Microbial Degradation of Pyridine by Co-culture of Two Newly Isolated Strains, Arthrobacter sp. Strain PDC-1 and Rhodococcus sp. Strain HPD-2

Abstract


Introduction
Pyridine and its derivatives are of major concern as carcinogenic, teratogenic, and mutagenic environmental pollutants.Pyridine is classified as a hazardous substance in the USEPA list of priority pollutants (Richards and Shieh, 1986), and is extensively used in industries as solvent and platform chemical for the synthesis of textiles, pharmaceuticals, agrochemicals and industrial manufacturing (Scriven and Murugan, 1996;Sims et al., 1989).Pyridine is volatile, the hetero-aromatic ring makes it persistent in the environment, and the nitrogen atom gives the hydrophilic properties to pyridine, which makes it better dissolved in water than its carbocyclic analogs.The pyridine and its oxidized derivatives may cause severe health implications (Sun et al., 2013).Therefore, removing pyridine from the environment or wastewater has long caught the researchersʼ attention (Bouyarmane et al., 2010;Li et al., 2017;Sims et al., 1989).The existing physicochemical purification methods are not very efficient when they treat nitrogen heterocyclics (Chu et al., 2018;Li et al., 2017).Therefore, the biological degradation of pyridine is considered to be an attractive approach compared with physical or chemical methods.Pyridine degradation by strain(s) individually or immobilized strain(s) on fixing agents in bioreactors is a subject of a good deal of research (Lin et al., 2010;Uma and Sandhya, 1998;Wen et al., 2013).In general, the pyridine strain with high pyridine degrading capacity will enhance the pyridine degradation capacity in the bioreactor through bioaugmentation (Jin et al., 2020;Liu et al., 2020;Lodha et al., 2008;Uma and Sandhya, 1998;Wen et al., 2013;Xue et al., 2020;Zhang et al., 2018).Therefore, the study of pyridine degrading strain(s) is one of the core issues of pyridine removal.To data, several strains have been isolated from the environment which could use pyridine as the sole source of carbon and nitrogen, including Achromobacter, Alcaligenes, Arthrobacter, Azparcus, Bacillus, Gprdpmoa, Micrococcus, Nocardioides, Nocardia, Paracoccus, Pseudomonas, Rhizobium, Rhodococcus, Rhodopseudomonas, Shewanella, Shinella et al (Jin et al., 2020;Liu et al., 2020;Mathur et al., 2008;Wang et al., 2018;Xue et al., 2020;Zhang et al., 2018).The isolates presented different degradation capacities.Some strains could grow with 5500 mg/L initial concentration of pyridine and completely removed the pyridine within 192 hours (Stobdan et al., 2008), while some strain could bear only 20 mg/L pyridine and the degradation efficiency was dropped significantly with 30-50 mg/L pyridine (Kaiser and Bollag, 1992;Lodha et al., 2008).
Therefore, the high pyridine tolerance and efficient pyridine degradation strain still to be elucidated.
In the wild, the pyridine degrading strain lives in a complex environment with other nutrients or other strains.All the elements will influence the pyridine degradation (Chandra et al., 2009).The pyridine degradation capability will be enhanced by adding some nutrients such as glucose as well as other pollutants such as phenol (Bai et al., 2009;Quan et al., 2017;Sun et al., 2011).Besides, other elements such as the products of pyridine degradation, ammonia, will also affect the pyridine degradation (Qiao and Wang, 2010;Wang et al., 2018).Pyridine degradation will also be influenced by fixation methods, as well as bioreactor and zeolite (Bai et al., 2010;Wen et al., 2013).
Co-cultures of bacteria for pyridine degradation has also been studied.In general, mixed culture was usually more effective for pyridine degradation compared with individual strain, which might due to the complementary metabolism of different strains (Chandra et al., 2010).However, the function of different strains in pyridine degradation was not fully understood.The research of the co-cultures for pyridine degradation will help us to elucidate the interaction between strains and the adaptation to the environment.
In this study, a pyridine-degrading bacterial consortium was obtained and two single strains with pyridine degrading capacity were isolated from the bacterial consortium.The degradation properties of the co-culture of the two isolates were investigated.The growth kinetics was investigated for better understanding the pyridine degradation.In addition, the metabolic products of the two strains were also analyzed.

