anaerobic conditions. A wide range of Vmax for nitrous oxide reduction rates was observed. Under anaerobic conditions, bacteria with clade I N2OR generally exhibited faster N2O reduction than those with clade II N2OR (Fig. 1 and 2). Under anaerobic conditions, S. stutzeri TR2 (clade I N2OR) (Fig. 1-B1) had the highest Vmax (8.37 ± 0.81 μM/s/OD), whereas G. aurantiaca T-27 (clade II N2OR) (Fig. 2-C1) had the lowest Vmax (0.13 ± 0.02 μM/s/OD). This general trend in kinetics, however, may not extend beyond the studied strains, especially given the diversity of microorganisms harboring clade II NosZ (12).

FIG 1.

FIG 1

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Michaelis-Menten kinetics of anaerobic (1) and aerobic (2) N2O reduction and transition of aerobic into anaerobic N2O reduction (3) from Stutzerimonas stutzeri ZoBell (A), Stutzerimonas stutzeri TR2 (B), Paracoccus denitrificans JCM 21484 (C), and Azospirillum brasilense Sp7 (D). Curve fitting results were plotted in dashed lines.

FIG 2.

FIG 2

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Michaelis-Menten kinetics of anaerobic N2O reduction (1) and the transition of aerobic respiration into anaerobic N2O reduction (2) from Pseudomonas aeruginosa PAO1 (A), Dechloromonas aromatica RCB (B), and Gemmatimonas aurantiaca T-27 (C). Curve fitting results were plotted in dashed lines.

The ability to reduce N2O in the presence of O2 varied by strain, and there was no overall trend between the tested strains with clade I and II N2OR. For example, S. stutzeri TR2 (Fig. 1-B2) reduced N2O under aerobic conditions with the Vmax of 6.65 ± 0.37 μM/s/OD, whereas Pseudomonas aeruginosa PAO1 (clade I N2OR) (Fig. 2-A1) could not reduce N2O in the presence of O2. D. aromatica RCB (clade II N2OR) (Fig. 2-B1) also could not reduce N2O in the presence of O2. In contrast, Gemmatimonas aurantiaca T-27 (clade II N2OR) (Fig. 2-C2) exhibited a very slow N2O reduction rate under aerobic conditions, which increased once O2 was depleted. Azospirillum brasilense Sp7 (clade I N2OR) (Fig. 1-D2) reduced N2O in the presence of O2 up to 180 μM; however, its Vmax could not be fitted to the Michaelis-Menten model.

The transition points from aerobic to anaerobic N2O reductions (i.e., the change of the slopes between two linear rates) were clearly observed after oxygen was depleted for all tested strains, except for G. aurantiaca T-27. For G. aurantiaca T-27, the N2O reduction rate gradually changed depending on the oxygen concentration (Fig. 2-C2). In order to further investigate the different oxygen inhibition kinetics observed for G. aurantiaca, nonlinear least square fitting with multiple variables was used to determine the inhibition constant (Ki). The noncompetitive inhibition model was found to best describe the changing Vmax against various O2 and N2O concentrations (see Fig. S1 in the supplemental material), with a Ki value of 7.86 ± 1.69 μM O2.

The fitted Km values for anaerobic N2O reduction ranged from 1.85 ± 1.25 μM (for D. aromatica) to 11.14 ± 6.04 μM (for S. stutzeri TR2). The Km values of aerobic N2O reduction for P. denitrificans and S. stutzeri TR2 and ZoBell strains did not significantly differ from those of anaerobic N2O reduction (Student’s t test, P > 0.05). This finding indicates that the affinity of clade I N2OR tested did not change with and without the presence of O2.

Classification of oxygen sensitivity of N2O reduction.

