Biodegradation characteristics and mechanism of quinoline by Ochrobactrum sp. strain C2

Quinoline, a recalcitrant heterocyclic compound, is widely used in dyeing, drug, and Coal Chemical industries (Qiao & Wang 2010; Zhao et al. 2012; Nainwal et al. 2019). Because of carcinogenic, teratogenic, mutagenic and bio-accumulating qualities quinoline and its derivatives have adverse effects on the growth and development of environmental receptors such as animals and plants (Neuwoehner et al. 2009; Bai et al. 2010; Wei et al. 2012; Gao et al. 2020). Various physicochemical treatment technologies such as catalytic oxidation, electro-chemical degradation, photocatalytic oxidation, ozone oxidation and adsorption have been used to remove quinoline from water. Compared with physical-chemical methods, biodegradation is an good alternative method due to the large capacity, low cost and a lack of secondary pollution (Fu & Zhao 2015).

Bacteria play an important role in quinoline biodegradation. In the past, a variety of quinoline-degrading strain had been found, such as Bacillus sp. (Tuo et al. 2012), Pseudomonas sp. (Griese et al. 2006; Zhang et al. 2016) and Thermovirga ornatilinea (Wu et al. 2020). The transformation mechanism of quinoline by these bacteria had four pathways, that is, the 5,6-dihydroxy-2(1H) quinolinone pathway (Schach et al. 1995), the anthranilate pathway (Bauer et al. 1994), the 7,8-dihy-droxy-2(1H) quinolinone pathway (Ruger et al. 1993), and the 8-hydroxycoumarin pathway (Luo et al. 2020). In cases with different degradation conditions, the biological metabolic pathways of quinoline were also different (Luo et al. 2020). Various aromatic compounds could be metabolized by catechol 1,2-dioxygenase or catechol 2,3-dioxygenase through an ortho- or meta-cleavage pathway (Murakami et al. 1997). The metabolic pathway could be inferred by the determination of enzyme activity. Previous studies mainly focused on the biodegradation pathway at alkaline condition while lack of the transformation mechanism at acid condition and the degradation mechanism from perspective of enzymatic reaction. These strains could degrade quinoline well only under alkaline conditions with poor adaptability under acid condition (Zhu et al. 2008; Tuo et al. 2012). Wastewater quality conditions fluctuate greatly in the actual treatment process resulting in lower efficiency (Bai et al. 2018). Therefore, it is of great significance to find more strains with a wide range of applications and strong environmental tolerance. The toxicity of quinoline to bacterial cells inhibits bacteria activity, resulting in low quinoline removal performance, which is also the main factor restricting the application of biodegradation in the actual treatment process. Immobilized strains with functional materials have recently been developed to overcome these problem (Wang et al. 2015; Ke et al. 2018). Immobilized strains have stronger adaptability to environmental alarms such as temperature, pH, or toxic compounds exposure as compared to suspended strains, which can improve the degradation capability (Kurade et al. 2018).

In this work, a quinoline-degrading strain, Ochrobactrum sp. strain C2, was isolated from the coking wastewater of a coking plant. The degradation process of quinoline by Ochrobactrum sp. was rarely reported. The optimum conditions for the strain to degrade quinoline were investigated. The biodegradation characteristics and metabolic mechanism of quinoline were conducted by enzymatic reaction. Furthermore, immobilized cells of bacteria were used to enhance the degradation of quinoline. These will provide theoretical and practical value for the treatment of quinoline-containing wastewater by biodegradation technology.

Quinoline (GR), pyrocatechol, bovine serum albumin, glycerol and peptone were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). Acetone, agar ethanol and sulfuric acid were from Beijing Chemical Works (China). All other chemicals were of analytical grade, commercially available.

Luria-Bertani (LB) medium (Weid et al. 2007) was used to enrich the strains. The mineral salt medium (MSM) (Tuo et al. 2012) was used to isolate the strain and to degrade quinoline, which contained (g/L): FeSO4·7H₂O 0.02, MgSO₄·7H₂O 0.20, Na₂HPO₄·12H₂O 4.26, KH₂PO₄ 2.65, CaCl₂ 0.006, NaCl 0.5, and trace element solution 1 ml, pH 7.0.

0.5 ml of activated sludge from coking wastewater was transferred into 50 ml of LB medium containing 50 mg/L quinoline and incubated for enrichment at 30 °C, 150 r/min for 3 days. Afterwards, 5 ml of the enriched medium was centrifuged at 10,000 r/min for 5 min. The deposition was mixed with normal saline and centrifuged twice to remove the residual medium and secondary metabolites. The precipitated bacteria was cultured for two months in MSM containing quinoline to acclimate. During acclimation, the concentration of quinoline was gradually increased until the concentration was 500 mg/L. Then the isolation was conducted according to the method described in the literature (Tuo et al. 2012). The isolated strain was named C2.