Chemicals
Pyridine, 2-hydroxypyridine, 3-hydroxypyridine, 4-hydroxypyridine and monocarboxylic pyridines were of analytical grade and purchased from Aladdin (Shanghai, China).Methanol and formic acid were LC-MS grade and purchased from Aladdin (Shanghai, China) and ThermoFisher, respectively.All other chemicals used in this study were of AR grade with more than 99% purity and purchased from commercial manufacturers.

Strains and cultivation conditions
Pyridine-degrading bacterial consortium was isolated from petroleum-contaminated soil taken near the Liao River estuarine wetland.Single colonies were obtained by plating the bacterial consortium on the MSM agar plate with particular substrates as previously described (Yu et al., 2018).The bacterial consortium/strains were grown in 250 mL Erlenmeyer flasks containing 50 mL MSM with different concentrations of pyridine unless otherwise stated.MSM was sterilized by autoclaving at 121°C for 20 min.Pyridine or other pyridinic compounds were added after filtered by a 0.22 μm sterile syringe filter (Jinteng, China).The strains were incubated at 30°C on a rotary shaker (150 rpm).Isolated strains were identified by 16S rRNA gene sequence analysis.The genomes of bacteria were extracted using Ezup pillar bacterial genome DNA extraction kit (Sangon Biotech, China) followed the instruction for Gram-positive strains.The 16S rRNA gene was amplified using primers 27F and 1492R.The PCR reaction was performed using the following cycling conditions: 95°C for 5 min, 35 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 90 s, followed by 72°C for 10 min.Nucleotide sequences similarities for phylogenetic analysis were obtained from the LPSN database.Phylogenetic analysis was performed by MEGA6.0 software.

Biodegradation of pyridine by Co-culture or isolated strains
In order to understand the degradation and growth pattern of bacterial co-culture or isolates, the effects of pH, temperature, and substrate concentration were determined in MSM.The samples were withdrawn at particular time interval and analyzed for bacterial growth.The cells were then removed by centrifugation at 10,000 × g, 4°C for 5 min, and the supernatant was transferred to a new centrifugation tube for further analysis.Control experiments were carried out using autoclaved MSM with the same pyridine concentration without microorganisms to determine the loss of substrate due to evaporation.

Degradation rate = (1 −
pyridine concentration in the sample pyridine concentration in the control ) × 100

Kinetics models
In order to understand the growth pattern of the co-culture.The effects of substrate concentration were performed in MSM with different concentrations of pyridine.In the batch system, the specific growth rate (μ) was defined as X is cell concentration (OD600nm), μ is a specific growth rate of the cell (h -1 ), and Kd is the endogenous decay coefficient (h -1 ).At the exponential growth phase, the Kd could be neglected, and the equation ( 1) was reduced to The value of μ was determined at the exponential phase of the growth curve according to equation (2).
Two models were used to fit the experimental data obtained from the batch experiments.Monod model represents bacterial growth under substrate-limited and non-inhibitory conditions as equation ( 3).
Haldane inhibitory growth model represents the growth kinetics of an inhibitory compound such as pollutants (Kumar et al., 2005;Park et al., 2002) as equation ( 4).
where S0 denotes the concentration of pyridine (mg/L).
The true maximum specific growth rate in the Haldane model occurred when dμ/dS =0 as below (Christen et al., 2012): The maximum specific growth rate can be obtained by replacing Sm in Eq. ( 4).
The values of the kinetic parameters were obtained from non-linear fitting using Origin software.