Based on the microsensor analysis, a broad range of N2O reduction kinetics was observed under aerobic and anaerobic conditions. As we plotted the extrapolated anaerobic and aerobic Vmax values (Fig. 3A), three distinct types of responses to oxygen were found in the studied strains, as follows: (i) strains with Vmax not affected by oxygen, including S. stutzeri ZoBell and TR2, are classified as oxygen tolerant; (ii) strains with much lower aerobic Vmax than anaerobic Vmax, including P. denitrificans, A. brasilense, and G. aurantiaca, are classified as oxygen sensitive; and (iii) strains that have no N2O reduction activity when oxygen is present, including P. aeruginosa and D. aromatica, are classified as oxygen intolerant. NosZ phylogeny seems to be not associated with the classification of oxygen sensitivity. Moreover, the half-saturation coefficients for N2O under anaerobic and aerobic conditions agree with previously reported observations. Bacteria harboring clade II NosZ generally have lower Km values than those with clade I NosZ, suggesting differentiating ecological niches for these two groups of N2O-reducing bacteria (14).

FIG 3.

FIG 3

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Vmax for aerobic N2O reduction rates versus Vmax for anaerobic N2O reduction rates (A) and Vmax for O2 consumption (B) of each studied strain.

NosZ amino acid sequence similarities among the strains.

The NosZ amino acid sequences of the strains studied were compared to examine whether the observed differences in oxygen sensitivity originate from the differences in the enzyme structures. Strains investigated in this study cover a variety of classes, including Alphaproteobacteria (A. brasilense and P. denitrificans) and Gammaproteobacteria (S. stutzeri and P. aeruginosa) for those having clade I NosZ and Betaproteobacteria (D. aromatica) and Gemmatimonadetes (G. aurantiaca) for those having clade II NosZ. Based on the NosZ phylogenetic analysis, clade I and clade II NosZ were clearly separated (Fig. 4), similar to the previous report (16). The two S. stutzeri strains, of which both showed oxygen-tolerant N2O reduction, shared a high similarity in the NosZ amino acid sequences (92.6%) (see Fig. S4 in the supplemental material). However, P. aeruginosa PAO1, which showed oxygen-intolerant N2O reduction, also has similar NosZ amino acid sequences to S. stutzeri (77.5% with the ZoBell strain and 79.7% with the TR2 strain). NosZ of oxygen-sensitive N2O reducers (P. denitrificans, A. brasilense, and G. aurantiaca) and oxygen-intolerant N2O reducers (P. aeruginosa and D. aromatica) were not clustered with each other. In addition, we could not identify amino acid residues that appeared specific to each of the oxygen-tolerant, -sensitive, and -intolerant groups.

FIG 4.

FIG 4

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Phylogenetic tree for selected NosZ sequence constructed by the neighbor-joining method.

Multiple sequence alignment showed that the candidate ligands of CuA and CuZ centers were found in all NosZ sequences (see Fig. S3 in the supplemental material). The CuZ catalytic site contains seven histidine ligands which were all conserved in the proposed CuZ center among clade I and clade II (Fig. S3). The candidate ligands of CuA (two cysteines at positions 618 and 622, two histidines at positions 583 and 626, and a methionine at position 629 in P. stutzeri NosZ) were also identified in all NosZ (Fig. S3).

NosZ structural similarities.

To identify the structural differences between oxygen-tolerant, -sensitive, and -intolerant NosZ, we predicted the enzyme structures based on the NosZ sequences by using Alphafold2 (17) with the ZoBell NosZ (18) as a query structure. We obtained high-confidence NosZ structures, as evaluated based on the sequence coverage and predicted per-residue confidence measure (pLDDT) scores from AlphaFold, with conserved CuA and CuZ catalytic domains (see Fig. S2 in the supplemental material). Slight structural differences were seen between clade I and II NosZ as measured by the Dali Z-scores, whereas no differences were seen between NosZ structures from oxygen-tolerant, -sensitive, and -intolerant strains. The Z scores for all clade I NosZ against the reference ZoBell NosZ were ≥59.8. In addition, the predicted structures for all clade I NosZ showed the root mean square deviation (RMSD) value of <2.0 and had no structurally dissimilar amino acid residues of longer than 80 amino acids (aa) to the reference NosZ. In contrast, the Z scores for the NosZ of D. aromatica and G. aurantiaca (clade II) were 50.8 and 49.3, respectively. Poor matches with the query sequence were obtained for the clade II NosZ with RMSD values of >2.0 and structurally dissimilar amino acid residues of >80 aa. Most of the structural heterogeneity was observed in the C and N terminals.

