Cyclohexanecarboxylic acid degradation with simultaneous nitrate removal by Marinobacter sp. SJ18

doi:10.21203/rs.3.rs-1844917/v1

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Cyclohexanecarboxylic acid degradation with simultaneous nitrate removal by Marinobacter sp. SJ18

https://doi.org/10.21203/rs.3.rs-1844917/v1

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Naphthenic acid (NA), a new kind of toxic pollutants, is labeled as the potential threat to human health. However, the transformations of NAs in marine environments are still unclear. In this study, the characteristics and pathways of cyclohexylcarboxylic acid (CHCA) biodegradation were explored in the presence of nitrate. Results showed that CHCA was completely degraded with pseudo-first-order kinetic reaction under aerobic and anaerobic conditions, accompanied by nitrate removal rates exceeding 70%, which was positively correlated with CHCA degradation(P<0.05). Degradation pathways of CHCA were proposed the cyclohexane was dehydrogenated to form cyclohexene, then ring-opened to generated fatty acid by dioxygenase under aerobic conditions, yet the cyclohexene was cleaved through β-oxidations under anaerobic condition. Whole genome analysis indicated that nitrate was removed via assimilation and dissimilation pathways and denitrification pathways under aerobic and anaerobic conditions, respectively. This study provides a basis for the combined pollution of NA and nitrate in marine environments.

cyclohexylcarboxylic acid

degradation pathway

nitrate metabolism

whole genome sequencing

marine environment

1.CHCA degradation in the presence of nitrate in marine environment was firstly studied

2.Strain SJ18 could rapidly degraded the combined pollutants of CHCA and nitrate

3.CHCA degradation pathways were proposed under aerobic and anaerobic conditions

4.Strain SJ18 contained functional genes for CHCA degradation and nitrate removal

Naphthenic acid (NA) is a ubiquitous functional group in the environment, mainly derived from crude oil, and also be synthesized in the incomplete degradation of petroleum hydrocarbons by microorganisms or in-situ degradation of petroleum (Brient et al. 2000, Whitby 2010). NAs and their metal salts are widely used in paint desiccants, corrosion inhibitors, emulsifiers, surfactants, lubricants and wood preservatives, as well as to catalyze the production of alkyl and polyester resins (Clemente &Fedorak 2005, Kannel &Gan 2012a). In recent years, NAs has been considered as a new type of toxic pollutants (Jie et al. 2015, Scarlett et al. 2013). NAs can penetrate the cell walls, destroy membrane lipid bilayers or change membrane properties due to their surfactant characteristics. It was reported that NAs are acutely and chronically toxic to a variety of organisms, such as fish, amphibians, phytoplankton and mammals (Frank et al. 2008, Kannel &Gan 2012a, Kannel &Gan 2012b, Melvin &Trudeau 2012).

In general, NAs can migrate to terrestrial, aquatic and marine environment via sewage discharge, crude oil leakage, precipitation runoff, and riverbank oil layer erosion (Kannel &Gan 2012a, Scarlett et al. 2012). As previously reported, the high concentrations of NAs have already been found in aquatic systems, such as the Athabasca river (0.1–0.9 mg L^− 1) and groundwater (55 mg L^− 1) (Whitby 2010). Huang Yi et al. found that the concentrations of NAs ranged from 2.29 to 132.91 mg/kg in contaminated soil from oilfields of China (Wang et al. 2015). Different types of NAs were detected in the seawater and sediments due to the leak of the Korean oil tanker in 2007 (Wan et al. 2014). Moreover, our previous research found the abundance of NAs reached 1–4 mg L^− 1 in the sediments of Dalian Bay, which was more than 10 times the abundance of PAHs (polycyclic aromatic hydrocarbons) (Zan et al. 2019).