Intermediates identification and phylogenetic analysis
Resting cell suspensions were prepared as previously described (Yu et al., 2018).The bacteria in the early stationary phase were collected by centrifugation at 8,000×g, 4°C for 5 min, washed twice with phosphate salt buffer, and resuspended with double distilled water.The resting cell reactions were performed in 250 mL flask at 30°C 150 rpm.The samples were collected at interval times.The cells were removed by centrifugation at 10,000 × g for 2 min.The supernatant was transferred to a new tube and stored at -20°C for further analysis.

Analytical methods
Growth was monitored spectrophotometrically by measuring the absorbance at wavelength 600 nm (OD600).Pyridine concentration was monitored by high-performance liquid chromatography (HPLC) (Waters Alliance HPLC, USA) equipped with a column of Waters Spherisorb ODS2 (4.6 mm × 250 mm) and a PAD detector.The mobile phase was 70% (v/v) methanol and 30% 0.05% (w/v) formic acid at a flow rate of 1 mL/min.The signal of 265 nm was monitored to calculate the pyridine concentration.The supernatant of the samples was treated by adding two-volume of methanol.The samples were precipitated at 4°C for 5 min, centrifuged at 10,000 × g for 2 min, and then filtered by a 0.22 μm sterile syringe filter.
To identify the degradation intermediates of the two isolated strains, samples from resting cell reactions were analyzed by LC-MS/MS.LC-MS/MS analysis was performed with UltiMate3000 UHPLC system (Thermo Fisher, USA) equipped with Orbitrap Fusion Lumos mass spectrometer and an electrospray ionization source (ESI), using reverse-phase column (Agilent ZORBAX RRHD Eclipse Plus 95Å C18 (2.1 × 100 mm, 1.8 µm) at 30°C.The mobile phase is 70% (v/v) methanol and 30% 0.05% (w/v) formic acid at a flow rate of 0.2 mL/min.Both positive and negative electrospray ionization analysis with the continuous full scanning from m/z 50 to 500 were performed.The data were analyzed using Compound Discovery software (Thermo Fisher).

Strains identification
A bacterial consortium, which could utilize pyridine as the sole source of carbon and nitrogen, was isolated from petroleum-contaminated soil in Liao River estuarine wetland.A series of pyridinic compounds were provided as growth substrates for this bacterial consortium, respectively.The bacterial consortium could grow with pyridine and 2-hydroxypyridine, but it could not use 3-hydroxypyridine, 4-hdyroxypyridine, picolinic acid, nicotinic acid, or isonicotinic acid as the growth substrate.After serial dilution, one dominant colony was observed on the MSM + pyridine agar plate.This strain was isolated and the 16S rRNA gene was sequenced.This strain was designated as PCD-1 and classified as Arthrobacter based on the 16S rRNA gene and morphology properties.However, PCD-1 could only grow with pyridine, it could not grow with 2-hydroxypyridine.Then, this bacterial consortium was cultivated with 2-hydroxypyridine as the sole substrate for 3 cycles.After serial dilution, one dominant colony was isolated from the plate.
This strain, namely HPD-2, was identified as Rhodococcus sp.Strain HPD-2 could grow with both pyridine and 2-hydroxypyridine as the substrate.

Effect of temperature and pH on the degradation of pyridine
The growth and pyridine degradation were assessed at different pH and temperature values for both Arthrobacter sp.PCD-1 and Rhodococcus sp.HPD-2.The results revealed that strain PCD-1 could grow well at a temperature ranging from 25°C to 37°C, and showed the highest growth rate at 30°C (Fig. 1a).Strain PCD-1 grew well at pH values ranging from 7.0 to 9.0 and showed the highest growth rate at pH 8.0 (Fig. 1b).However, there was almost no growth at pH 6.0 or pH 10.0 for strain PCD-1.The optimum pH values were observed to be 7.0-9.0for Rhodococcus sp.HPD-2.
The lag phase at pH 7.0 was slightly shorter than those at pH 8.0 and 9.0, and the maximum cell density at pH 9.0 was a little bit higher than those at pH 7.0 and 8.0 (Fig. 1c).The optimum temperature for strain HPD-2 was 30°C (Fig. 1d).The results revealed that both strains could grow well with pyridine as the substrate, and the optimum growth conditions for the two strains were very similar.Rhodococcus sp.strain HPD-2 could use both pyridine and 2-hydroxypyridine as the growth substrate.2-Hydroxypyridine is a better growth substrate for strain HPD-2 than pyridine (Fig. 2a).The culture color of strain HPD-2 with pyridine was white, while the culture color with 2-hydroxypyridine was yellow-brown (Fig. 2b).

