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Kinetic evaluation for rapid degradation of dimethylamine enriched with Agromyces and Ochrobactrum sp.
Journal of Environmental Management 245 (2019) 322–329
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Research article
Kinetic evaluation for rapid degradation of dimethylamine enriched with Agromyces and Ochrobactrum sp.
T
Ishan Raja,d,∗, Amit Bansiwalb, A.N. Vaidyac a
Environmental Biotechnology and Genomics Division, CSIR-NEERI, Nagpur, India Environmental Material Division, CSIR-NEERI, Nagpur, India c Solid and Hazardous Waste Management Division, CSIR-NEERI, Nagpur, India d Academy of Scientific and Innovative Research, CSIR-NEERI, Nagpur, 440020 Maharashtra, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Agromyces sp Ochrobactrum sp Whole genome analysis Dimethylamine Bioremediation Biodegradation kinetics
Dimethylamine (DMA) possesses an obnoxious odor which has resulted in public concern during the past several decades. A rare bacterial species proficient to degrade DMA, designated IR-26, was isolated from Indian Oil Corporation Limited (IOCL) and identified as Agromyces and Ochrobactrum sp., which has presented a rapid degradation when compared to other bacterial species which were capable to degrade DMA. The removal efficiency of 100% has been calculated in different concentration of DMA. The kinetic study reveals the maximum reduction rate of DMA was 0.11 per hour and the maximum growth rate of biomass was 0.013 per hour respectively. The saturation constant of DMA was around 1.96 mg/L which shows a high affinity of DMA. The importance of these analyses is offered and conversed in this paper.
1. Introduction Dimethylamine (DMA) belongs to an alkyl amines class which possesses carcinogenic and teratogenic effect at a very low concentration (Tate and Alaxander., 1976). DMA exhausts from various chemical, pharmaceutical, dye, petrochemical, and pesticide industries, which has developed as a serious problem (Rappert and Muller., 2005; Ye et al., 2013). DMA comprises of a strong, pungent and fishy smell which is highly unavoidable and creates a public nuisance (Zhang et al., 2012). DMA is also reported to cause headaches, dermatitis, and conjunctivitis and has been identified as a potential neurotoxin in uremic patients (Ye et al., 2013). DMA is also a prevalent precursor of N-nitrosodimethylamine (NDMA) which is a potent carcinogen and mutagen (Wang et al. (2014)). With the eradication of DMA, the chances for the formation of NDMA reduces which is ideal precautionary management approach, as once it is formed it is very difficult to eliminate it (Kristiana et al., 2013). Presently, extensive study is going on for the removal of NDMA precursor (He and Cheng. (2016)). Hence, it is suitable to explore methods for the eradication of DMA discharged from various sources. Several treatment technologies were reported for the eradication of DMA (Raj et al., 2018; Wysocka et al., 2019). Recently Gao et al. (2019) have reported the adsorption of DMA from the chemical industry to an adsorbent (Beta-Zeolite). Such options are not feasible as there is no treatment of the DMA has been presented, it
∗
merely transfers the pollutant to another medium which further requires attention. Microbial degradation is an important tool to eliminate the toxic and hazardous pollutant from the environment. DMA has been oxidized by the bacteria and results in monomethylamine and finally to ammonia (Liffourrena et al., 2010). Several microorganisms have been identified and characterized for the degradation of DMA like Arthrobacter sp., Hyphomicrobium sp Bacillus sp Pseudomonas aminovorans, Methylobacterium sp Methylophilus methylosporus, Mycobacterium sp Paracoccus sp Pseudomonas sp. Although more microbial flora has to be investigated and check which could yield a superior removal efficiency of DMA and other alkyl amines. Recently, Lidbury et al. (2017) have identified R. pomeroyi which is a marine bacterium capable of utilizing DMA along with other alkyl amines as a sole nitrogen source. In present study two bacterial strains were reported, capable of biological degradation of DMA with greater efficiency and studied degradation kinetic parameters of DMA. Enriched biomass was developed from Indian Oil Corporation Limited (IOCL), Assam, India and the kinetic parameters involved in the degradation has been calculated with Monod model which is presented and discussed further in the paper.
Corresponding author. Environmental Biotechnology and Genomics Division, CSIR-NEERI, Nagpur, India. E-mail address: [email protected] (I. Raj).
https://doi.org/10.1016/j.jenvman.2019.05.074 Received 18 February 2019; Received in revised form 13 May 2019; Accepted 19 May 2019 Available online 31 May 2019 0301-4797/ © 2019 Elsevier Ltd. All rights reserved.
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2. Material and methods
performing BLAST against KEGG genes database using KEGG automated annotation server (KAAS), respectively (Moriya et al., 2007; Huerta-Cepas et al., 2015). The KEGG orthology database of prokaryotes was used as the reference for pathway mapping. A total of 4,602 genes were processed in KAAS, of which 1,893 genes were classified into 23 functional pathway categories.