To date, the behavior of NAs in the environment includes physical adsorption, natural mineralization, plant degradation and microbial degradation, of which microbial degradation is considered the main way (de Oliveira Livera et al. 2018, Huang 2011, Lu et al. 2011, Quesnel et al. 2011). The biodegradation of NAs by a variety of microorganisms has been explored (Alessandro et al. 2018, Del Rio et al. 2006b, Johnson et al. 2012, Wang et al. 2015). In aerobic condition, cyclohexanoic acid has been found to be degraded by Alcaligenes, Arthrobacter and Corynebacterium (Ougham &Trudgill 1982). Pathways proposed for the aerobic degradation of NAs mainly includes β oxidation, α and β combined oxidation and aromatization pathways, and β oxidation was the majority of biodegradation pathway (Quagraine et al. 2005, Whitby 2010). For example, Quagraine et al. found the existence of α- and β-oxidation pathways during the aerobic transformation of cycloacetic acid by Alcaligenes sp. (Quagraine et al. 2005). The sedimentary microorganisms were used to degrade hydronaphthoic acid and inferred that the degradation process of decahydronaphthoic acid was completed by β-oxidation and Baeyer-Vil-liger oxidation (Del Rio et al. 2006a). Besides, the aromatization pathway was first described in the microbial of Arthrobacter (PRL-W15) which degrades cyclohexanoic acid by attacking alicyclic rings and form p-hydroxybenzoic acid through para-hydroxylation (Whitby 2010). In the aspect of anaerobic biodegradation, the effects of different conditions (nitrate reduction, sulfate reduction, iron reduction and methanogenesis) on the anaerobic biodegradation of NAs were also investigated (Clothier &Gieg 2016, Ghattas et al. 2017, Misiti et al. 2013). However, these studies are focused on terrestrial areas, most of which are aerobic biodegradation and the anaerobic degradation pathway is still unclear. Besides, the biotransformation of NAs is even less understood in marine environment (Zan et al. 2022).

At the same time, nitrate has been confirmed to be widely distributed in the offshore environment and coexists with pollutants such as petroleum hydrocarbons and aromatic compounds (Capone &Bautista 1985, Hee-Sung Bae 2002, Laufer et al. 2016). It has been found in the Pearl River Delta that nitrate may affect the biotransformation properties of microorganisms by interacting with a range of enzymes involved in metabolism or biodegradation (Su et al. 2018, Xu et al. 2014). Therefore, further investigation is thus required for the biotransformation of NAs in marine environment.

Here, a facultative NA-degrading bacterium was obtained from marine sediment, which is widespread distributed in marine environment. In the presence of nitrate, the transformation of surrogate NAs (CHCA) under aerobic and anaerobic conditions was studied. The experiments of nitrate conversion, degradation intermediates and the draft genome sequencing were carried out to explore (1) the biodegradation characteristics of CHCA in marine environment, (2) the biodegradation and nitrate removal pathway of CHCA. This subject provides a reference for perfecting and understanding the biodegradation features of NAs by marine bacterium.

1.1 Surrogate NAs and strains

Cyclohexanecarboxylic acid (CHCA, C₇H₁₂O₂, CAS: 98-89-5, purity: 99%, Meilunbio, China) was chosen as the representative NA. The CHCA was dissolved in 0.1 mol L^− 1 sodium hydroxide solution (NaOH) and prepared a 10.0 g L^− 1 stock solution for later use. Strain was derived from the sediments of Dalian Bay (39°05′ N, 121°66′ E), China. Supplementary Text S1 for specific methods of strain enrichment and screening.

1.2 Degradation of CHCA by strain

CHCA degradation experiments were set up as follows, strain was pre-cultured in sterile 2216E medium at 25°C for 24 hours, and centrifuged (10000 g, 10 mins) in the logarithmic phase of growth (OD₆₀₀ = 1.5). The cell pellets were washed twice with sterile artificial sea water (ASW). Then, cell pellets were resuspended in 50 mL ASW with 0.1 mL trace medium (see Text S2 for ASW and trace medium composition) of 100 mL erlenmeyer flasks for aerobic degradation (erlenmeyer flask was sealed with cotton stopper for oxygen exchange), and 100 mL serum bottle for anaerobic degradation (high-purity helium exposure for 10–15 mins to ensure the system was in an oxygen-free environment), both of the degradation system initial OD₆₀₀ = 0.05. The CHCA degradation initial concentration was 20 mg L^− 1, nitrate concentration was 50 mg L^− 1, and performed at 25°C in darkness, pH was adjusted to 8.0 ± 0.2. Control groups were inactivated by high temperature (121℃ for 20 mins), and all the experiment in triplicate.

