The Respiratory Way without Microbial Growth of Paracoccus denitriﬁcans

: This study elucidates the effects of Carbon/Nitrogen (C/N) ratios on the respiratory behavior of Paracoccus denitriﬁcans PD1222, a microorganism noted for its metabolic adaptability. We explored its ability to undergo dissimilative denitriﬁcation, a less understood process where energy is harnessed from nutrient consumption without resultant growth. By manipulating the C/N ratios and available nitrogen sources in our experimental design, we were able to demonstrate signiﬁcant shifts in P. denitriﬁcans metabolic behavior. At a C/N ratio of 1.34, with nitrate as the sole nitrogen source, dissimilative denitriﬁcation occurred with no observable increase in biomass. Succinate, the provided carbon source, was quickly metabolized without contributing to cell growth. Our results contribute to the understanding of environmental microbiology, speciﬁcally denitriﬁcation processes, and indicate P. denitriﬁcans’s potential for wastewater treatment scenarios, where pollutant consumption without biomass proliferation is desired.


Introduction
Microbial respiration, a vital process that supports the life of microorganisms, hinges upon the presence of a final electron acceptor, which is crucial for maintaining energy processes involved in ATP production.The role of this final electron acceptor in respiration is to alleviate the excess reducing potential derived from oxidation [1].This reducing potential, an overabundance of electrons, is a result of organotrophic or chemotrophic catabolism and the regeneration of NAD + [2,3].The oxidative strength of the final electron acceptor is dictated by its redox potential.In this context, oxygen emerges as the quintessential oxidizer with a redox potential of O 2 /H 2 O +1.23 V vs, marking it as the most potent oxidizer involved in respiration found in nature, a process identified as aerobic respiration [4,5].
Conversely, several oxidizers with redox potentials lower than that of O 2 correspond to anaerobic respiratory processes, such as those involving NO 3 − , SO 4 − , Fe, Mn, and others [1,6].Microbial respiration, whether aerobic or anaerobic, can be interpreted from two perspectives: assimilative or dissimilative.Metabolic cycles involve assimilatory and dissimilatory processes related to elements such as C, N, and P. Assimilatory processes incorporate these elements into biomass or release them during catabolism.Moreover, many microbes engage in dissimilatory coupling, driving energy-releasing reactions linked to changes in the oxidation state of elements, subsequently releasing these transformed elements into the environment.The various inorganic or organic compounds yield differing amounts of energy, and the interaction between these processes forms a microbial energy economy.Notably, dissimilatory reactions, including those concerning C, N, Fe, and S, exemplify how microbially mediated oxidation-reduction (redox) transformations impact nutrient availability for the metabolism [7].The former takes place alongside microbial growth, while the latter transpires when microorganisms sustain respiration without entering cell duplication.In the case of aerobic respiration, the mechanisms of dissimilative metabolism are less clear, in stark contrast to anaerobiosis.In the latter scenario, various electron acceptors not only partake as oxidizing agents in respiration but also often act as sources of micro-or macronutrients, such as NO 3 − , SO 4 − , PO 4 − , and so forth [8].Dissimilative respiration, although a little-known concept in general microbiology, is of paramount importance in environmental microbiology.It is especially desirable in wastewater treatment systems where the ideal scenario is for microorganisms to consume pollutants without proliferating.Biological denitrification, a specific process wherein NO 3 − is reduced to N 2 in four steps, couples this respiratory process with the oxidation of organic matter [9][10][11].There is extensive documentation on how this biogeochemical process can oscillate between assimilative and dissimilative respiration.The Carbon/Nitrogen (C/N) ratio is fundamental in determining whether the metabolic process will be assimilative or dissimilative [12,13].Investigations by Jia et al., [14] and Sobieszuk et al. [15] underscore the crucial role of the C/N ratio in denitrification.They propose that the nature of the C/N sources significantly influences the rate of reduction of NO 3 − and NO 2 − [14].The precise dosage of both sources is pivotal, given that an extra source of carbon may induce excessive biomass growth, thereby elevating the consumption and assimilation of nitrogen [16].
Complete denitrification is typically attained when working with C/N ratios that are close to or at stoichiometric equivalence.Hence, if the goal is to operate under dissimilative conditions, low C/N ratios are optimal.However, if microbial growth accompanying denitrification is desired, high C/N ratios should be implemented.This shift from assimilative (with growth) to dissimilative denitrification has been frequently observed in environmental processes with consortia.In contrast, this transition has not been reported in axenic strains, as most studies [17][18][19] aim to foster microbial growth during the anaerobic respiratory process.
Paracoccus denitrificans is one of the quintessential microorganisms, known for its remarkable metabolic versatility.Being facultative, it can respire with a wide array of final electron acceptors.It is a gram-negative prokaryote, possessing a respiratory system akin to that of mitochondria [20][21][22].The objective of this study was to facilitate dissimilative denitrification by P. denitrificans, a feat not yet reported in an axenic strain.