2.1. Inoculum preparation An inoculum was developed from activated sludge collected from IOCL, Assam. The biomass was enriched with DMA as the only carbon and nitrogen source along with other trace nutrients. The enrichment process was carried out in 1 L reagent bottle with deionized water. Regular maintenance was carried with centrifugation (6500 rpm for 8 min) of the biomass which was washed three times with NaCl and supplementing it with fresh minimal nutrient media and cumulative DMA. Microbial growth was determined by evaluating the optical density (OD) at 600 nm (OD600). A microbial colony was isolated by diluting the culture in deionized water and the microbes were selected by developing on two kinds of agar media for 24 h–96 h: Luria agar medium and Minimal medium supplemented with 50 mg/L of DMA. Bacterium for the degradation of DMA was designated and the strains were recognized by 16s rRNA amplification and sequencing tracked by sequence resemblance examines with BLAST program. In the present study whole genome analysis of Ochrobactrum intermedium has been discussed and genomic analysis of Agromyces indicus will be determined and discussed in the next communication.
2.5. Phylogenetic analysis The phylogeny at the chromosomal level has been analyzed for Ochrobactrum sp. Strain_AV using the final scaffold sequences against some of the closely related strains through neighbor-joining (NJ) algorithm of AAF phylogeny tool v2016104.1 (Fan et al., 2015) and the phylogeny plot was generated using MEGA (Kumar et al., 2016). 2.6. Data deposition and availability This Whole Genome Shotgun project has been placed at DDBJ/ENA/ GenBank under the accession SADZ00000000. The description defined in this paper is version SADZ01000000. 2.7. Biodegradation experiments
2.2. Genomic DNA extraction and microbial identification
The bacterial culture was prepared in 500 ml reagent bottles incubating in the minimal media at 30 °C on a horizontal shaker (150 rpm). The ingredients used for the minimal media are KH2PO4, K2HPO4, MgSO4.7H2O4, KCl, CaCl2, NaCl and FeSO4 (Liao et al., 2015). Five sets were arranged for the evaluation of biomass growth rate and DMA reduction rate which was operated in a 500 ml reagent bottles containing 200 ml of media: 10 mg/L DMA + Biomass 19.25 mg/L; (ii) 20 mg/L DMA + Biomass 19.25 mg/L; (iii) 30 mg/L DMA + Biomass 19.25 mg/L; (iv) 40 mg/L DMA + Biomass 19.25 mg/L; (v) 50 mg/L DMA + 19.25 mg/L.
Genomic DNA was extracted using the phenol-chloroform extraction method from the pure culture. The bacterial isolate was identified using 16S rRNA gene amplification and its subsequent Sanger sequencing. A similarity search against the NCBI database was performed for the 16S rRNA sequence using BLASTn (Altschul et al., 1990). 2.3. Whole genome sequencing The genomic libraries were prepared using the Illumina TruSeq Nano DNA library preparation kit (Illumina Inc USA) as per the Illumina protocol. The libraries were evaluated on the 4200 Tape Station system using High Sensitivity D5000 screen tape (Agilent Technologies, Santa Clara, CA, USA) to calculate the collection size. They were then measured on a Qubit 2.0 fluorometer by Qubitds DNA HS kit (Life Technologies, USA) consequent to the Illumina recommended procedure. A library was then introduced on Illumina Next Seq 500 platform (Illumina Inc USA) and 150 bp paired-end sequencing was performed by the Eurofins Genomics India Pvt. Ltd Bengaluru, India.
2.8. Analytical methods Bacteria were filtered from the minimal media with 0.22 μ membrane filter (Millex GV, Millipore, Ireland) before determining DMA and other derivatives. The filtrate was analyzed for DMA and ammonia in Ion chromatography (DIONEX- AS 500, Thermo Fisher, USA) equipped with the CS17 column. The flow rate of the gas in the instrument was sustained at 0.7 ml/min and suppressor temperature was adjusted at 30 °C. Methanesulfonic acid (6 mM) was prepared and used as an eluent to quantify DMA and ammonia. Bacterial growth was observed with UV-VIS spectrophotometer (Shimadzu, Japan) at OD600. An OD600 result obtained was changed to dry cell weight by a correlation acquired from a calibration curve. The curve was established by plotting dry weight of bacteria per liter against OD600 for the evaluation of growth and degradation rate. The total organic carbon was analyzed with TOC analyzer equipped with ASI-V autosampler (Shimadzu, Japan). The regular evaluation of pH was done with the help of the pH meter (Cyberscan, Eutech, USA) which was first calibrated with different pH standards. The bacterium mineralizing the DMA was isolated and amplified on a thermocycler (Bio-Rad T100, USA) which was further studied with the help of the Sanger sequencer and BLAST tool from NCBI.