DISCUSSION

Biological N2O reduction is generally believed to occur under strictly anaerobic conditions. The oxygen sensitivity of N2O reduction can be explained by (i) the transcriptional regulation of nos and (ii) the inactivation of N2OR by molecular oxygen. The transcription of nosZ can be regulated directly or indirectly by O2-sensing transcriptional regulators. For instance, the transcription of nosZ is directly regulated by fumarate and nitrate reductase protein (FnrP) in response to oxygen depletion in P. denitrificans (19). P. aeruginosa also has similar FNR-type sensing regulators; the cascading regulation of anaerobic regulator of arginine deiminase and nitrate reductase (ANR) and dissimilatory nitrate respiration regulator (DNR) indirectly controls the synthesis of N2OR (20). Another potential explanation of oxygen sensitivity points to the inactivation of N2OR upon exposure to oxygen. The N2OR isolated under aerobic and anaerobic conditions exhibited various redox and spin states of copper in active sites. Under limited exposure to oxygen, the enzyme shifted in electron paramagnetic resonance spectra but retained its N2O-reducing activity (21). In contrast, aerobic incubation caused loss of copper content and inactivation of the catalytic site. Inactivation of N2OR by oxygen was also reported due to irreversible confirmation changes. A sulfur atom binding to the active site of N2OR isolated from S. stutzeri ZoBell was lost during aerobic enzyme isolation, leading to irreversible inactivation (6).

However, these two mechanisms do not explain the occurrence of O2-tolerant N2O reducers. The cells used for the microsensor experiments were incubated under anaerobic conditions with the addition of nitrite or N2O to induce the expression of N2OR. One exception is for G. aurantiaca T-27T. This strain was incubated under aerobic conditions, as G. aurantiaca T-27T is an obligate aerobic bacterium that can express nosZ in the presence of O2 (11, 22). The same cell cultures were used for aerobic and anaerobic N2O reduction rate measurements; therefore, the initial level of N2OR expressed in the cells should be the same between the two conditions (i.e., aerobic versus anaerobic N2O reduction). Consequently, the transcriptional regulation of nos is not contributing to the O2 tolerance during N2O reduction of each tested strain.

In addition, the structures of NosZ, including the active sites, were highly similar between O2-tolerant, -sensitive, and -intolerant N2O reducers. Based on the structural similarity and the presence of conserved residues in the active sites, all of the active sites of NosZ and copper cofactors examined most likely receive similar inhibitory effects upon exposure to oxygen (6, 21). Despite similar N2O respiration and bioenergetics in clade I and clade II NosZ, other accessory proteins encoded by the nos cluster are expected to function differently (23). These auxiliary processes could be involved in the maintenance and repair of NosZ, with detailed mechanisms remaining unclear.

Another mechanism that may explain the observed occurrence of O2-tolerant N2O reduction is the scavenging of O2 in the cells. A whole-cell assay (as opposed to the assay done with isolated enzymes) was used in this study to calculate the N2O and O2 consumption rates. When both N2O and O2 are present, facultatively anaerobic bacteria (e.g., denitrifiers) usually prioritize the respiration of O2 over N2O because aerobic respiration is more favorable from both bioenergetic and kinetic perspectives (24). A rapid O2 consumption rate can potentially lower the in situ O2 concentration in the periplasm, where N2OR is located. From a simplified estimation shown in the supplemental materials, an O2 consumption rate of 1 μM/s/OD can cause a significant decrease in O2 concentration across cell membranes. When the O2 respiration rate is comparable to the O2 diffusion rate that replenishes dissolved oxygen in the periplasm, the local oxygen minimum could protect N2OR from inhibition in O2-tolerant N2O reducers. From the tested strains, we indeed observed that bacteria with higher oxygen consumption rates generally have greater oxygen tolerances (Fig. 3B). A threshold of O2 consumption rate could potentially exist, where a lower rate could not emulate the diffusion rate of O2 sustaining an anaerobic zone for N2OR. Such a protection mechanism could be analogous to the respiration of O2 in Azotobacter protecting O2-sensitive nitrogenase (25).