1.3 Extraction and detection of NAs

Samples were separately collected at 0, 12, 36, 60, 84 and 96 hours during aerobic and anaerobic degradation. Then, collected sample with concentration of 1 mL was further added into a 2 mL centrifuge tube containing 50 µL NaOH (1 mol L^− 1), shaking for 1 min to mix thoroughly, then centrifuged (8000 g for 10 min) and collected the supernatants. The supernatants were acidified with H₂SO₄ (1 mol L^− 1) to pH < 2, and extracted with dichloromethane (v/v = 1:1) for three times. The extracts were concentrated, dried with anhydrous Na₂SO₄, and quickly evaporated at 35°C. Finally, the extracts were transferred to a 2 mL vial, and the solvent was evaporated in a gentle stream of nitrogen. By adding 100 µL of derivatization reagent tert-butyldimethylsilyl (Sigma-Aldrich, St. Louis, Missouri, USA) to 100 µL of extractive concentrates and water bath at 60°C for 20 minutes. Finally, the derivatized samples were quantified by gas chromatography-mass spectrometry (GC-MS, Shimadzu Corporation GCMS-QP2020, Japan) (detailed method in Text S3).

1.4 Detection methods of chemical indices

Nitrate, nitrite, and ammonia nitrogen were determined by sulfamic acid spectrophotometry, N-(1-naphthyl)-2-ethylenediamine spectrophotometry and Nessler reagent spectrophotometry. The total organic carbon (TOC) was measured by an organic carbon meter (multifunctional N/C 3100, Germany), and the strain optical density was measured by an ultraviolet spectrophotometer (SP-756P, Spectrum of Shanghai, China), and the wavelength was 600 nm (OD₆₀₀). The gaseous nitrogen was analyzed by gas chromatograph (GC/TCD). A total of 1mL of headspace gas was injected, the temperature of injection port and detector was 120℃, and the carrier gas was high-purity helium with 1.0 mL min^− 1 flow rate.

1.5 Strain identification and genome sequencing

The 16S rRNA of the strain was amplified by the general PCR method of prokaryotic bacteria, and the gene primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used. PCR conditions: pre-denaturation at 94°C for 5 min, then at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, a total of 35 cycles for this process, and finally repair and extension at 72°C for 10 min. The 16S rRNA gene sequence was compared in NCBI (http://www.ncbi.nlm.nih.), and the phylogeny was constructed using the MEGA5 software program.

The Illumina HiSeq 2500 sequencing platform and PE150-100X library-building sequencing strategy were selected for draft genome sequencing. The clean data was obtained by removing the adapters and processing low-quality sequences from the raw data obtained. SOAP denovo version 2.04 software were used to assemble the sequence, select the best assembly result and fill the hole and optimize the assembly result (Lim et al. 2016). Those above process were completed by Novogene Technology Co., Ltd. (see Text S4 for specific methods).

2.1. CHCA degradation and nitrate removal properties

Strain SJ18 was grown and isolated from 2216E medium, the colony was round, translucent and white on 2216E medium solid agar plate containing 50 mg/L NAs (Fig. S1). The 16S rRNA sequencing results showed that the similarity between the degrading strains and the genus Marinobacter profundi was > 99%. The gene size of strain SJ18 was 4364528 bp, a total of 3951 genes were annotated, and the average GC content was 59.84%. Based on these characteristics, we termed this strain as Marinobacter sp. strain SJ18 (Genebank accession number: MH458950.1), and the phylogenetic tree was displayed in Fig. S2 of supplementary material.