2.4. De novo assembly and annotation A total of 6,872,010 reads were generated after sequencing which resulted in ∼2.06 Gb of sequenced data. The pre-processing i.e. ambiguity filtration and quality filtration, of the raw reads obtained from Illumina sequencing, were performed using Trimmomatic v0.35 (Bolger et al., 2014). The filtered reads obtained with a cut-off value for PHRED quality score of 20 were selected for de novo assembly which was carried out using SPAdes genome assembler (Nurk et al., 2013). A total of 1,152 scaffolds generated after assembly was then subjected to Contiguator, which is a finishing tool for bacterial genomes (Galardini et al., 2011). By applying prodigal from the assembled scaffolds genes were predicted, with default parameters, which resulted in the prediction of 4,062 genes (Hyatt et al., 2010). Functional annotation was accomplished by BLASTx program, which is a part of the NCBI-BLAST2.3.0 + standalone tool. Blast 2 GO platform was used to evaluate the gene ontology (GO) annotations (Conesa et al., 2005). GO assignments were applied to categorize the utilities of the predicted genes and a WEGO (Web Gene Ontology Annotation) plot was generated. Predicted genes were assigned to COG categories and KOs by
2.9. Theory and calculation The bacterial growth rate and DMA removal rate was evaluated in the present study by correlating the log of the bacterial culture (lnb) and time (t) by applying the Monod equation (Metcalf and Eddy, 2003)
μ = (Inb2 − Inb1)/(t2 − t1)
(1)
where, b1 and b2 are the bacterial concentrations (mg/L) at time t1 and t2 (h), respectively. The constraints of growth kinetics μ varies with (S) was observed 323
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hence, a double-reciprocal plot i.e. Lineweaver berk's plot was applied between the inverse of μ and the inverse of (S) which brings an association between μmax and KS as shown below (Metcalf and Eddy, 2003)
1/ μ = Ks / μmax (S ) + 1/ μmax
Table 2 Summary of KEGG pathway analysis for Ochrobactrum sp. Strain AV.
(2)
Here, μmax (per hour) is the maximum specific growth rate and Ks (mg/ L) is the half saturation constant for DMA. From 1st equation growth rate possibly be transformed and applied for determining the specific removal rate of DMA (VDMA), simultaneously resulted in the media is shown below:
V DMA = −(InDMA2 − InDMA1 )/(t2 − t1)C
(3)
where, DMA1 and DMA2 are the DMA concentrations at time t1 and t2, correspondingly, C is the initial microbial concentration in both situations. The specific removal rate (V) evaluated by equation (3) which contrasts with the initial DMA concentration. Therefore, values of maximum specific removal rate, Vmax (per hour) and half saturation constant, KS (mg/L) for DMA was estimated by applying Lineweaver berk's plot was drawn between the inverse of (V) and the inverse of (S), which brings an association between Vmax and KS as shown below: (4)
1/ V = K S /(Vmax . S ) + 1/ Vmax
3. Results and discussion 3.1. Microbial genomic analysis High quality of bacterial strain was enriched and developed from activated sludge sampled from Indian Oil Corporation Limited (IOCL), Assam. After regular maintenance of 120 days, DMA oxidizing bacterial strain was cultured and isolated. The isolate designated as IR-26 was gram-positive, rod-shaped cell bacteria. Further, the isolated colony was sequenced through 16s rRNA Sanger sequencing and found to be Agromyces and Ochrobactrum sp. The isolated strain has been submitted with accession number MF156974 and MG696218 in the NCBI database. The reported DMA degrading cultures comprised of genera Arthrobacter, Mycobacterium, Hyphomicrobium, Bacillus, Methylobacterium, Paracoccus, Pseudomonas, Microccocus, and Methylophilus (Rappert and Muller, 2005). Information on isolates of the alkyl amine degrading microbial community was quite limited. Till date, Agromyces and Ochrobactrum sp. has not been reported in the degradation of DMA and as per the author's best knowledge, the present study was first to report this new culture. The 16S rRNA study showed that the genome affiliated to Ochrobactrum species. The assembly of the draft genome sequence consisted of 1,152 scaffolds amounting to 4,958,020 bp with 78 bp and 666,518 bp as the minimum and maximum size of the scaffold, respectively (4,303 bp average size of the scaffolds). The scaffold N50 value was 383,647 bp. The genome features are summarized in Table 1. A total of 4,062 protein-coding genes were determined from the contigs with an average size of genes as 924 bases, and 90 and 8655 bases as a minimum and maximum size of genes, respectively. The distribution of genes into KEGG pathways is presented in Table 2. The
Pathway Metabolism
Number of genes
Amino acid metabolism Carbohydrate metabolism Metabolism of cofactors and vitamins Energy metabolism Nucleotide metabolism Metabolism of other amino acids Lipid metabolism Xenobiotics biodegradation and metabolism Biosynthesis of other secondary metabolites Metabolism of terpenoids and polyketides Glycan biosynthesis and metabolism Enzyme families Genetic information processing Transcription Translation Folding, sorting and degradation Replication and repair Environmental information processing Membrane transport Signal transduction Cellular processes Transport and catabolism Cell growth and death Cellular community - prokaryotes Cell motility Organismal systems Environmental adaptation
234 228 164 153 88 70 70 49 36 31 30 1 4 81 39 51 261 94 12 35 125 34 3
genome contained genes involved in basic metabolic pathways, such as pyruvate metabolism, glycolysis and TCA cycle, and was concluded to carry out aerobic respiration. Majority of the genes were observed to be associated with amino acid and carbohydrate metabolism. The GO assignments were used to classify the functions of the predicted genes and a WEGO (Web Gene Ontology Annotation) plot was generated, which suggested the pathways to majorly belong to the cellular component, molecular functions and biological processes (Fig. 1). The phylogenetic analysis for Ochrbactrum sp. Strain AV at the chromosomal level suggested the strain to be closest to Ochrobactrum sp. Strain UBA 7634 (Fig. 2). This suggests that the strain AV could be a novel member of the genus Ochrobactrum. Circular representation of Ochrobactrum strain_AV genome by CGView server (Fig. 3) demonstrates high proportion regions of the genome being similar to Ochrobactrum sp. UBA7634, Ochrobactrum intermedium, and Ochrabacterum A44. The figure also enunciates that there are large regions which are unique to only Ochrobactrum strain AV and not found in other Ochrobactrum sp. UBA7634, Ochrobactrum intermedium and Ochrobactrum Ochrobactrum intermedium strain_AV
Table 1 Genome features of Ochrobactrum intermedium strain_AV. Attribute
Value
NCBI accession number Genome size Total no. of scaffolds Shortest scaffold Longest scaffold N50 average length Total no. of protein-coding genes
SADZ00000000 4,958,020 bp 1,152 78 bp 666,518 bp 383,647 bp 4,303 bp 4,062
Fig. 1. Graphical representation of GO terms via WEGO plot Ochrobactrum intermedium strain_AV. 324
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Fig. 2. Phylogeny of the isolated bacteria among Ochrobactrum species.