Our results have some implications for N2O removal applications. N2O-reducing bacteria, including some of the strains examined in this study, have been used for N2O mitigation in natural and engineered systems (2). For instance, bioaugmentation of S. stutzeri TR2 to denitrifying activated sludge has been demonstrated to mitigate N2O emissions (26, 27). Azospirillum brasilense strains were also used as a microbial inoculant for N2O mitigation in soil (28). Nevertheless, engineering applications of biological N2O mitigation face major challenges, including the oxygen sensitivity of N2O reduction due to the coexistence and fluctuations of dissolved oxygen and N2O concentrations commonly observed in natural and engineered systems. Based on the classification of O2 tolerance in this study, kinetic parameters can be used as selection criteria for microorganisms in environmental applications. Oxygen-tolerant N2ORs were identified only in S. stutzeri in this study. S. stutzeri also exhibited some interesting kinetics when both electron acceptors (O2 and N2O) are present. The TR2 strain showed preferred N2O respiration over oxygen, contrary to predictions based on electron supply rate to the electron transport chain (29). In addition, the ZoBell strain can reduce N2O fast and in the presence of O2, making it promising for N2O bioremediation applications. Besides N2O reduction rates, microorganisms with low Km values, such as P. denitrificans and D. aromatica, could be useful in scavenging low concentrations of dissolved N2O. It is important to note, however, that the kinetics and O2 sensitivity of N2O reducers can be influenced by environmental factors, such as the type of organic carbons (30) and temperature (31). Therefore, when selecting appropriate N2O reducers for engineering applications, their N2O reduction kinetics and O2 sensitivity should be measured under environmentally relevant conditions.

MATERIALS AND METHODS

Bacterial strains.

Stutzerimonas stutzeri strain TR2 was kindly provided by Otsubo, Miyauchi, and Endo at Tohoku Gakuin University, Japan. Stutzerimonas stutzeri strain ZoBell (=ATCC 14405) and Dechloromonas aromatica strain RCB (=ATCC BAA-1848) were obtained from the American Type Culture Collection (ATCC). Pseudomonas aeruginosa PAO1 (=JCM 14847), P. denitrificans JCM 21484T, and A. brasilense Sp7T (=JCM 1224T) were obtained from the Japan Collection of Microorganisms (JCM). Gemmatimonas aurantiaca T-27T (=NBRC 100505T) was obtained from Biological Resource Center (NBRC; Kisarazu, Japan).

These strains, except for D. aromatica RCB and G. aurantiaca T-27T, were grown on R2A agar plates amended with 10 mM acetate and 5 mM nitrite under aerobic conditions. After 48 h of incubation at 30°C, single colonies were picked and transferred to 10 mL of R2A broth with 10 mM acetate and 5 mM nitrite. Each liquid culture was incubated in a sealed tube with an N2 atmosphere at 30°C until harvested during the exponential growth phase. D. aromatica RCB was grown on Trypticase soy agar (TSA) supplemented with 5% defibrinated sheep blood under anaerobic conditions at 30°C for 10 days. Single colonies were transferred to 10 mL of R2A broth supplemented with 20 mM lactate and incubated under a 1.39% N2O atmosphere (in N2) at 30°C until harvested. G. aurantiaca T-27T was grown on R2A agar under aerobic conditions. Single colonies were transferred to 10 mL of R2A broth and aerobically incubated at 25°C until harvested. The addition of nitrite inhibited the growth of G. aurantiaca, which was expected to have an incomplete denitrification pathway (32).

Microsensor experiments.

