Alicyclic amines are widely used in several types of industries, and considerable attention has been devoted to possible environmental pollution by alicyclic amines in hypersaline industrial wastewater. In this study, a new hypersaline tolerant bacterial TYUT067 capable of growing in liquid basal salt medium with cyclohexylamine (CHAM) as the sole carbon source and energy source, was isolated from soil, and discovered with highly efficient CHAM degrading ability. The strain TYUT067 was identified as Paenarthrobacter sp. based on 16S rDNA gene sequence, and its degradation characteristic was examined. The results revealed that the isolated TYUT067 could grow well under pH range of 6.5–10.0, temperature from 20 °C to 30 °C. For degradation of 60 mM of cyclohexylamine, 100% degradation could be finished within 120 h. The TYUT067 could degrade 10 mM CHAM under hypersaline conditions (3–5% NaCl, w/v), revealed the hypersaline tolerance of TYUT067. Different type of amines was also tested with TYUT067, the degradations of >90% were achieved toward several alicyclic amines. The current results suggested that TYUT067 was a potential species could be efficiently used for the degradation of alicyclic amines and might be applicable to a hypersaline wastewater treatment system for the removal of alicyclic amines.
A new cyclohexylamine degradation bacterial strain TYUT067 was isolated from soil environment.
The isolated TYUT067 is an alkali-tolerant strain and mesophilic microorganisms.
The isolated TYUT067 can degrade CHAM under hypersaline environment.
The isolated TYUT067 can degrade various alicyclic amines.
Alicyclic amines are nonaromatic amines that have a cyclic alkyl moiety attached to the nitrogen atom. They are widely used as precursors or reagents for manufacturing several industrial chemicals, such as dyes, pesticides, rubber and foods (Cavender 2001; Lawrence 2004). Owing to their broad usage, alicyclic amines can reach the environment directly from industrial effluents or indirectly from pesticide degradation. The industrial wastewater with alicyclic amines will lead to high level of nitrogen and may become poisonous to environment and human health. Among the alicyclic amines, cyclohexylamine is widely used in the synthesis of antiseptic and insecticide, and considerable attention has been attracted to its potential toxicity toward human beings and aquatic ecosystems (Price et al. 1970; Petersen et al. 1972; Kroes et al. 1977).
Considering the great harm of amines to environment and human health in wastewater, various methods have been developed to degrade amines in wastewater. Conventional methods such as electrochemical degradation (Slein & Sansone 1980), chemical oxidation (Casero et al. 1997), photocatalytic oxidation (Maldotti et al. 1994) and thermal desorption–degradation of cyclohexylamine (Breen et al. 2000) were used for degrading amines. Recently, photocatalytic hydrogen production under visible light with simultaneous organic amine degradation was also reported (Wu et al. 2004; Peng et al. 2014; Yang et al. 2019). However, these methods usually have many limitations such as huge investments, complex processes and secondary pollution. Comparing the conventional physicochemical methods, biodegradation as a promising method with the advantages of low cost, practical feasibility, and eco-friendly, has become a hot spot worldwide in the field of wastewater treatment (Wei et al. 2003; Chanthamalee et al. 2013). In the past few decades, biodegradation have been widely used in the degradation of organic amines in wastewater. In 1979, Tokieda et al. reported anaerobic deamination of CHAM by the intestinal microorganisms of rabbits (Tokieda et al. 1979). In 1980, Calamari et al. reported the short-term toxicity of several aliphatic and aromatic amines on three species of organisms (Selenastrum capricornutum, Daphnia and Salmo gairdneri); simultaneously, biodegradation of these amines including the CHAM was investigated with three different inoculums (Calamari et al. 1980). In 1999, a pure culture of gram-positive bacterium (Brevibacterium oxydans IH-35A) with highly efficient CHAM degrading ability was isolated from soil, the CHAM degradation pathway in this strain was proposed as the following route: CHAM was first deaminated to cyclohexanone (CHnone) by the cyclohexylamine oxidase, CHnone was then oxidized to 6-hexanolactone, which was hydrolyzed to 6-hydroxyhexanoate and further converted into adipate (Iwaki et al. 1999a). Later, a novel cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans IH-35A responsible for the initial step of the degradation of CHAM to cyclohexanone was purified and characterized (Iwaki et al. 1999b; Mirza et al. 2013). In the last 10 years, two gram-negative bacteria Pseudomonas plecoglossicida NyZ12 (Shen et al. 2008) and Acinetobacter sp. YT-02 (Yan et al. 2018) capable of utilizing CHAM as the sole source of carbon and nitrogen, have been isolated from soil and the sludge in wastewater treatment pool, respectively. Their genomes were sequenced, open reading frames (ORFs) predicted to encode CHAM degradation were identified by bioinformatics analysis, the genes encoding cyclohexylamine oxidase and the gene cluster responsible for cyclohexanol and cyclohexanone degradation were proposed (Li et al. 2015; Yan et al. 2017).