shown to utilize nitrogen from methylamine and not the carbon for its energy and growth. More such microorganism needs to be explored for finding a high metabolic oxidation rate of DMA and other alkyl amines. In the present study, Agromyces sp. and Ochrabacterum sp. has been reported for the first time for the biodegradation of DMA as per the author's best knowledge.
A44. Phylogenetic analysis of Ochrobactrum strain AV with other Ochrobactrum species shows similar trends as obtained by the CG View result. Different microorganisms of genera Pseudomonas, Arthobacter and Paracoccus have been reported degrading TMA and DMA both in water and air (Chang et al., 2004). Recently Dziewit et al. (2015) have reported Paracoccus aminophilus JCM 7686 which was proficient to consume different kinds of carbon composites as the only energy source. The gene responsible for oxidizing DMA and other alkyl amines were identified. Chen et al. (2010) have also documented the degradation of methylamine by nonmethylotrophs (eg. Agrobacterium tumefaciens) has
3.2. Kinetics study of DMA Biological degradation of DMA by different microbial culture has been documented (Sharp et al., 2010; Weidhaas et al., 2012; Liao et al.,
Fig. 3. Graphical comparison Ochrobactrum Intermedium strain_AV (SADZ00000000) genome with Ochrobactrum intermedium LMG 3301, Ochrobactrum UB7634 and Ochrabactrum A44 retrireved from NCBI database using CGView server using default parameters. 325
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different previous reports. The degradation kinetics of DMA was analyzed, seeded with biomass concentrations 19 mg/L. The results of the microbial growth are shown in Fig. 4a. Agromyces sp. and Ochrobactrum sp. initiate its growth in 1.5 h of the study. DMA as a carbon substrate dependent growth periods includes lag phase, log, stationary were observed during the incubation period of 45 h. The microbial growth was determined very slow though high degradation capacity was observed. Hence, it can be inferred that the bacterial culture possesses a high metabolism which could consume DMA as a sole energy source. Various literature has reported the degradation kinetics by different microbial culture on alkyl amine especially TMA but very less literature on biodegradation kinetics of DMA was found. The growth rate of Methylophilus methylotrophus and Methylobacterium sp. was determined for TMA degradation (Roseiro et al., 1999). The growth kinetics parameters were shown by Agromyces sp. and Ochrobactrum sp. was calculated by applying Eqs (1) and (2) and represented in Fig. 4b and c. The μmax of the reaction was calculated using Eq. (3) was 0.013 per hour and the saturation constant (Ks) of the system has been calculated 0.63 mg/L. The kinetics studies have shown and demonstrated a very slow growth of microbial culture and growth rate slow down with a rise in the concentration which was a standard observation in the kinetic modeling. The inhibition effect on the growth was negligible which shows the ability of the microbial culture to degrade the higher DMA concentration. No inhibition effect on growth was noticed during the incubation period which established the capability of these bacteria in degrading TMA at much higher concentration. Biodegradation study of DMA was evaluated by analyzing the degradation of different concentration of DMA (Fig. 5a). Rapid biodegradation of DMA was observed when enriched with Agromyces and Ochrobactrum sp. A DMA concentration of 10 mg/L has been completely oxidized in 20 h of study. Liao et al. (2015) has reported DMA degradation from drinking water biofilter and has shown the degradation of 10 mg/L in 7 days supplementing with an additional carbon source. The present study has shown a high removal rate of DMA and high metabolic rate of bacteria with DMA as an only nitrogen and carbon source to the isolated degraders. The DMA biodegradation mechanism
Fig. 4. (a) Bacterial growth curve (b) specific growth (c) maximum growth rate is evaluated by lineweaver-berk plot.