Culture Media
In this study, a denitrifying culture medium was employed, consisting of Na 2 HPO 4 (29 mM), NaH 2 PO 4 (11 mM), NH 4 Cl (5.4 mM), MgSO 4 (0.4 mM), EDTA (342 µM), µM), and CoCl 2 •6H 2 O (13.5 µM); this medium was employed to assess C/N ratios of 1.22, 1.46, and 1.83, respectively.For an additional experiment evaluating a ratio of 1.34, the culture medium underwent changes, as indicated in Table 1.These modifications included the removal of NH 4 Cl and the utilization as the sole N source (NaNO 3 ).It is important to note that all the tested ratios commenced from a pre-inoculum of P. denitrificans of 18 h in Luria-Bertani (28 • C, agitation of 200 rpm) with an absorbance of 0.6 (ABS 600 nm) that served as the starting point; the strain was donated by Dr. José de Jesus García Trejo (Universidad Nacional Autónoma de México).The biomass was centrifuged (4000 rpm, 10 min) and washed with a sterile physiological saline solution (0.9% NaCl).Following this, the biomass was resuspended in the culture medium, which was devoid of both the C source and the N source, in 10 mL serological bottles, each containing 5 mL of the culture medium (sans C and N) and 1 mL of biomass.Subsequently, the bottles were sealed and purged with helium for 5 min.The C and N sources were then reintroduced in varying proportions (1.22, 1.34, 1.46, and 1.83 mg C/mg N) by injecting 1 mL of the respective solutions and were then incubated (28 • C, 200 rpm, 36 h).The value of different relations of C/N is based on Equation (1).
The N sources were eliminated (Table 1), with NO 3 − being the sole exception and media modification.Each experiment was conducted in triplicate.

−
The quantification of NO 3 − , NO 2 − and succinate was carried out in HPLC equipment (Dionex 3000, Thermo Cientific, CA, USA).For NO 3 − and NO 2 − , a WATERS IC-Pak Anion HC 4.6 × 150 mm column was used with a retention time of 22 min, and the sample was read at 214 nm.As the mobile phase, the one provided by the manufacturer was used at a flow of 2 mL/min.The sample injection volume was 10 µL.
For succinate, a GRACE Alltech OA-1000 Organic Acids 300 mm × 6.5 mm column was used with a retention time of 11 min.The samples were acidified with a drop of concentrated phosphoric acid and read at 210 nm.As the mobile phase, 25 mM KH 2 PO 4 pH 2.5 was used.The sample injection volume was 10 µL.

Quantification of N 2
For the quantification of N 2 , 9 µL were taken directly from the reactor and injected into a gas chromatograph (SRI 310C, Atlanta, GA, USA), equipped with an electrical conductivity detector (TCD) and a ShinCarbon ST, 100/120 mesh column, 2 m, 1/16 in.OD, 1.0 mm ID.Helium was used as carrier gas at a flow of 10 mL/min.Each experiment was conducted in triplicate.