The aerobic and anaerobic degradation of CHCA were shown in Fig. 1 (a, b). In aerobic condition, CHCA was rapidly degraded after the experiment started, and completely degraded within 60 hours. The maximum degradation rate appeared around 24 h (degradation rate was about 0.43 mg h^− 1). There was no decrease of CHCA in heat-killed group, indicating abiotic factors have negligible influence on this degradation. At the same time, the TOC content dropped from 18.9 ± 0.5 to 7.0 ± 0.1 mg L^− 1, and the OD₆₀₀ value gradually increased with the progress of degradation, then reaching a maximum of 0.74 when the CHCA degradation was complete (about 60 h). Subsequently, the OD value gradually decreased, suggesting that strain SJ18 could utilize CHCA as the only carbon source, the cell density gradually decreased after CHCA were completely consumed. Besides, the concentration of nitrate in the experimental group gradually decreased, while the ammonium concentration increased, and no significant changes were detected on the nitrite concentration. Nitrate removal rate was > 70% throughout the process, the rate of nitrate consumption was consistent with the rate of CHCA aerobic degradation, and no nitrogen was captured during the degradation, indicating that the nitrate was reduced to ammonium, which was utilized by cells. The corresponding indicators of the blank control group did not change significantly. Under the anaerobic degradation, the CHCA was completely degraded within 84 hours, and the maximum degradation rate was about 0.37 mg h^− 1 (around 36 h). It could be seen that the anaerobic degradation efficiency was lower than that of aerobic conditions. Meanwhile, the TOC concentration decreased from 18.7 ± 0.3 to 8.5 ± 0.1 mg L^− 1 and the change of OD₆₀₀ value was consistent with that of the aerobic group. However, the nitrite was observed to continue rise with the consumption of nitrate, and no significant changes were detected in the ammonium. In addition, the nitrite concentration level was observed to gradually decrease after the end of degradation, meanwhile, the production of nitrogen was detected at the end of the degradation (data not shown). Nitrate removal rate reached 70% and the rate of nitrate consumption was also consistent with the rate of anaerobic degradation. It could be speculated that denitrification may occurred during the anaerobic degradation of CHCA.

The fitting results of degradation curves showed that aerobic and anaerobic degradation of CHCA conformed to the pseudo-first-order kinetic reaction (Fig. S3). The reaction rate constant k = 0.0342 (R² = 0.936) for aerobic degradation, and k = 0.0292 (R² = 0.946) for anaerobic degradation. Pearson correlation analysis showed that both aerobic and anaerobic degradation of CHCA were significantly positively correlated with nitrate removal, with correlation coefficients of 0.871 (P < 0.05) and 0.843 (P < 0.05), respectively.

2.2 Degradation intermediates and pathways of CHCA

In order to explore the aerobic and anaerobic degradation pathway of CHCA, samples at 0, 36, 60 and 84 hours were selected for intermediate products detection by GC-MS. Results showed that several intermediate products were captured, the cyclohexenecarboxylic acid (m/z = 198, 183) were found in both aerobic and anaerobic groups based on mass spectral database. The appearance of cyclohexenecarboxylic acid product indicated that CHCA might lose two electrons through dehydrogenation and form a carbon-carbon double bond on the cyclohexane ring. This degradation method is generally considered to be typical β-oxidation and appears in most aromatic or cycloalkane pollutants (Kung et al. 2014, Whitby 2010). Moreover, the presence of glycerol (m/z = 205, 147, 73) was detected in the aerobic group, and the production of lactic acid (m/z = 191, 147, 117) was found in the anaerobic group. Glycerol and lactic acid are presumed to be the oxidative hydrolysis products of fatty acids under anaerobic conditions, respectively. This would imply that cyclohexenecarboxylic acid could open the ring by hydrolysis and eventually form short-chain fatty acids. Unfortunately, no specific fatty acid products were captured during the degradation process. This might be due to the fact that fatty acids were readily available to microorganisms, resulting in their short-lived during degradation, and this speculation has been confirmed in some reports. Quesnel et al. found that the intermediate product cyclohexanoic acid existed for a short period of time during the study of the biodegradation process of cyclohexanoic acid and could not be captured (Quesnel et al. 2011). Although the product after ring opening was not captured, it could be basically confirmed that CHCA was mainly biodegraded through β-oxidation.