Based on literature (Dong & Cai 2001), physiological and biochemical properties of strain C2 were examined, containing gelatin liquefaction test, starch hydrolysis test, methyl red test, citrate utilization test and indole test.

The genomic DNA of C2 was extracted by DNA extraction kit (Solarbio bacterial DNA Kit). The primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGCTACCTTGTTACGACTTT-3′) were used to amplified the 16 rDNA by PCR. The obtained PCR products were sequenced using Miseq Illumina by Sangon Biotech Co., Ltd (Shanghai, China). The measured sequence was submitted to the GenBank. The phylogenetic free of the strain C2 was constructed using the neighbor-joining method by MEGA6.0.

Different from single-factor experiments, comprehensive effects of factors could be given by multi-factor orthogonal experiment (Yu et al. 2021). To obtain the suitable conditions for the strain C2 to degrade quinoline, the effect of four factors on the degradation of quinoline was studied by the orthogonal experiment. Four factors including rotational speed (70 r/min, 150 r/min, 200 r/min), pH (5, 7, 9), temperature (28 °C, 30 °C, 37 °C) and inoculation amount (3%, 6%, 10%) were investigated, and three levels were set for each factor for the orthogonal experiment. Removal rate of 250 mg/L quinoline was determined after 24 h at different condition, three parallel experiments were conducted in each group.

1% glucose and 0.5% ammonium sulfate were added into MSM as additional carbon source (C) and nitrogen source (N) separately for evaluating the effects of additional carbon sources and nitrogen sources on degradation of quinoline.

Sodium alginate was used as the embedding material to embed strain C2 to make immobilized microspheres SA + C2: 10% of bacteria was add into the sterilized 4% of sodium alginate, then was added into the sterilized 4% of CaCl₂ drop by drop with pipette. The prepared microspheres were put into the refrigerator for crosslinking at 4 °C for 12 hours, and then washed by the sterilized water for standby. C2, SA and SA + C2 were respectively inoculated in the coking wastewater containing 400 mg/L of quinoline and 2,500 mg/L of chemical oxygen demand (COD) for evaluating the effects of immobilized cells on degradation of quinoline.

The strain C2 was cultured in LB medium for 18 hours. Then 10% of the medium was centrifuged and inoculated in MSM medium with different concentration of quinoline and cultured for 24 h at 30 °C, 150 r/min. The growth of strain C2 and degradation of quinoline were measured every 3 hours. Cell growth was observed by determining optical density of the medium at 600 nm (OD₆₀₀) and the specific growth rate was defined as an increase in OD₆₀₀ during the logarithmic growth phase (Zhu et al. 2008).

The concentration of quinoline was measured at 313 nm by ultraviolet spectrophotometer (Li & Zhao 2001). COD was analyzed by dichromate method (Ministry of Ecology & Environmental of the People's Republic of China 2017), Metabolites of quinoline was analyzed with GC-MS after Strain C2 was cultured in MSM with 250 mg/L quinoline for 24 hours. After nitrogen blowing, the pretreated sample was put into GC/MS instrument (Tuo et al. 2012), and the product was analyzed by qualitative analysis. The structure of metabolites was confirmed by comparing with the mass spectrometric cleavage patterns of known compounds.

100 ml of bacterial solution that was cultured for a period of time was centrifuged in a centrifugal tube at 4 °C, 12,000 r/min for 10 min. Then the precipitation was mixed with 10 mL, 50 mM Tris-HCl and pH 8.0 buffer for cleaning to collect bacterial cells. The centrifugation process was repeated for several times. Cells pellet was lysed at the ultrasonic cell breaker in ice bath for 30 cycles at 150 W, working for 5 seconds followed by cooling for 6 seconds. Finally, the centrifugation of broken cells was taken out at 4 °C, 12,000 r/min for 30 min, and the supernatant fraction was crude enzyme.

The unit of enzyme activity A (U) was defined as the amount of enzyme required to catalyze the formation of 1 u mol of product per min (Nadaf & Ghosh 2011). The activity of catechol-1,2-dioxygenase (C12O) was measured by absorbance of the the formation of cis, cis-muconic acid at 260 nm. The activity of catechol-2,3-dioxygenase (C23O) was measured spectrophotometrically by following the formation of 2-hydroxymuconic semialdehyde from catechol at 375 nm (Takeo et al. 2007). The protein concentration was determined by the Bradford method (Bradford 1976).

In the formula:

M – The quality of protein, mg;

ε – Molar extinction coefficient of catechol at 260 nm, mmol/(L·cm);

ΔA – Variation of optical density at 260 nm per minute;

V – Volume of enzyme activity determination system, L.