Growth kinetics of co-culture
Both Arthrobacter sp.PCD-1 and Rhodococcus sp.HPD-2 could use pyridine as the growth substrate, thus the two strains were re-mixed to form the co-culture CoPD.The growth with co-culture and individual strain were compared.It was observed that the growth of individual strain Arthrobacter sp.PCD-1 and Rhodococcus sp.HPD-2 was slower compared to the co-culture of the two strains (Fig. 3).Under optimal conditions, the growth of co-culture, CoPD, was measured with different concentrations (from 100 mg/L to 5000 mg/L) of pyridine.The co-culture could grow in all the tested concentrations.The lag phase with high initial pyridine concentration ( ≧ 2000 mg/L) was significantly prolonged, which may be due to substrate inhibition (Fig. 4a).
Figure 4 shows a typical trend that the value of specific growth rate increases with the increase of initial pyridine concentration up to a certain concentration level, then it begins to decrease with the increase of pyridine concentration.The results confirmed that pyridine was an inhibitory substrate.
The growth kinetics are essential for understanding the capacities of the bacteria for pollutant degradation.Therefore, the determination of the growth kinetics of microorganisms has been one of the main issues in these studies.Two growth models were used to describe the growth rate of co-culture CoPD for a wide pyridine concentration.In general, the Haldane's growth model is used to describe the growth kinetics data of an inhibitory compound.The correlation coefficients (R 2 ) also showed that the Haldane inhibition model is the better kinetic model for the co-culture CoPD growth with pyridine(Fig.4b).The value of maximum specific growth rate, μmax, is 0.141 h -1 at 380 mg/L (Sm) initial pyridine concentration, and the substrate inhibition coefficient, Ki, is 3830 mg/L.
The value of the substrate half-saturation coefficient, Ks, is 37.9 mg/L.
The effects of initial concentration on the degradation behavior of pyridine were conducted.The initial concentration of pyridine was varied from 100 to 5000 mg/L.The degradation data are given in Figure 4c, d.Co-culture CoPD could completely degrade all the tested pyridine concentrations ranging from 100 to 5000 mg/L to an efficiency of >98%.

Degradation profile of pyridine by two strains
To investigate the degradation pattern of pyridine by two strains, resting cell reactions were performed with different substrates including pyridine, 2/3/4-hydroxypyridine or 2/3/4-carboxypyridine.The samples were analyzed using HPLC, and the results showed that strain PCD-1 could only convert pyridine, while strain HPD-2 could only convert pyridine and 2-hydroxypyridine.A single peak of intact pyridine compound was observed at a retention time of 3.56 min in the HPLC chromatogram, and the peak disappeared gradually with the reaction time.
However, no new peak could be detected in the HPLC signal.
The intermediates were then identified using high-resolution accurate mass measurements by Orbitrap Fusion Lumos (Thermo Scientific) (Fig. 5).Two main intermediates detected in the resting cell reaction of strain PCD-1 were presented.The base peak of M1 with m/z of 98.06020 in the positive ion ESI mass spectrum in Figure 5a was identified as 2,5-pyrrolidione, which matched with formula C4H3NO2.Compounds M2 and M3 which were detected with an m/z of 87.08062 and 101.09622, respectively, were identified as butyrolactone (C4H6NO2) and maleic semialdehyde (C4H4NO3), each.For strain HPD-2, the intermediate M2 was also detected in the LC-MS spectra as shown in Figure 5c.Besides, a peak of M4 with an m/z of 85.02858 in the positive ion ESI mass spectrum was identified as furanone (C4H4O2) as shown in Figure 5d.It can be seen that M2 could be produced by both strain PCD-1 and HPD-2.Besides, M3 and M4 are the substrate and product of a catalyzing reaction.Thus, it can be proposed that the two strains use similar pyridine degradation pathway, which was proposed as Figure 6 based on the intermediates' identification and previous studies.