Bacterial cultures were harvested during the early to mid-exponential growth phase as determined by the optical density at 600 nm (OD600) measurement. Cultures were washed twice with a sterile 10 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 7.5) and resuspended in a PIPES buffer supplemented with 10 mM sodium acetate. The cell suspensions were purged with a gas mix of N2O (1.39%, vol/vol) in N2 for 10 min to achieve targeted levels of dissolved N2O concentrations (300 μM). The cell suspensions were then diluted with PIPES buffer to the desired concentration (~106 CFU/mL; OD600, ~0.1) and transferred to a double chamber containing mini stirrer bars (Unisense, Aarhus, Denmark) (see Fig. S5 in the supplemental material). The chamber was capped and placed in a sensor rack with built-in stirrers and submerged in a 30°C water bath. An N2O microsensor and an O2 microsensor (Unisense) were inserted into the chamber via small halls to measure dissolved N2O and O2 concentrations every second for up to 2 h or until O2 depletion. The N2O and O2 microsensors were two-point calibrated with zero and saturated solutions (300 μM for N2O and 236 μM for O2) at 30°C. No cross interference was observed between N2O and O2 on respective microsensors (see Table S1 in the supplemental material). The OD600 of the cell suspension was recorded at the end of each microsensor test. At least three independent microsensor measurements were done for each strain.

The measured concentrations of N2O and O2 were averaged over time intervals of 100 to 1000 s depending on the duration of microsensor tests. This step is useful to minimize the noise generated by the microsensors. Linear rates for N2O consumption were extrapolated within each time interval. The Michaelis-Menten plots were then constructed using the rates and corresponding N2O concentrations. A nonlinear least square method with the Levenberg-Marquardt algorithm (33) was used for curve fitting on Origin 2021 (version 9.8.0.200) to determine kinetic parameters, including the maximum rate (Vmax) and the Michaelis constant (Km). Similarly, Vmax for O2 was linearly extrapolated from O2 concentrations measured by the microsensor.

Bioinformatics and comparative protein structure modeling.

The NosZ sequences of the selected strains (GenBank accession numbers WP_011287329, EHY76008, BAM68548, NP_252082, QEL93987, WP_156798935, and Q51705 for D. aromatica RCB, S. stutzeri ZoBell, S. stutzeri TR2, P. aeruginosa PAO1, A. brasilense Sp7, G. aurantiaca T-27, and P. denitrificans JCM 21484, respectively) were retrieved from National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov). Multiple sequence alignment and phylogenetic tree construction were done using the neighbor-joining method without distance correction by using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). NosZ structures were predicted through the nondocker implementation of AlphaFold2 version 2.1.1 via the Minnesota Supercomputing Institute (MSI). The NosZ sequence of the selected strains was used as the input with the default prediction parameters to run on a Linux environment. The best-predicted protein models were selected for each sequence and loaded into PyMOL (Schrödinger, Inc., New York, NY). All models were colored based on their predicted local distance difference test (pLDDT) that are stored in the B-factor fields of the PDB files. All predicted structures were compared against each other using DaliLite.v5 (http://ekhidna2.biocenter.helsinki.fi/dali) (34).

Supplementary Material

Reviewer comments
reviewer-comments.pdf (265.9KB, pdf)

ACKNOWLEDGMENTS

We thank Wakako Otsubo, Keisuke Miyauchi, and Ginro Endo Tohoku Gakuin University, Japan for providing Stutzerimonas stutzeri strain TR2. We also thank Sujin Yeom and Mike Blazanin for their help with the initial experimental setup and Carrie Wilmot for valuable comments.

This work was supported by the Biocatalysis program and the MnDRIVE Initiative of the University of Minnesota.

Footnotes

Supplemental material is available online only.

Supplemental file 1
Supplemental material. Download spectrum.04709-22-s0001.pdf, PDF file, 5.6 MB (5.6MB, pdf)

Contributor Information

Satoshi Ishii, Email: ishi0040@umn.edu.

Victor Gonzalez, CCG-UNAM.

Maria Delgado, Consejo Superior de Investigaciones Cientificas.

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