Effect of Different Ratio C/N
The findings pertaining to the evaluated C/N ratios (1.22, 1.34, 1.46, and 1.83) are illustrated in Figure 1.
Regarding the window of C/N ratios in which dissimilative denitrification occurs, our study suggests that there could be a specific range around the 1.34 C/N ratio where this phenomenon is observed.However, we recognize that this might not be the sole ratio for such an occurrence.Expanding the study to other close ratios, both below 1.22 and above 1.46, could provide more comprehensive insights and constitutes a direction that we are considering for future research.
As depicted in Figure 1A-C, the biomass of P. denitrificans increases in response to an additional source of nitrogen in the form of NH 4 Cl, which is especially noticeable at the ratios of 1.22, 1.46, and 1.83, while the Carbon/Nitrogen (C/N) ratio commences at the stoichiometric value (1.22).
As depicted in Figure 2, a clear demonstration of dissimilative denitrification is presented.The entirety of the consumed nitrate is recovered at diverse sampling intervals, exclusively as N 2 or NO 2 − .At the culmination of the five-hour cultivation period, the nitrate is fully converted into molecular nitrogen.This implies that without an auxiliary nitrogen source, cell duplication was impossible, a fact substantiated by the nitrogen mass balance.
Microbiol.Res.2023, 14, FOR PEER REVIEW As depicted in Figure 1 (A, B, C), the biomass of P. denitrificans increases in response to an additional source of nitrogen in the form of NH4Cl, which is especially noticeable a the ratios of 1.22, 1.46, and 1.83, while the Carbon/Nitrogen (C/N) ratio commences at the stoichiometric value (1.22).As depicted in Figure 2, a clear demonstration of dissimilative denitrification is pre sented.The entirety of the consumed nitrate is recovered at diverse sampling intervals exclusively as N2 or NO2 − .At the culmination of the five-hour cultivation period, the ni trate is fully converted into molecular nitrogen.This implies that without an auxiliary nitrogen source, cell duplication was impossible, a fact substantiated by the nitrogen mass balance.Figure 1 characterizes an assimilative behavior brought about by the assimilation of the ammonium source, which is tied to the growth of the microorganism.Conversely, Figure 2 depicts dissimilation: it involves the utilization of the carbon source to liberate ATP without the inclusion of organic matter for growth.In this latter scenario, P. denitrificans solely maintains respiration.An electron balance reveals that the entire reducing potential, acquired from the oxidation of succinate, was accepted by nitrate (NO3 − ), reducing it completely to nitrogen (N2).At the C/N ratio of 1.34 (Figure 2) and in the absence of an additional N source, conditions shift, resulting in no observable increase in microbial growth.The growth curve remains nearly linear, with no statistically significant differences (CI 95%, p > 0.05).

Discussion
The C/N ratio holds a significant influence over the expression of dissimilative metabolism.This is typically associated with microorganisms that are constrained by nutrient availability, including bacteria present in terrestrial and aquatic habitats.These organisms utilize this metabolism as an effective survival strategy, enabling them to flourish in environments that other microorganisms find challenging.In broad terms, conditions conducive to the manifestation of this metabolism include limited nutrient availability and the presence of high-energy substrates, such as carbon-or nitrogen-rich compounds.These compounds offer energy without necessitating additional nutrients for cell growth.Another contributing factor is the presence of electron acceptors, such as nitrate (NO3 − ).Additionally, inducing anaerobic conditions may compel the microorganism to utilize an electron donor distinct from oxygen (O2)-a behavior evidenced in this study.Figure 1 characterizes an assimilative behavior brought about by the assimilation of the ammonium source, which is tied to the growth of the microorganism.Conversely, Figure 2 depicts dissimilation: it involves the utilization of the carbon source to liberate ATP without the inclusion of organic matter for growth.In this latter scenario, P. denitrificans solely maintains respiration.An electron balance reveals that the entire reducing potential, acquired from the oxidation of succinate, was accepted by nitrate (NO 3 − ), reducing it completely to nitrogen (N 2 ).At the C/N ratio of 1.34 (Figure 2) and in the absence of an additional N source, conditions shift, resulting in no observable increase in microbial growth.The growth curve remains nearly linear, with no statistically significant differences (CI 95%, p > 0.05).