Discussions
Environmental pollution has become one of the serious problems in modern society.Millions of tons of pollutants are discharged every day, among which pyridine and its derivatives are one of the most distributed contaminants.Microbial bioremediation is a promising method for pyridine removal from the environment.In this study, two newly isolated strains, Arthrobacter sp.PCD-1 and Rhodococcus sp.HPD-2, which could use pyridine as the sole source of carbon and nitrogen, were isolated from the petroleum pollutant soil in Liao River estuarine wetland.The two isolates were mixed to form bacterial consortium CoPD.Both Arthrobacter and Rhodococcus were widely studied because of their properties of degradation for recalcitrant aromatic and pyridinic compounds (Arora and Sharma, 2015;Presentato et al., 2018;Ren et al., 2018;Sengupta et al., 2019;Sun et al., 2011;Yao et al., 2012;Zefirov et al., 1994).Compared with reported Arthrobacter and Rhodococcus, the co-culture CoPD showed more excellent pyridine degradation capacity.
Besides, CoPD showed better pyridine biodegradation capacity than each of the two isolated strains.CoPD was able to completely degrade pyridine at an initial concentration up to 5,000 mg/L.This is a high reported pyridine concentration among all the reported pyridine degrading bacteria, and only a few strains have been reported grown with such high pyridine concentration.The calcium alginate gel beads immobilized Pimelobacter sp. could remove nearly 90% pyridine at an initial concentration of 10000 mg/L within 164 h (Lee et al., 1994).Gordonia terrea IIPN1 could completely remove 5530 mg/L pyridine within 192 days (Stobdan et al., 2008).Achromobacter sp.
DN-06 could grow with up to 4,300 mg/L pyridine and the μmax fitting to Haldane's inhibitory growth kinetics was 0.161 h -1 , similar to that of co-culture CoPD (Deng et al., 2011).The excellent pyridine capacity of CoPD might due to the efficient pyridine catalyzing enzymes and the high pyridine tolerance.The growth kinetics studies have proved that high pyridine concentration will inhibit the growth of co-culture CoPD.It has been observed that the lag phase in high pyridine concentration is long.This was probably due to the RNA and protein involved in pyridine degradation that should be synthesis in this phase, therefore high inoculation will reduce the lag phase (Lee et al., 1994).The toxic and inhibitory effect toward the bacterial cells is another reason for the long lag phase.Pyridine is volatile compounds, and the concentration will decrease during the cultivation in both of the control and inoculated culture, especially with high pyridine concentration.At high concentrations, the decrease of pyridine concentration in early-stage mainly due to evaporation.The growth of the strain after a long lag phase may be due to the pyridine concentration has decreased to a relatively low concentration by volatilization.Nevertheless, the co-culture CoPD was able to tolerate and grow on pyridine up to 5000 mg/L.
It is important to analyze the degradation pathway of the pyridine, because microbial degradation may produce some carcinogenic intermediates.Several different pathways have been investigated for microbial degradation of pyridine (Khasaeva et al., 2011;Shukla, 1984;Shukla and Kaul, 1986;Wang et al., 2018;Zefirov et al., 1994).The reported microbial degradation pathways could be classified into two different strategies.One strategy is hydroxylation strategy, in which a hydroxylate pyridinic intermediate, such as 2-hydroxypyridine, 2,5-dihydroxypyridine, 2,3-dihydroxypyridine, or 2,3,6-trihydroxypyridine, was produced (Fig. 6a).In this strategy, at least two hydroxyl groups were added into the pyridine-ring, which subsequently undergo ring cleavage catalyzed by ring-cleavage dioxygenases.The hydroxylate pyridine will polymerize or oxidize to form pigment intermediates, therefore, this strategy can be easily identified by the culture color.The other strategy is dehydrogenation strategy, in which the pyridine ring is hydroxylated to form pyridinic quinones, such as 1,4-dihydroxypyridine, 2,3-dihydro-pyridine-2,3/2,4-diol, or piperidine-2-ol, and the pyridinic quinone intermediates are usually unstable, which will automatically form ring cleavage product (Fig. 6b).Thus, in most cases, the cultures of strain have no color and the intermediates could not be detected by the HPLC analysis with an ultraviolet detector.