2015). Moreover, DMA is among the transitional product in the TMA breakdown (Ho et al., 2008). Biological degradation of different alkyl amines by isolated inoculum has been well described in different studies (Rappert and Muller, 2005). Recently Oyarzun et al. (2019) has shown the degradation of alkyl amine from the fishmeal industry and DMA with a removal efficiency of 83% was reported. Different reports specified that biotreatment reactors inoculated with various microbial cultures could efficiently treat alkyl amine in gaseous form (Chang et al., 2004; Ho et al., 2008; Yin and Xu, 2012). However, there has been very less study on biodegradation kinetics of DMA or other aliphatic amines. Survey of the literature reveals as first report on the evaluation of the degradation rate of DMA by the A. indicus and O. intermedium. In the present study, the rapid degradation of DMA was established by the enriched culture. This result proposed that the degradation kinetics of DMA has been highly potent when enriched with Agromyces and Ochrobactrum sp. treatment when compared with
Fig. 5. DMA degradation is evaluated with the activity of microbial culture. (A) DMA degradation and carbon consumption (B) specific degradation of DMA and halflife of DMA (C) maximum degradation rate is evaluated by lineweaver-berk plot. 326
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mainly depends upon the intake of carbon present in the DMA by the biomass which degraded DMA into MMA which finally mineralizes to ammonia. The bacterial culture contains Agromyces and Ochrobactrum sp. has shown a normal sigmoidal growth phase in the study. Growth kinetics was calculated by applying equations (1) and (2) to the observation obtained from the proliferation of bacteria in the presence of DMA (Fig. 5). The maximum growth rate has been calculated by applying equation (3) which has been observed around 0.013 per hour. The growth rate was very less and it confirms that the bacterial species Agromyces and Ochrobactrum sp. grows very slowly in presence of DMA which was the only energy source. The saturation constant Ks of the bacterial growth was 0.6 mg/L which was quite high and show the low affinity of the bacteria. Similarly, the degradation rate has been evaluated of different concentration of DMA and 100% removal efficiency was observed in all setup. Specific degradation rate (VDMA) for different concentration of DMA has shown a declining pattern with the increase in the concentration and half-life (T-half) of DMA has also shown an integrated pattern with the escalation of DMA (Fig. 5b). From the study, the maximum degradation rate has been evaluated with the help of equation (4), the Lineweaver berk's plot has shown maximum degradation rate in higher concentration. The Vmax of degradation study was observed around 0.11per hour and a saturation constant of DMA (Km) degradation was 1.96 mg/L. The Vmax in the study was quite low but Km has shown a good affinity of DMA (Fig. 5c). The degradation rate has been checked, compare and evaluated using different models presented in Table 3. Biodegradation of DMA has been described in various literature (Webster et al., 2013; Liao et al., 2015). Rappert and Muller (2005) have shown different bacterial species capable to degrade DMA. Recently, Santawee et al. (2019) has also shown the rapid degradation of TMA when inoculated with Bacillus Thuringiensis. Also, Lobo et al. (1999) have reported the biological degradation kinetics of TMA with the help of Aminobacter aminovorans in which the μmax reported was 0.08 g/L/h which were quite higher when we compare it to the present study and the Vmax reported was 1.79 mM/h. There were no reports which have elaborated the DMA biodegradation kinetics so far as per the author's best knowledge. Subsequently, Ho et al. (2008) have calculated Vmax of DMA along with other alkyl amines with the application of Paracoccus sp. CP2 resulted in Vm = 0.9g-N Kg-1 and Km = 64 ppm which has shown a slow rate of degradation when compared to the degradation with Agromyces and Ochrobactrum sp. The saturation constant (Km) in Ho et al. study was very high which depicts a very low affinity of Paracoccus with the substrate. DMA was a limited energy source for the biomass to nurture. DMA offers a carbon and nitrogen source to the bacteria. The nitrogen was a vital component to the bacteria to proliferate (helps in replication). Hence, the nitrogen consumption by the bacteria from the DMA to the ammonia has been estimated in varied concentration (Table 4). The nitrogen content present in the initial study phase of TMA and final degradation product ammonia has been calculated and the difference among them has been assumed to be taken up by the bacteria. It has been observed that Agromyces sp. and Ochrobactrum sp. was consuming nitrogen source for its growth and metabolic activity. Thus, the isolated biomass was capable of DMA degradation. As per best of the author's knowledge, this is the first study to evaluate the biodegradation kinetic parameters of
Table 4 Nitrogen utilization by the isolated degraders from DMA degradation to ammonia formation. S.No
DMA concentration (mg/L)
Initial N content in DMA (mg/L)
Final N content in ammonia (mg/L) (PPM)
N utilized by the bacteria (mg/L)(P
1. 2. 3. 4. 5.