Discussion
The C/N ratio holds a significant influence over the expression of dissimilative metabolism.This is typically associated with microorganisms that are constrained by nutrient availability, including bacteria present in terrestrial and aquatic habitats.These organisms utilize this metabolism as an effective survival strategy, enabling them to flourish in environments that other microorganisms find challenging.In broad terms, conditions conducive to the manifestation of this metabolism include limited nutrient availability and the presence of high-energy substrates, such as carbon-or nitrogen-rich compounds.These compounds offer energy without necessitating additional nutrients for cell growth.Another contributing factor is the presence of electron acceptors, such as nitrate (NO 3 − ).Additionally, inducing anaerobic conditions may compel the microorganism to utilize an electron donor distinct from oxygen (O 2 )-a behavior evidenced in this study.
Equation ( 1) displays the calculations for the amount of carbon needed to fully reduce the associated nitrate to N 2 .Based on these calculations, the molar C/N ratio essential for the complete reduction of nitrate stands at 0.357 mol C/mol N. For the purposes of conducting N mass balances, this was expressed as mg C/mg N.This means that a molar ratio of 0.357 mol C/mol N corresponds to a mass ratio of 1.22 mg C/mg N.Moreover, it is a widely accepted fact that the reducing power derived from the catabolism of carbon compounds is not entirely recuperated; a portion is lost as metabolic heat [23].Thus, at any given point, there would not be sufficient reducing power to completely reduce NO 3 − to N 2 using only a C/N ratio of 1.22.Drawing from this understanding and our research group's prior experience, we chose to assess carbon/nitrogen ratios surpassing the stoichiometric value, suggesting a 10% carbon increase (C/N = 1.34 and 1.47).A 25% increase (C/N = 1.83) was also put forward.We selected the C/N ratio of 1.83 to ascertain that the microorganism had the potential to grow and to ensure that the lack of biomass growth was attributed solely to the C/N ratio and not to other factors.
In Figure 1, it is evident that despite having a low C/N ratio, growth was still observed at 1.22, 1.46, and 1.83.These C/N values only consider NO 3 as the nitrogen source.However, the culture medium used in this kinetics included an additional nitrogen source, ammonium chloride (NH 4 Cl).This explains the observed biomass, as there are two distinct nitrogen sources, and ammonium assimilation is typically preferred over nitrate because it requires less energy.It has been reported that for Paracoccus denitrificans PD1222, nitrate assimilation is negatively controlled by ammonium through the two-component NtrBC system [24].As it is a completely anaerobic process, nitrate had to be used as the final electron acceptor.Once the ammonium was consumed, a portion of the nitrate was directed towards growth.This was confirmed with the nitrogen yields (Y N2 ) of 0.88 for a C/N of 1.22, 0.88 for a C/N of 1.46, and 0.85 for a C/N of 1.83.As a result, the culture medium was reformulated by removing the ammonium source to eliminate interferences.Yet, it is clear that the C/N ratio influences the denitrification process based on the observed nitrogen yields, as nitrate consumption efficiencies were 100% for those three C/N ratios.After removing the ammonium source and based on observations with a C/N of 1.22, which might be too limited in reducing power for a complete NO 3 reduction, a C/N of 1.34 was tested.As seen in Figure 2, the molecular nitrogen yield (Y N2 ) was 1, indicating that all the nitrogen in nitrate form was reduced to N 2 .This left no available nitrogen to support culture growth, which is supported by the biomass trend in the same figure.
The static growth (Figure 2) can be attributed to the fact that a microorganism does not accumulate biomass when nurtured in an environment deficient in nitrogen [25].Considering that nitrate was the sole nitrogen source available and was subsequently recovered entirely as molecular nitrogen, there was an inherent constraint on any potential for biomass growth.Without an available nitrogen source, an essential component for amino acid and nucleotide synthesis, Paracoccus would face significant challenges in augmenting its biomass.
Furthermore, with the limitation of the carbon source in our experimental design, it would also curtail the potential for excess carbon assimilation, which is often channeled into storage molecules such as polyhydroxyalkanoate (PHA).
Thus, while PHA production could theoretically attenuate the optical density readings, the restrictive conditions under which the experiment was conducted reinforce our findings towards a predominantly dissimilative metabolic behavior.The entirety of our data, when viewed in light of these conditions, underscores the likelihood that Paracoccus, in this specific setting, utilized a predominantly dissimilative pathway, with a predisposition to use NO 3 − as the terminal electron acceptor [21].The inclusion of a carbon source, in the form of succinate, facilitated its swift consumption by P. denitrificans.This is due to the presence of succinate dehydrogenase being constitutive in the microorganism, which aids in converting succinate to fumarate.This conversion enables a rapid entrance into the truncated Krebs cycle [26] for efficient consumption.Upon depletion, succinate may be released into the environment as carbon dioxide.However, under our experimental design, the microorganism generates energy without incorporating carbon into its biomass.
Since the 1980s, it has been suggested that P. denitrificans can adapt flexibly to changes in growth conditions, with this adaptation involving the redirection of electrons through an extant redox network.Yet this network modifies itself in response to the presented changes, leading to the generation of signal transduction pathways that are either individually organized or networked.This regulation includes the enzymes that participate in the denitrifying process.
While Paracoccus denitrificans is known to possess three distinct nitrate reductases, it is the enzyme anchored to the internal membrane, oriented towards the cytoplasm, that predominantly exhibits dissimilative characteristics.According to studies by Olaya-Abril et al. [21] and Kučera et al. [27], this particular enzyme is the key director of the denitrification process.Although the other nitrate reductases within the organism have the potential to participate in the assimilative reduction of nitrate, their contribution towards growth utilization is minimal or potentially negligible under the conditions of our study.Thus, based on the cited research and our collected data, we postulate that the observed activity is primarily attributed to this specific dissimilative nitrate reductase, while the roles of the others in growth-related assimilation are largely overshadowed or discarded.
On the other hand, a balance must be ensured in the electron flow of the rest of the participating enzymes to avoid the accumulation of intermediate compounds toxic to the cells.The levels of these intermediaries must be kept to a minimum to optimize the efficiency of the process [28,29].
In this context, we have yet to determine a concentration range that indicates inhibition by any of the intermediaries, such as NO 2 and NO, suggesting that this is dependent on experimental conditions.Specifically, in our study, we did not observe an accumulation of NO 2 .However, with a 1.42 ratio (Figure 1), a higher concentration was noted at 24 h, concurrently with a notable decrease in NO 3 consumption.These behaviors are at odds with those observed in the other two tested ratios.The accumulation of the intermediaries assumes a significant importance, as inhibitory effects have been reported during NO 3 reduction.N 2 O exhibits a dual effect by both inhibiting the reduction of NO 3 through electron competition and stimulating its consumption via the NO 3 /NO 2 antiport system.After the reduction of NO 3 , this system has been demonstrated to not require additional energy for NO 3 adsorption [30].
The redox state of intermediary compounds in the denitrification process directly influences the reduction of NO 3 , prompting a shift in the distribution of electron flow from the substrate (in this case, succinate) to NO 3 .This shift correlates with respiratory regulation signals through NADH production and substrate feedback [31].This intricate balance is what P. denitrificans seeks in order to achieve homeostasis and execute its metabolic activities, presenting itself as a microorganism capable of adapting to stressful circumstances to ensure survival.Consequently, this report serves as the first to highlight the denitrifying respiration of P. denitrificans without biomass growth.