Four degradation intermediates were identified in the present study, including 2,5-pyrrolidione, butyrolactone, maleic semialdehyde, and furanone.Butyrolactone and furanone were only detected in the pyridine degrading strain using hydroxylation strategy (Fig. 6).N-formylmaleamic acid was the upstream substrate of butyrolactone and furanone (Fig. 6) and it was produced through degradation of 2,5-dihydroxypyridine or 2,3,6-trihydroxypyridine (Khasaeva et al., 2011;Yu et al., 2015).As was reported by other researchers, 2,3,6-trihydroxypyridine, also called nicotine blue, is a blue pigment, therefore, the culture will turn to blue or brown with a trace amount of 2,3,6-trihydroxypyridine (Yao et al., 2012).Besides, 2,5-dihydroxypyridine will also bring blue and brown color for the culture (Tang et al., 2013;Yu et al., 2015).However, the cultures of PCD-1, HPD-2 and CoPD in MSM+pyridine were all white.No new peak could be observed in resting cell reactions by HPLC analysis, which indicated that no hydroxylated pyridine intermediate was accumulated.Moreover, the co-culture CoPD could not grow with pyridine N-oxide.The results indicated that strain PCD-1 and HPD-2 use dehydrogenation strategy to degrade pyridine.
Combined with the analysis result of Compound Discovery software,we proposed that the two strains utilize a different degradation pathway from the reported strains.The pyridine degradation was supposed to be initiated via a oxidization reaction to form 2,3-dihydro-pyridine-2,3-diol.
The butyrolactone was converted to form succinic semialdehyde and succinic acid, which enters the TCA cycle.
The better pyridine degradation capacity for CoPD compared with individuals indicated that there are interactions between the two strains in CoPD.The two strains have similar adaptive capacity to the environment and the same degradation pathway.Therefore, they could exchange and use each other's intermediates freely.The higher efficiency might be due to that rate-limiting steps of the two strains are different, therefore, they could share the accumulated metabolic intermediates with each other during pyridine degradation to break the rate-limiting steps.The high pyridine degradation capacity indicated that the two strain has efficient catalyzing enzymes and a special pyridine tolerant system.Therefore, further study will focus on the molecular mechanism of pyridine degradation and tolerance.

Declaration
Ethics approval and consent to participate: This article does not contain any studies with human participants or animals performed by any of the authors. The

Figure 1 .
Figure 1.The temperature and pH influence on PCD-1 and HPD-2 growth were performed

Figure 2
Figure 2 Growth of Rhodococcus sp.strain HPD-2 with 1000 mg/L pyridine and 1000 mg/L

Figure 3
Figure 3 Growth of co-culture CoPD and the individual two strains in MSM with 1000 mg/L

Figure 4
Figure 4 Growth kinetics of co-culture.(a) Effects of pyridine concentration on the growth of

Figure
Figure 5 LC-MS analysis of pyridine degradation intermediates of Arthrobacter sp.PCD-1 (a,

Figure 6 482 Figure
Figure 6 Proposed microbial pyridine degradation pathways.(a) The hydroxylation strategy of temperature and pH in uence on PCD-1 and HPD-2 growth were performed in MSM with 1000 mg/L pyridine.(a) Effects of temperature for growth of strain PCD-1; (b) Effects of pH for growth of strain PCD-1; (c) Effects of temperature for growth of strain HPD-2; (d) Effects of pH for growth of strain HPD-2.Each value is the mean from three parallel replicates ±SD.