Besides the alicyclic amines in industrial wastewaters, the high salinity is also a component of many industrial wastewaters. It is estimated that 5% of industrial wastewater is saline or hypersaline (Borgne et al. 2008). The treatment of this kind of wastewater poses a serious challenge, since the high ionic strength of wastewater can disrupt most of the bacteria present in activated sludge systems, blocking the degradation of organic pollutants. Halotolerant microorganisms are promising in bio-treatment of hypersaline industrial wastewater because their metabolism requires sodium chloride (Ventosa & Nieto 1995). At present, successful examples for biodegradation of aromatic acids, alkanes, chlorinated hydrocarbons, PAHs and phenol under the condition of high salinity have been documented (Margesin & Schinner 2001; Deng et al. 2017). However, only one document was reported for aerobic biodegradation of amines by activated sludge in industrial saline wastewaters with 3–7% NaCl (w/v) (Campo et al. 2011), while no pure cultures of hypersaline tolerant bacterium were reported for biodegradation of alicyclic amines. Then, isolation of halotolerant microorganisms for alicyclic amines degradation is highly required in industrial wastewater treatment.
In this study, a new hypersaline tolerant CHAM degradation strain was isolated for the first time from the soil environment. The strain was identified by 16SrDNA sequencing. The factors affecting the strain growth and CHAM degradation, including the pH, temperature, salinity, and the concentration of CHAM were examined. The degradation of different types of amines was also investigated with the isolated strain, and excellent degradation effect on alicyclic amines was observed.
MATERIALS AND METHODS
Aniline, benzylamine, phenethylamine, CHAM, cycloheptylamine, alpha-methylbenzylamine, trans-2-aminocyclohexanol, N-methyl-cyclohexylamine, cyclohexanol, cyclohexanone, ε-caprolactone and adipate were from Aladdin (Shanghai, China). All other chemicals and organic solvents were commercially available.
Enrichment and isolation of CHAM degradation bacteria
Soil samples were collected from the lawn and garden of Taiyuan University of Technology (TYUT) (Taiyuan, Shanxi province, China), where the insecticide may be sprayed for vegetation protection. CHAM are widely used as precursors or reagents for manufacturing insecticide, and the CHAM can reach the environment indirectly from insecticide degradation. Then, the soil from these places may contain the efficient CHAM degrading bacteria. Samples were collected in sterile 50-mL polyethylene tube, and stored at 4 °C for further use. Modified basal salt medium (MBSM) (pH 7.0) was utilized in the enrichment culture of CHAM-degrading bacteria from soil, which contained 10–20 mM CHAM-HCl as the sole source of carbon and energy, and was composed of (g/L): NH4NO3 1.0; KH2PO4 0.5; K2HPO4·3H2O 5.24; MgSO4·7H2O 0.2; 2.0 mL of 1% of CaCl2; 200 μL of 1% FeCl3; 5.0 mL of 1% yeast extract and 2.0 mL of trace elements solution. A 5.0 g of the soil sample was inoculated into the 50.0 mL of the CHAM-supplemented MBSM (10 mM CHAM-HCl). The cultures were incubated at 30 °C and 150 rpm. After 7 days, aliquots of the cultures (1 mL) were transferred to fresh CHAM containing MBSM (20 mM CHAM-HCl, 1% (w/v) NaCl). After five consecutive enrichment cultures, 1 mL of bacterial culture broth was serially diluted and 0.1 mL plated on CHAM containing agar medium, further incubated for 24–48 h at 30 °C for isolation of pure cultures. Single colonies grow on the agar medium were selected as the candidate CHAM-degrading strains, and their ability to grow in liquid medium with 20 mM CHAM as the sole source of carbon and energy were tested.