The whole genome sequencing was also used to explore the degradation process of CHCA. The COG, GO and KEGG databases of strain SJ18 were analyzed to predict the biological process, function genes and degradation pathways for the CHCA under aerobic and anaerobic conditions. The COG analysis results (see Fig. S4) showed the function genes classification including energy production and conversion, amino acid transport and metabolism, carbohydrate transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, cell wall/membrane/envelope biogenesis were significantly enriched. Based on GO analysis (Fig. S5), the enriched genes mainly include cellular process, metabolic process, binding, catalytic activity, nitrogen utilization, antioxidant activity, enzyme regulator activity, transporter activity and other functions in the genome of strain SJ18. The KEGG annotation results (Fig. 3) showed that the pathway of membrane transport, coenzyme metabolism, carbohydrate metabolism and amino acid metabolism were also enriched. In addition, the more detailed pathway such as ABC transporters, fatty acid degradation and biosynthesis, degradation of ketone bodies, pyruvate metabolism and styrene degradation, chlorocyclohexane and chlorobenzene degradation, benzoate degradation, toluene degradation, naphthalene degradation pathway were also obviously enriched in the strain SJ18. Some genes associated with CHCA degradation in these pathways were analyzed, the long-chain acyl-CoA synthase (fadD), benzoate 1,2-dioxygenase (benA), catechol 1,2-dioxygenase (catA), 3-hydroxyacyl-CoA dehydrogenase (HADH), hydroxycyclohexene carboxylic acid dehydrogenase (benD), alcohol dehydrogenase (adh) and 3-hydroxy-3-methylglutaryl coenzyme a lyase (hmgL) genes involved in the genome of strain SJ18, which have been confirmed to participated in the β-oxidation and the metabolism process of alkanes and polcyclic aromatic hydrocarbons in aerobic and anaerobic conditions (Boll et al. 2014, Cameron et al. 2019, Kung et al. 2013, McKew et al. 2021). It could be inferred that those function genes may play an important role in the biodegradation of CHCA.

According to the above results, the aerobic and anaerobic degradation pathways of CHCA were inferred (Fig. 4). First, two hydrogen atoms were removed by the dehydrogenase, forming a carbon-carbon double bond (cyclohexenecarboxylic acid) on the cyclohexane ring. For aerobic degradation, the cyclohexenecarboxylic acid was ring-opened to formed fatty acids by specific dioxygenases (such as benzoate 1,2-dioxygenase or catechol 1,2-dioxygenase) with the participation of oxygen. Under anaerobic conditions, cyclohexenecarboxylic acid was hydrolyzed and dehydrogenated to sequentially generate 1-hydroxy-cyclohexanecarboxylic acid and cyclohexanonecarboxylic acid, and those products were subsequently hydrolyzed and opened to form short-chain fatty acids. Finally, glycerol and lactic acid were formed through multiple β-oxidation under aerobic and anaerobic conditions, respectively.

2.3 Nitrate metabolism pathway during CHCA degradation

The changes of nitrate, nitrite and ammonium during CHCA degradation were described in section 3.1. Nitrate reduction to ammonia and denitrification were considered to be involved in the aerobic and anaerobic degradation, respectively. Hence, the transformation of nitrate was further elucidated with the assistance of genome sequencing. The genome sequence of the strain was compared with the KEGG database, and the nitrogen metabolism pathway of strain SJ18 was obtained, as shown in Fig. 5. The results showed that a total of seven of nitrogen-related genes were found, include nitrate reductase (NarGHI), nitrate assimilation reduction catalytic subunit (NasAB), nitrite reductase (NirS), nitrite reductase subunits (NirBD), nitric oxide reductase (NorBC), nitrous oxide reductase (NosZ) and nitrite oxidoreductase (NxrAB). Among them, NarGHI, NirS, NorBC and NosZ genes were considered as common denitrification genes that could carry out the complete denitrification process, and the genes NarGHI and NirBD could convert nitrate to ammonium by dissimilatory reduction. (Lu et al. 2011, Marchant et al. 2017). However, NarGHI gene was oxygen-sensitive and the expression of NarGHI was inhibited under aerobic conditions (Zhang et al. 2018). Thus, the nitrate cannot be converted to nitrite under aerobic conditions due to the missing of NapAB gene (periplasmic nitrate reductase, which can reduce nitrate to nitrite under aerobic conditions). Instead, the reduction of nitrate was achieved by the catalytic subunit of nitrate assimilation reduction (NasAB). Referring to the inorganic nitrogen chemical indicators and genomic results, the transformation process of nitrate in CHCA aerobic and anaerobic biodegradation was proposed: in aerobic condition, nitrate as an electron donor was transported from extracellular to intracellular through nitrate transporter (Nrt), then NasAB reduces nitrate to nitrite, and subsequently, nitrite was reduced to ammonium with the action of NirBD. Finally, ammonium could be processed into glutamate by glutamine synthase (glnA) and glutamate dehydrogenase (gudB/rocG), and subsequently participated in glutamate metabolism. Under anaerobic condition, NarGHI catalyzes the reduction of nitrate to formed nitrite, the NirS was involved in the reduction of nitrite to nitric oxide (NO), and then, NorBC was participated in the reduction of NO to nitrous oxide (N₂O), which in turn reduced N₂O to nitrogen (N₂) by NosZ.