10 20 30 40 50
3.10 6.21 9.31 12.4 15.5
1 2 2.93 3.68 4.61
2.1 4.21 6.38 8.72 10.9
DMA by Agromyces and Ochrobactrum sp. 3.3. TOC reduction, ammonia formation, and change in pH The reduction of TOC of different concentration inoculated with Agromyces and Ochrobactrum sp. has been presented in Fig. 5a. The reduction in carbon concentration from the initial period towards the completion of the study has shown the depletion of DMA by the bacterial inoculum. The higher concentration of DMA has high TOC value and provided more carbon to the bacterium which has given maximum bacterial growth in the study. Liao et al. (2015) have reported the TOC reduction of DMA by a microbial consortium. Although the degradation activity of the isolated degraders was far way slower when compared to the present study. DMA metabolizes into ammonia and formaldehyde under aerobic condition and follows the same degradation pathway of trimethylamine (TMA) (Kim et al., 2001). TMA could be primarily dissolved to DMA and to methylamine (MA). MA finally dissolved to ammonia under a specific environment (Ho et al., 2008). In the present study, arise in ammonia was noticed in the presence of A. indicus and O. intermedium during the incubation, endorsing that DMA was finally mineralized to ammonia. The increase in ammonia has similarly been described in the earlier studies in the degradation of alkyl amine (Ding et al., 2007; Liao et al., 2015).Ammonia formation was observed with the biodegradation of different concentration of DMA. Ammonia as the end product of DMA removal was calculated (Fig. 6) and the higher concentration of 50 mg/L of DMA has resulted in the high concentration of ammonia i.e. 5.5 mg/L. Ho et al. (2008) subsequently degraded ammonia formed by the degradation of alkylamine by employing Arthobacter sp. (ammonia oxidizer) alongside Paracoccus sp. (amine oxidizer) results finally to nitrite and nitrate as a residual in a bioreactor system. However, ammonia was in ion form in the residual left after the amine degradation and hence it was not toxic but eradicating it alongside with alkyl amine is a needful approach. The pH of the biodegradation study was presented in Fig. 7. The value of pH has shown a reduction with the reduction of DMA or TOC in the sample. DMA was having a higher pKa value and hence, a high concentration of DMA will have high pH. Therefore, with the degradation of DMA when inoculated with Agromyces and Ochrobactrum has resulted in the reduction of pH. The value of all the different concentration of DMA has come close to neutral pH which helps in ammonia nitrogen present in the residual to be stable which was present in the ionic form. 4. Conclusion
Table 3 Comparative model study for maximum degradation rate (Vmax) and saturation constant (Km). Vmax (per h)
The present study comprises of oxidation for DMA with investigating the kinetic parameter and specifies the ideal condition for establishing the biological process. Genome information could be suitable for determining the potential of Ochrobactrum sp. and validated its application for the degradation of DMA and other alkyl amines. Slow bacterial growth with a high metabolic activity was observed. Monod model was successfully applied in evaluating the kinetic parameters in the present study. The observation of TOC and ammonia has further validated the degradation capacity of the isolated degraders on DMA.
Km (mg/L)
Model 3.24 Hanes Plot Lineweaver-Burk Plot Eadie-Hofstee Plot Results
0 .1083 0.1144 0.1148
1.96 1.92
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Fig. 6. DMA of cumulative concentration mineralizes into aggregated ammonia nitrogen. org/10.1016/S0022-2836 (05) 80360-2. Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics btu170. https://doi.org/10.1093/bioinformatics/ btu170. Chen, Yin, Scanlan, Julie, Song, Lijiang, Crombie, Andrew, Tanvir Rahman, M., Schäfer, Hendrik, Colin Murrell, J., 2010. γ-Glutamylmethylamide is an essential intermediate in the metabolism of methylamine by Methylo cellasilvestris. Appl. Environ. Microbiol. 76 (13), 4530–4537. https://doi:10.1128/AEM.00739-10. Conesa, Ana, Götz, Stefan, García-Gómez, Juan Miguel, Terol, Javier, Talón, Manuel, Robles, Montserrat, 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21 (18), 3674–3676. https://doi.org/10.1093/bioinformatics/bti610. Ding, Y., Shi, J.Y., Wu, W.X., Yin, J., Chen, Y.X., 2007. Trimethylamine (TMA) biofiltration and transformation in biofilters. J. Hazard Mater. 143, 341–348. https://doi. org/10.1016/j.jhazmat.2006.09.031. Dziewit, Lukasz, Czarnecki, Jakub, Prochwicz, Emilia, Daniel, Wibberg, Schlüter, Andreas, Pühler, Alfred, Bartosik, Dariusz, 2015. Genome-guided insight into the methylotrophy of Paracoccus aminophilus JCM 7686. Front. Microbiol. 6, 852. https://doi.org/10.3389/fmicb.2015.00852. Fan, H., Ives, A.R., Surget-Groba, Y., Cannon, C.H., 2015. An assembly and alignment-free method of phylogeny reconstruction from next-generation sequencing data. BMC Genomics 16 (1), 522. https://doi.org/10.1186/s12864-015-1647-5. Galardini, M., Biondi, E.G., Bazzicalupo, M., Mengoni, A., 2011. CONTIGuator: a bacterial genomes finishing tool for structural insights on draft genomes. Source Code Biol. Med. 6 (1), 11. https://doi.org/10.1186/1751-0473-6-11. Gao, K., Wang, Q., Du, X., Wei, Q., Huang, Y., 2019. Efficient adsorption and eco-environmental oxidization of dimethylamine in Beta zeolite. J. Microb. Meso 282, 219–227. https://doi.org/10.1016/j.micromeso.2019.03.023. He, Yuanzhen, Cheng, Hefa, 2016. Degradation of N-nitrosodimethylamine (NDMA) and its precursor dimethylamine (DMA) in mineral micropores induced by microwave irradiation. Water Res. 94, 305–314. https://doi.org/10.1016/j.watres.2016.02.065. Ho, K.L., Chung, Y.C., Lin, Y.H., Tseng, C.P., 2008. Biofiltration of trimethylamine, dimethylamine, and methylamine by immobilized Paracoccus sp. CP2 and Arthrobacter sp. CP1. Chemosphere 72, 250–256. https://doi.org/10.1016/j.chemosphere.2008. 01.044. Huerta-Cepas, J., Szklarczyk, D., Forslund, K., Cook, H., Heller, D., Walter, M.C., Rattei, T., Mende, D.R., Sunagawa, S., Kuhn, M., 2015. A hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res. 44, D286–D293. https://doi.org/10.1093/nar/gkv1248. Hyatt, D., Chen, G.-L., Locascio, P.F., Land, M.L., Larimer, F.W., Hauser, L.J., 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinf. 11, 119. https://doi.org/10.1186/1471-2105-11-119. Kim, S.G., Bae, H.S., Lee, S.T., 2001. A novel denitrifying bacterial isolate that degrades trimethylamine both aerobically and anaerobically via two different pathways. Arch. Microbiol. 176 (4), 271–277. https://doi.org/10.1007/s002030100319. Kristiana, I., Tan, J., Joll, C.A., Heitz, A., von Gunten, U., Charrois, J.W.A., 2013. Formation of N-nitrosamines from chlorination and chloramination of molecular weight fractions of natural organic matter. Water Res. 47, 535–546. https://doi.org/ 10.1016/j.watres.2012.10.014. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. https://doi.