Single colonies grow on the CHAM containing agar medium were selected as the candidate CHAM-degrading strains. These colonies were then tested for their ability to grow in liquid MBSM with 20 mM CHAM as the sole source of carbon and energy. An isolate showing the maximum growth rate and the highest CHAM degradation was selected for further studies. The morphological properties (such as colony characteristics, shape of cells and gram reactivity) of the isolate were initially examined. The phylogenetic analysis of 16S rDNA gene was further performed for identification of the isolate. The genome DNA of selected strain was extracted using QIAGEN DNA extraction kit according to the manufacturer's instructions. The part of the 16S rDNA gene from the isolated strain was amplified by polymerase chain reaction (PCR) using a pair of forward (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse (5′-GGTTACCTTGTTACGACTT-3′) primers. The PCR amplification was performed in 30 μL reaction mixture containing 15 μL 2× Taq PCR Master Mix, 12 μL ddH2O, 1.0 μL each primer and 1.0 μL template genome DNA. The PCR amplification procedure were as follows: initial denaturation for 95 °C for 5 min, followed by 30 cycles of 94 °C for 40 s, 55 °C for 40 s and 72 °C for 1.0 min, and final extension at 72 °C for 10 min. The amplified products were purified by QIA Quick PCR Purification Kit (QIAGEN) and the sequencing was carried out at Sangon Biotech Co., Ltd (Shanghai, China). The 16S rDNA sequence was analyzed using the National Center for Biotechnology Information (NCBI) BLASTN programs (http://www.ncbi.nlm.nih.gov/BLAST). A phylogenetic tree was constructed by neighbor-joining method with MEGA 7.0.
Effect of pH and temperature
The effect of pH on microbial degradation of cyclohexylamine was determined by incubating (temperature at 30 °C) the pure bacterial culture in MBSM (50 mL) at pH values range from 4.0 to 10.0, 20 mM CHAM-HCl and 1% (w/v) NaCl were contained in the medium. The effect of temperature on microbial degradation of cyclohexylamine was evaluated by incubating the pure bacterial culture in MBSM (pH 7.0, 50 mL) at the temperature from 20 to 45 °C, 20 mM CHAM-HCl and 1% (w/v) NaCl were included in the medium. After 48 h of incubation, a 0.5 mL aliquot of the suspensions were taken and centrifuged and basified (pH > 10.0) by adding NaOH (10 μL, 10 N), then the samples were saturated with NaCl and extracted with 0.5 mL ethyl acetate containing 20 mM of n-dodecane as an internal standard. The organic phase was dried over sodium sulfate and subjected to GC analysis. The growth of isolate was measured by monitoring optical density (OD) at 600 nm using a spectrophotometer. The assays were performed in triplicate.
Degradation of different concentration of CHAM
The effect of different concentration of CHAM on the growth of microbial and degradation of CHAM were evaluated by incubating the pure bacterial culture in MBSM (pH 7.0, 50 mL) containing 10.0–60.0 mM CHAM-HCl and 1% (w/v) NaCl at 30 °C. The flasks were shaken on the rotary shaker at 200 rpm for 48 h. Then, a 0.5 mL aliquot of the suspensions was taken and the samples were extracted as described above. The growth of isolate was measured as described above. The assays were performed in triplicate.
Effect of different concentration of NaCl on cyclohexylamine degradation
The effect of different concentration of NaCl on the growth of microbial and degradation of cyclohexylamine were evaluated by incubating the pure bacterial culture in MBSM (pH 7.0, 50 mL) containing 10 mM CHAM-HCl and 1–7% (w/v) NaCl at 30 °C. The flasks were shaken on the rotary shaker at 200 rpm for 72 h. At interval times, a 0.5 mL aliquot of the suspensions was taken and the samples were extracted as described above. The growth of isolate was measured as described above. The assays were performed in triplicate.