In general, strain SJ18 not only has the ability to reduce nitrate to ammonia, such as dissimilated nitrate reduction to ammonia pathway (DNRA) and assimilated nitrate reduction to ammonia pathway (ANRA), but also has the denitrification. During the process of nitrate removal, the first step of the DNRA pathway was denitrification, which relied on NarGHI to reduce nitrate. In ANRA pathway, the NasAB was used to assimilate and reduce nitrate to nitrite. However, due to the NirA or NIT-6 coding gene missing, the strain SJ18 was unable to reduced nitrate to ammonium through the ANRA pathway. Therefore, the nitrite produced in the first step of ANRA need to be further metabolized through other nitrogen metabolism pathways, such as the denitrification or the DNRA pathway. In addition, strain SJ18 also has the ability to encode nitrite oxidoreductase, and the nitrite could be nitrified to form nitrate.

Overall, the metabolic pathways of nitrate in aerobic and anaerobic biodegradation were obviously different. Under aerobic conditions, nitrate was gradually consumed with the degradation of CHCA, while the cell growth (OD value increased) was observed. Indicating the nitrate was converted to ammonium through ANRA and DNRA pathways, subsequently, the produced ammonium was used for cells growth. During anaerobic degradation, the concentration of nitrite increased rapidly as nitrate was consumed, and nitrogen was detected in the end. The denitrification was speculated to be an important pathway of nitrate metabolism under anaerobic conditions. The accumulation of nitrite in the degradation system might be due to the rate of nitrate reduction (coding gene, NarGHI) step was much faster than the nitrite reduction (NirS), nitric oxide reduction (NorBC) or nitrous oxide reduction (NosZ) steps.

This study demonstrated that CHCA was completely degraded within 60 and 84 hours under aerobic and anaerobic conditions, respectively, and a significant positive correlation was found between nitrate removal and CHCA degradation. The CHCA degradation pathways were speculated that the cyclohexane ring was firstly dehydrogenated to form cyclohexene. Then, cyclohexene carboxylic acid ring-opened to generated fatty acid with the action of dioxygenase under aerobic conditions, and the cyclohexene carboxylic acid was ring-opened through β-oxidations under anaerobic condition. In addition, the nitrate could be utilized through ANRA and DNRA pathways in aerobic condition, and the nitrate might be removed by denitrification under anaerobic condition. Although the behavior and biotransformation of NAs in marine environment requires more further exploration, this study provides a basis for the combined pollution of NA and nitrate in the marine environment.

Availability of data and materials

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Author Contributions

Shuaijun Zan: Investigation, Experimental design, Methodology, Writing-original draft, Writing-review/editing.

Jing Wang: planned research, involved in supervised all analyses, data interpretation and discussion as a Project Leader.

Jingfeng Fan: participated in project cooperation.

Yuan Jin: assisted in conducting some experiments.

Zelong Li: assisted in conducting some sample analyses and writing editing.

Miaomiao Du: assisted in conducting some experiments.

Statements and Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This study was supported by the National Natural Science Foundation of China (21876018 and 42177375).

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Editorial decision: Major Revision
23 Sep, 2022
Reviewers agreed at journal
17 Aug, 2022
Reviewers invited by journal
04 Aug, 2022
Editor invited by journal
03 Aug, 2022
Editor assigned by journal
14 Jul, 2022
First submitted to journal
10 Jul, 2022

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Cyclohexanecarboxylic acid degradation with simultaneous nitrate removal by Marinobacter sp. SJ18

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Version 1

Abstract

Figures

Highlights

Introduction

1. Materials And Methods

1.1 Surrogate NAs and strains

1.2 Degradation of CHCA by strain

1.3 Extraction and detection of NAs

1.4 Detection methods of chemical indices

1.5 Strain identification and genome sequencing

2. Results And Discussion

2.1. CHCA degradation and nitrate removal properties

2.2 Degradation intermediates and pathways of CHCA

2.3 Nitrate metabolism pathway during CHCA degradation

3. Conclusion

Declarations

References

Supplementary Files

Status:

Version 1