Five kinds of biochemical and physiological characteristics of strain C2 were identified (Table 1). The starch was blue-purple after adding iodine solution, which indicated that strain C2 could not hydrolyze starch completely. In the methyl red test, methyl red was positive showed that strain C2 could decompose glucose to pyruvate, and pyruvate could further decompose to formic acid, acetic acid, lactic acid, etc., and reduced the pH value of medium below 4.5. The indole test of strain C2 showed no red ring at the interface of two layers of liquid, which indicated that strain C2 did not decompose tryptophan in peptone. In gelatin test, the medium could not liquefy, which indicated that strain C2 could not hydrolyze gelatin. When the medium became alkaline, the indicator of bromothymol blue changed from green to dark blue, indicating that strain C2 could grow with sodium citrate as carbon source to form carbonate.

The effect of rotational speed, inoculation, temperature and pH on the degradation of quinoline was studied by orthogonal experiment. The result showed at Table 2, Kn was the average value of each factor at the same level, indicating the quality of the value. According to the value of Kn, the best combination of degradation conditions was A2B1C2D3, that is, 30 °C, rotating speed 150 r/min, pH 5.0 and inoculation amount 10%. The actual best degradation rate was sample 4, which was consistent with the above analysis.

The effect of each factor in the experiment could be reflected by solving the range (R). The greater the Range, the greater influence of this factor on the degradation. Therefore, the order of factors in this experiment was CDAB, that is, temperature > inoculation quantity > rotational speed > pH. Analysis of variance was used to test whether various factors had influence on the degradation rate. Rotational speed, temperature and inoculum amount had extremely significant effects on the degradation of quinoline, and pH also had significant effects on the degradation of quinoline. Various studies found quinoline-degrading bacteria could degrade quinoline well under alkaline or neutral conditions (Sun et al. 2009; Tuo et al. 2012; Zhang et al. 2016; Khudhair 2018). The strain C2 could degrade quinoline well in an acidic environment, which was possible that different pH affected the activity of different enzyme and produced different products. This experiment also verified this hypothesis, because different color products were observed at different pH (Tuo et al. 2012).

The Monod kinetic constants were obtained from the experimental data by double reciprocal plot method (Table 3), The concentration of quinoline was much less than the saturation constant at 150–250 mg/L, that is, when s < K_S, the Monod equation could be transformed into μ = μ_max/K_s. The results showed that when the concentration of quinoline was 150–250 mg/L, the growth of strain C2 was a first-order reaction. The growth rate of strain in this concentration range was not inhibited by concentration of quinoline, and its specific growth rate increases with the increase of concentration. When the concentration of quinoline was more than 250 mg/L, the correlation coefficient was too low to be described by the Monod equation, which means that there was an inhibition effect.

Table 3

Growth kinetic of strain C2

Quinoline (mg/L) .	Monod .	.	.	R2 .
150	μ = 2.87S/(1,375 + S)	2.87	1,375	0.988
250	μ = 0.26S/(3,009 + S)	0.26	3,009	0.929
300	/	/	/	0.550
400	/	/	/	0.579

Quinoline (mg/L) .	Monod .	.	.	R2 .
150	μ = 2.87S/(1,375 + S)	2.87	1,375	0.988
250	μ = 0.26S/(3,009 + S)	0.26	3,009	0.929
300	/	/	/	0.550
400	/	/	/	0.579

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The characteristics of catechol dioxygenase in crude enzyme solution were studied as shown in Table 4, the activity of catechol-1,2 dioxygenase (C12O) could be detected in the medium contained quinoline, but catechol-2,3 dioxygenase (C23O) could not be detected. When there was no quinoline in the medium, the activity of catechol-1,2 dioxygenase (C12O) and catechol-2,3 dioxygenase (C23O) could not be detected. It could be preliminarily speculated that C12O in strain C2 played an important role in the degradation of quinoline, and the enzyme was an inducible enzyme (Guzik et al. 2011).

Strain Ochrobactrum sp. C2 capable of degrading quinoline was isolated. The strain C2 could utilize quinoline as the only source of carbon and nitrogen to grow and degrade quinoline well under acidic conditions. Future research may focus on the use of C and N in quinoline instead of being removed. The degradation pathway of quinoline was that the hetero-nitrogen ring was oxidized to quinolone, and then the hetero-nitrogen ring was opened to form intermediate products such as benzaldehyde, 2-pentanone and hydroxyphenyl propionic acid. Immobilized cells of bacteria were used to enhance the degradation of quinoline. These will provide theoretical and practical value for the treatment of quinoline-containing wastewater by biodegradation technology.

This work was funded by the National Natural Science Foundation of China (No. 41977029).

The authors declare that they have no conflict of interest. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

Written informed consent for publication was obtained from all participants.

Caihong Yu contributed to the conception of the study; Qiancheng Zhao and Qiaoyu Hu performed the experiment; Qiancheng Zhao and Qiaoyu Hu contributed significantly to analysis and manuscript preparation; Qiancheng Zhao, Ziliang Qiu and Caihong Yu performed the data analyses and wrote the manuscript; Qiancheng Zhao and Qiaoyu Hu helped perform the analysis with constructive discussions.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.