Degradation of different type of carbon sources
The effect of different type of carbon sources on the growth of microbial were evaluated by incubating the pure bacterial culture in MBSM (pH 7.0, 50 mL) containing 10.0 mM carbon sources (aniline, benzylamine, phenethylamine, cycloheptylamine, cyclohexylamine, alpha-methybenzylamine, trans-2-aminocyclohexanol, N-methyl-cyclohexylamine, cyclohexanol, cyclohexanone, ε-caprolactone, adipate) and 5% (w/v) NaCl, respectively. The flasks were shaken on the rotary shaker at 200 rpm for 72 h. Then, a 0.5 mL aliquot of the suspensions was taken and the samples were extracted as described above. The growth of isolate was measured as described above. The assays were performed in triplicate.
The concentrations of amines, cyclohexanol, cyclohexanone and ε-caprolactone were quantified using gas chromatograph (GC-14C, Shimadzu, Japan) with a flame ionization detector (FID) and an HP-5 column (30 m × 0.320 mm × 0.25 mm; Agilent Technologies, Inc.). The details of instrumental conditions were as follows: injection temperature, 250 °C; detector temperature, 250 °C; column temperature, 120 °C.
RESULTS AND DISCUSSION
Enrichment and isolation of the CHAM-utilizing bacteria
Initially, 200 bacterial strains grown on the MBSM agar medium containing 20 mM CHAM-HCl and 1% NaCl were isolated. The potential bacterial isolates were selected according to the cell density (OD600) of the strain and degradation rate of CHAM. The results showed that few selected pure cultures grew well (cell density (OD600) > 2.0) in MBSM containing 10 mM CHAM-HCl and 1% NaCl, while a great number of the isolates grew less than 1.0 (OD600). After the first-round screening, 45 isolates with more than 50% of CHAM degradation were kept. In the second-round screening, 45 isolated strains were separately incubated in CHAM (20 mM) containing MBSM (1% NaCl included) for 24 h, the cell growth (OD600) and degradation of CHAM were examined, respectively. At last, seven bacterial strains with fastest growing and highly efficient CHAM degradation (>60%) were selected for further studies. As shown in Figure 1, strain TYUT067, which showed the highest degradation rates (100%) of CHAM and fastest growing in MBSM (OD600 3.1) among the isolated strains, was selected as the best candidate for further tests.
Identification of isolated strain
The strain TYUT067 showed as light yellow, opaque, smooth and moist on the surface of the cells, when it was growing on the MBSM agar medium (Figure 2(a)). The cells of strain TYUT067 was observed as gram positive and rod shaped by using an optical microscope. Based on the 16S rDNA sequencing and taxonomic analyses, strain TYUT067 was confirmed to belong to the genus Paenarthrobacter sp., showed 99.72% identity to Arthrobacter sp. TS-15 and Paenarthrobacter sp. EB58, respectively. Arthrobacter sp. TS-15 is a gram-positive soil bacterium with the ability to degrade ephedrine. This strain is closely related to Arthrobacter aurescens (Shanati & Ansorge-Schumacher 2020). Recently, these kinds of strains (Arthrobacter aurescens) were reclassified as Paenarthrobacter aurescens (Busse 2016). Therefore, strains TYUT067 was designated as Paenarthrobacter sp. TYUT067 (GenBank Accession No. MW320521) (Figure 2(b)). To our best knowledge, Paenarthrobacter sp. is one of the most studied degraders of herbicide (Deutch et al. 2018), nicotine (Mihăşan et al. 2018) and iprodione (Yang et al. 2018), while no relevant documents were reported on the degradation of CHAM with the strain of Paenarthrobacter sp.