Fig. 7. Effect on pH on the degradation of DMA with different concentration.
On comparing the various aspects of the degradation study, it is suggested that the rapid biodegradation of DMA can be observed when seeded with Agromyces indicus and Orchobactrum intermedium. Such investigation in the future will help the biofiltration industry to flourish in the eradication of such hazardous pollutants. Acknowledgment Authors are grateful to Dr. Rakesh Kumar, Director, CSIR- NEERI, INSPIRE program of Department of Science and Technology, and Government of India for the financial support in carrying out this research work and Knowledge Resource Center (KRC), CSIR- NEERI for checking the plagiarism (KRC.:CSIR-NEERI/KRC/2019/FEB/EBGDSHWMD-EMD/1). References Altschul, Stephen F., Gish, Warren, Miller, Webb, Myers, Eugene W., Lipman, David J., 1990. Basic local alignment search tool. J. Mol. Biol. 215 (3), 403–410. https://doi.
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org/10.1093/molbev/msw054. Liffourrena, Andrés, S., Salvano, Mario A., Lucchesi, Gloria I., 2010. Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways. Arch. Microbiol. 192 (6), 471–476. https://doi: 10.1007/s00203-010-0577-5. Liao, X., Chen, C., Zhang, J., Dai, Y., Zhang, X., Xie, S., 2015. Dimethylamine biodegradation by mixed culture enriched from drinking water biofilter. Chemosphere 119, 935–940. https://doi.org/10.1016/j.chemosphere.2014.09.020. Lidbury, I., Mausz, M.A., Scanlan, D.J., Chen, Y., 2017. Identification of dimethylamine monooxygenase in marine bacteria reveals a metabolic bottleneck in the methylated amine degradation pathway. ISME J. 11 (7), 1592. https://doi:10.1038/ismej. 2017.31. Metcalf, E.E., Eddy, H.P., 2003. Wastewater Engineer Treatment Disposal, Reuse. McGRaw, New York. Moriya, Y., Itoh, M., Okuda, S., Yoshizawa, A.C., Kanehisa, M., 2007. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 35, W182–W185. https://doi.org/10.1093/nar/gkm321. Nurk, S., Bankevich, A., Antipov, D., Gurevich, A.A., Korobeynikov, A., Lapidus, A., Prjibelski, A.D., Pyshkin, A., Sirotkin, A., Sirotkin, Y., Stepanauskas, R., Clingenpeel, S.R., Woyke, T., Mclean, J.S., Lasken, R., Tesler, G., Alekseyev, M.A., Pevzner, P.A., 2013. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J. Comput. Biol. 20, 714–737. https://doi.org/10.1089/cmb.2013.0084. Oyarzun, P., Alarcón, L., Calabriano, G., Bejarano, J., Nuñez, D., Ruiz-Tagle, N., Urrutia, H., 2019. Trickling filter technology for biotreatment of nitrogenous compounds emitted in exhaust gases from fishmeal plants. J. Environ. Manag. 232, 165–170. https://doi.org/10.1016/j.jenvman.2018.11.008. Raj, I., Vaidya, A.N., Pandey, R.A., Bansiwal, A., Deshmukh, S., Purohit, H.J., 2018. Recent advancements in the mitigation of obnoxious nitrogenous gases. J. Environ. Manag. 205, 319–336. https://doi.org/10.1016/j.jenvman.2017.09.064. Rappert, S., Muller, R., 2005. Microbial degradation of selected odorous substances. Waste Manag. 25, 940–954. http://doi.org/10.1016/j.wasman.2005.07.015. Roseiro, J.C., Partidário, P.J., Lobo, N., Marcal, M.J., 1999. Physiology and kinetics of trimethylamine conversion by two methylotrophic strains in continuous cultivation systems. Appl. Microbiol. Biotechnol. 52, 546–552. https://doi.org/10.1007/
329
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Research article
Kinetic evaluation for rapid degradation of dimethylamine enriched with Agromyces and Ochrobactrum sp.