Conclusions
In our study, we endeavored to unravel the complex metabolic pathways and adaptability of P. denitrificans under varying conditions.Our findings provide robust evidence that under specific C/N ratios, this microorganism predominantly engages in dissimilative denitrification.This distinct shift from assimilative processes underscores the organism's resilience and adaptability to fluctuating environmental inputs.Of particular note was the C/N ratio of 1.34, which emerged as a potent facilitator of this dissimilative metabolic pathway.This ratio starkly halted significant biomass growth, suggesting a unique metabolic response under such conditions.However, it is imperative for future research to determine if this is confined to a narrow C/N range or if similar behaviors manifest outside the parameters we examined.
Diving deeper into the enzymatic mechanisms, it is evident that while P. denitrificans possesses three nitrate reductases, not all play a role in this dissimilative process.Our data aligns with previous literature, highlighting the enzyme anchored to the internal membrane, oriented towards the cytoplasm, as the primary driver of dissimilative denitrifi-cation.Concurrently, the other nitrate reductases appear more attuned to growth-related assimilation, especially under the conditions we investigated.
One of the pivotal challenges we encountered revolved around biomass measurements.While optical density offered a lens into the organism's metabolic responses, potential variances such as the production of PHA might influence readings.However, our nitrogen source conditions and nitrate recovery data resonate with a compelling narrative: despite potential variations in optical density, P. denitrificans predominantly leaned towards a dissimilative metabolism in our experimental setup.
In sum, the adaptability showcased by P. denitrificans in this study, marked by its ability to undergo denitrifying respiration without notable biomass growth, not only accentuates its ecological significance but also positions it as a potential asset in biotechnological applications.As we pave the way for future research, we believe a deeper exploration into C/N ratios, the intricate roles of nitrate reductases, and alternative biomass measurement techniques will be instrumental in refining our understanding and harnessing the potential of this remarkable microorganism.