Effect of temperature and pH on CHAM degradation
The optimum pH of TYUT067 for the degradation of CHAM was determined by incubation of the strain from pH 4.0 to 10.0. As shown in Figure 3(a), TYUT067 showed the highest degradation of CHAM (>99%) at pH 7.0–9.0, and the cells grown very well at these pH range, the highest OD600 can reach to 3.0 at pH 7.0. When the pH was below 7.0 or above 9.0, the degradation of CHAM was evidently decreased, and the TYUT067 stop growing at the pH below 6.0. The effect of temperature on the degradation of CHAM was examined at the temperatures from 20 to 45 °C. As shown in the Figure 3(b), TYUT-067 grow well in the MBSM medium at the temperatures from 20 to 35°C, with the CHAM degradation of 55–100% at 24 h cultivation. When the TYUT067 was incubated at the temperature above 35°C, the cells were growing very slowly and stopped growing at 45°C. The optimum temperature for the CHAM degradation and TYUT-067 growing was determined at 30°C, with the degradation of 100% and cell density (OD600) of 3.0, respectively. These results revealed that the isolated TYUT067 was an alkali-tolerant strain and mesophilic microorganisms. The genus Paenarthrobacter are widely distributed in soils from various environments all over the world. However, alkaliphilic or alkali-tolerant Paenarthrobacter have rarely been reported, and the strain TYUT067 may be particularly suitable for bioremediation under certain environmental stresses.
Growth curve and degradation of CHAM
The growth curve of TYUT067 and the degradation of CHAM were then analyzed by inoculating TYUT067 in MBSM medium with 10 mM CHAM-HCl and 1% NaCl. As shown in Figure 4, the TYUT067 grew very slowly at the lag phase. After 6 h, the TYUT067 entered into exponential growth phase, and the highest cell density (OD600) of TYUT067 could reach to 2.1 after cultivation for 26 h; after that, the cells entered into stationary phase. At the same time, 100% degradation of CHAM could be achieved. During the CHAM degradation, cyclohexanone was evidently detected in the growth process. The concentration of cyclohexanone (CHnone) intermediate product was increased at the first 12 h cultivation, then started to decrease, and completely degraded after 24 h cultivation. The accumulation of intermediate CHnone in the CHAM degradation process suggested that the biodegradation pathway of CHAM in this strain involves an initial deamination step. This phenomenon is similar to the previous reported strains (such as Brevibacterium oxydans IH-35A and Pseudomonas plecoglossicida NyZ12) which can degrade CHAM (Iwaki et al. 1999a; Shen et al. 2008). In addition, a trace amount of cyclohexanol was detected in the CHAM degradation process, and it was then metabolized transiently. We speculated that there may be an alcohol dehydrogenase (or ketoreductase) in the TYUT067 which is involved in the reduction of accumulated CHnone.
Degradation of different concentration of CHAM
Degradation of different concentration of CHAM was examined by inoculating TYUT067 in MSBM with CHAM-HCl from 10 to 60 mM for 48 h. As shown in Figure 5(a), the degradation of CHAM was gradually decreased (from 100% to 38%) with the increased concentration of CHAM (from 10 to 60 mM). For degradation of 10 and 20 mM CHAM, 100% degradation of CHAM could be finished within 48 h, with the OD600 of 2.1 and 3.0, respectively. For degradation of 30 mM CHAM, 80% degradation of CHAM could be finished after 48 h cultivation, and trace amount of cyclohexanone was observed. Further prolonging the cultivation time to 72 h, 100% degradation of CHAM could be finished. For 40 and 60 mM CHAM, the degradations of CHAM were decreased to 50 and 35% after 48 h cultivation, respectively, while 120 h cultivation time were needed for consumption of such high concentration (60 mM) of CHAM completely (Figure 5(b)).
Effect of different concentration of NaCl on CHAM degradation
The treatment of hypersaline wastewaters represents a challenge since the high salt concentrations can change the morphology of strain and decrease their metabolism of some carbon compounds. However, limited information was documented on amines biodegradation in hypersaline industrial effluents. Then the effect of different concentrations of NaCl on the degradation of CHAM by strain TYUT067 was investigated. As shown in Figure 6, with 1% (w/v) NaCl included in the medium, the degradation of CHAM could reach to 100% after 16 h cultivation, and the cell density (OD600) of TYUT067 could reach to 2.1 after 26 h cultivation. With 3% NaCl included in the medium, a slow degradation of CHAM was evidently observed, and 100% degradation of CHAM could be finished after 36 h cultivation. With 5% NaCl added, 100% degradation of CHAM could be finished after 72 h cultivation. Further increasing the NaCl to 7%, the TYUT067 stop growing, and trace amount of CHAM was degraded (<2%). As we know, waters containing dissolved salts (mainly NaCl) >1% (w/v) and >3.5% (w/v) are called saline water and hypersaline water (Lefebvre & Moletta 2006; Gayathri & Namasivayam 2010). The strain TYUT067 could grow in CHAM containing MSBM with high salt concentration (5% NaCl), indicated that the TYUT067 was a hypersaline tolerant bacterial which can be utilized to degrade CHAM in hypersaline wastewater.
