Biodegradation of 3,5,6-trichloro-2-pyridinol by Cupriavidus sp. DT-1 in liquid and soil enviroments CURRENT STATUS: UNDER REVIEW

The bactrial strain Cupriavidus sp. DT-1 can degrade 3,5,6-trichloro-2-pyridinol (TCP) and transform it into 2-hydroxypyridine (2-HP). This is a unique degradation pathway of TCP but incomplete. In the present study, strain DT-1 could degrade 2-HP at a high concentration 500 mg/L and use it as sole carbon source for growth. Three metabolites (nicotine blue, maleamic acid and fumaric acid) were detected in the medium and the complete degradation pathway of TCP was derived. Inoculation of TCP-contaminated soils with strain DT-1 resulted in a degradation rate 94.4% and 86.7% as compared to 20.4% and 28.4% in uninoculated soils, respectively. Fluorescent marker gene gfp was introduced into strain DT-1 and a new strain DT-1-gfp was created, viability test showed the strain could survive well in soils for more than 35 d. This finding suggests that strain DT-1 has potential for use in bioremediation of TCP-contaminated environments.


Introduction
TCP is a typical representative of organochlorine pollutant with a long half-life ranging from 65 to 360 days in the natural environment (Fang et al., 2019). It is the primary degradation product of chlorpyrifos but more mobile than the parent compound due to its greater water solubility (Yang et  2-HP is a class of N-heterocyclic organic pollutants with the characteristics of biological toxicity and long existed in the environment (Lataye et al., 2006). It is more water soluble, more mobile, and more leachable into groundwater and soils, which causes widespread contamination and threatens to ecological environment and human health (Petkevicius et al.,2018;Chu at al., 2018 ). More importantly, 2-HP is a primary intermediate metabolite in the degradation of N-heterocyclic organic compounds such as TCP and nicotine (Lu et al., 2013;Yu et al., 2015). Therefore, remove the residual of 2-HP from environment has great significance for eliminating the pollution casued by these toxic compounds.
Microbial degradation with the characteristics of high efficiency, low cost and no secondary pollution is an environmentally favorable decontamination method to remove the pollution of TCP (Wang et al., 2012). To date, several microorganisms with capacity to degrade TCP have been isolated from different genera (Table 1), and the biodegradation pathway of TCP has been researched recent years. Results show the intermediate metabolite during TCP degradation most commonly detected is 3,6-dihydroxypyridine-2,5-dione (Li et al.,2010;Bhuimbar et al.,2011;Cao et al., 2012). Different from other strains, strain Cupriavidus sp. DT-1 has a unique metabolic pathway for degradation of TCP with the metabolite product 2-HP, and further mineralizes 2-HP completely (Lu et al., 2013). However, we have only deduced the metabolic pathway from TCP to 2-HP in previous research, the subsequent degradation pathway has not been studied thoroughly. Therefor we have not understood the complete metabolic pathway of TCP degraded by strain DT-1. Table 1 The reported TCP-degrading microorganisms In this work, degradation of TCP and 2-HP in liquid culture was further researched and characterized. The degradation metabolites were indentified and the complete metabolic pathway of TCP was deduced. Additionally, the abilities of strain DT-1 for TCP degradation and survival in soil were also studied to evaluate its efficiency in bioremediation of TCP-contaminated soil.

Growth of strain D-2 in MSM culture
Diluents ranging from 10 − 4 -10 − 1 were obtained by tenfold gradient dilution.
According to dilution plate counting method, 0.2 mL diluent was spread on LB plate and cultured at 30℃ for 48 h. Plates with a colony number from 30-300 were selected for counting. All samples were in triplicate.

Extraction and Analytical methods
High-performance liquid chromatography (HPLC) was used to analyze the concentration of TCP in liquid samples. Organic matter was determined by potassium dichromate volumetric method (Ciavatta et al.,1991). Select characterisitics of the soil samples were showed in Table 2.

Degradation of TCP and 2-HP in liquid culture
The result of degradation of TCP and its metabolite 2-HP was shown in Fig. 1. There was no significant change in the concentration of TCP and 2-HP during the first 2 h. This is attributed to the fact that the strain need adaptation to a new environment and enzymes relevant to degradation have not been synthesized. During next 2-10 h, the concentration of TCP decreased while which of 2-HP increased rapidly, indicating strain DT-1 began to show its degradation activity, and 2-HP was the major degradation product. However, the total concentration of 2-HP was less than the reduction of TCP, indicating 2-HP was degraded further due to strain DT-1 could use 2-HP as the sole carbon source for growth (Fig. 2). During 10-18 h, TCP was completely degraded, and the concentration of 2-HP was decreased rapidly until disappeared.
Strain DT-1 could degrade 2-HP at a high concentration of 500 mg/L within 10 h and use it as the sole carbon source for growth. Growth and degradation occured mainly between 2 and 8 hours (Fig. 2). The optimal initial concentration of TCP was lower in the research due to its toxic effect to microorganism at a higher concentration.
However, 2-HP had less toxicity to strain DT-1, the initial concentration in the experiment was 500 mg/L. Which made the metabolites of 2-HP easier to be detected due to their higher concentrations.
Indentification of 2-HP metabolites 2-HP and its metabolites were preliminarily detected by HPLC. 2-HP was used as a standard which showed a retention time of 3.58 minutes (Fig. 3A). For the sample  (Fig. 4d), respectively. The result showed the degradation pathway of 2-HP by strain DT-1 was the same as which in Nocardia sp. (PNO) (Shukla and Kual, 1986). But this represented the first discovery of the degradation pathway in a Gram-negative bacterium. Combined with the previous conclusion, the complete metabolic pathway of TCP degradation by strain DT-1 was obtained (Fig. 5).

Degradation of TCP and 2-HP in different soils
As shown in Fig. 6a . 6b), while in uninoculated soil the degradation rate 28.4% was higher. That was because the proliferation of indigenous microorganisms due to much abundanter nutritional materials in the farmland soil, which enhanced the self-  Table 3 The content of DT-1-gfp in campus and farmland soils.

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

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Availability of data and materials
The dataset supporting the conclusions of this article is included within the article.
All data are fully available without restriction

Competing interests
The authors declare that they have no competing interests.    The complete metabolic pathway of TCP degradation by strain DT-1.