Degradation of different substrates by TYUT067
The ability of TYUT067 to grow on various substrates was tested in liquid MBSM containing the carbon sources at a concentration of 10 mM. As shown in the Figure 7, TYUT067 grew very well in alicyclic amines containing MBSM, such as CHAM, cycloheptylamine, N-methycyclohexylamine, trans-2-aminocyclohexanol, with the degradation of 80–100% after 72 h cultivation. With aniline, benzylamine, and phenethylamine as sole carbon sources, TYUT067 grew very slowly and trace amounts of amines (<10%) were degraded. TYUT067 stop growing at the initial growth stage with adipate as the sole carbon source and energy source, which indicated that the TYUT067 could not utilize it for growing. As best as we know, assimilation of adipate by Paenarthrobacter aurescens is usually very weak (Busse 2016). Interestingly, TYUT067 grew very well on cyclohexanol, cyclohexanone and ε-caprolactone, and complete degradation of these substrates could be finished within three days. These results are consistent with the previous reported strains Pseudomonas plecoglossicida NyZ12 (Yan et al. 2017) and Acinetobacter sp. YT-02 (Yan et al. 2018), which can utilize cyclohexanol, cyclohexanone and ε-caprolactone as the sole carbon sources and energy sources. Then, a possible CHAM degradation pathway of TYUT067 was deduced as follows (Figure 8): CHAM was first deaminated to CHnone by the cyclohexylamine oxidase, CHnone was oxidized to 6-hexanolactone by the CHnone monooxygenase, 6-hexanolactone was then hydrolyzed to ring-cleaved product 6-hydroxyhexanoate by a hydrolase. Considering the TYUT067 cannot use adipate as the sole source of carbon and energy for growing, we speculate that there may be a novel degradation pathway for cyclohexylamine included in TYUT067. The cyclohexanol could be degraded by TYUT067, and CHnone was also detected in the cyclohexanol degradation process, suggesting that CHnone was a metabolic intermediate of cyclohexanol, we speculated that there may be a cyclohexanol dehydrogenase (or cyclohexanone reductase) was included in the cyclohexanol degradation pathway responsible for oxidation of cyclohexanol to cyclohexanone. Further analysis of the genome sequence information and identification of metabolites could provide the insight into metabolic mechanism of CHAM in TYUT067.
In conclusion, a new halotolerant strain TYUT067 was isolated from the soil environment with highly efficient cyclohexylamine degradation ability. The strain was confirmed as belonging to the genus Paenarthrobacter sp. by 16SrDNA sequencing and 16SrDNA similarity analysis. The strain TYUT067 was effective under pH range of 6.5–10.0, and temperature from 20 °C to 35 °C, revealed that the isolated TYUT067 was an alkali-tolerant strain and mesophilic microorganisms. The TYUT067 can degrade CHAM under hypersaline conditions (3–5% NaCl), and showed that TYUT067 is a hypersaline tolerant microorganism. Degradation of different types of amines was conducted with the isolated TYUT067, and excellent degradation effect was observed toward alicyclic amines. To our best knowledge, this is the first report on pure culture of hypersaline tolerant microorganism for alicyclic amine degradation. With the characteristic of fast growth, alkali resistance, hypersaline tolerant and highly efficient alicyclic amines degradation ability, the isolated TYUT067 may be a potential candidate to be used for the removal of alicyclic amines in hypersaline industrial wastewater treatment system. Further investigations on the degradation pathway of CHAM and cloning of corresponding enzyme genes from TYUT067 responsible for CHAM degradation are currently underway.
This work was financially supported by the Key Research and Development (R&D) Project of Shanxi Province (201803D31050).
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.