T
Ishan Raja,d,∗, Amit Bansiwalb, A.N. Vaidyac a
Environmental Biotechnology and Genomics Division, CSIR-NEERI, Nagpur, India Environmental Material Division, CSIR-NEERI, Nagpur, India c Solid and Hazardous Waste Management Division, CSIR-NEERI, Nagpur, India d Academy of Scientific and Innovative Research, CSIR-NEERI, Nagpur, 440020 Maharashtra, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Agromyces sp Ochrobactrum sp Whole genome analysis Dimethylamine Bioremediation Biodegradation kinetics
Dimethylamine (DMA) possesses an obnoxious odor which has resulted in public concern during the past several decades. A rare bacterial species proficient to degrade DMA, designated IR-26, was isolated from Indian Oil Corporation Limited (IOCL) and identified as Agromyces and Ochrobactrum sp., which has presented a rapid degradation when compared to other bacterial species which were capable to degrade DMA. The removal efficiency of 100% has been calculated in different concentration of DMA. The kinetic study reveals the maximum reduction rate of DMA was 0.11 per hour and the maximum growth rate of biomass was 0.013 per hour respectively. The saturation constant of DMA was around 1.96 mg/L which shows a high affinity of DMA. The importance of these analyses is offered and conversed in this paper.
1. Introduction Dimethylamine (DMA) belongs to an alkyl amines class which possesses carcinogenic and teratogenic effect at a very low concentration (Tate and Alaxander., 1976). DMA exhausts from various chemical, pharmaceutical, dye, petrochemical, and pesticide industries, which has developed as a serious problem (Rappert and Muller., 2005; Ye et al., 2013). DMA comprises of a strong, pungent and fishy smell which is highly unavoidable and creates a public nuisance (Zhang et al., 2012). DMA is also reported to cause headaches, dermatitis, and conjunctivitis and has been identified as a potential neurotoxin in uremic patients (Ye et al., 2013). DMA is also a prevalent precursor of N-nitrosodimethylamine (NDMA) which is a potent carcinogen and mutagen (Wang et al. (2014)). With the eradication of DMA, the chances for the formation of NDMA reduces which is ideal precautionary management approach, as once it is formed it is very difficult to eliminate it (Kristiana et al., 2013). Presently, extensive study is going on for the removal of NDMA precursor (He and Cheng. (2016)). Hence, it is suitable to explore methods for the eradication of DMA discharged from various sources. Several treatment technologies were reported for the eradication of DMA (Raj et al., 2018; Wysocka et al., 2019). Recently Gao et al. (2019) have reported the adsorption of DMA from the chemical industry to an adsorbent (Beta-Zeolite). Such options are not feasible as there is no treatment of the DMA has been presented, it
∗
merely transfers the pollutant to another medium which further requires attention. Microbial degradation is an important tool to eliminate the toxic and hazardous pollutant from the environment. DMA has been oxidized by the bacteria and results in monomethylamine and finally to ammonia (Liffourrena et al., 2010). Several microorganisms have been identified and characterized for the degradation of DMA like Arthrobacter sp., Hyphomicrobium sp Bacillus sp Pseudomonas aminovorans, Methylobacterium sp Methylophilus methylosporus, Mycobacterium sp Paracoccus sp Pseudomonas sp. Although more microbial flora has to be investigated and check which could yield a superior removal efficiency of DMA and other alkyl amines. Recently, Lidbury et al. (2017) have identified R. pomeroyi which is a marine bacterium capable of utilizing DMA along with other alkyl amines as a sole nitrogen source. In present study two bacterial strains were reported, capable of biological degradation of DMA with greater efficiency and studied degradation kinetic parameters of DMA. Enriched biomass was developed from Indian Oil Corporation Limited (IOCL), Assam, India and the kinetic parameters involved in the degradation has been calculated with Monod model which is presented and discussed further in the paper.
Corresponding author. Environmental Biotechnology and Genomics Division, CSIR-NEERI, Nagpur, India. E-mail address: [email protected] (I. Raj).
https://doi.org/10.1016/j.jenvman.2019.05.074 Received 18 February 2019; Received in revised form 13 May 2019; Accepted 19 May 2019 Available online 31 May 2019 0301-4797/ © 2019 Elsevier Ltd. All rights reserved.
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2. Material and methods
performing BLAST against KEGG genes database using KEGG automated annotation server (KAAS), respectively (Moriya et al., 2007; Huerta-Cepas et al., 2015). The KEGG orthology database of prokaryotes was used as the reference for pathway mapping. A total of 4,602 genes were processed in KAAS, of which 1,893 genes were classified into 23 functional pathway categories.
2.1. Inoculum preparation An inoculum was developed from activated sludge collected from IOCL, Assam. The biomass was enriched with DMA as the only carbon and nitrogen source along with other trace nutrients. The enrichment process was carried out in 1 L reagent bottle with deionized water. Regular maintenance was carried with centrifugation (6500 rpm for 8 min) of the biomass which was washed three times with NaCl and supplementing it with fresh minimal nutrient media and cumulative DMA. Microbial growth was determined by evaluating the optical density (OD) at 600 nm (OD600). A microbial colony was isolated by diluting the culture in deionized water and the microbes were selected by developing on two kinds of agar media for 24 h–96 h: Luria agar medium and Minimal medium supplemented with 50 mg/L of DMA. Bacterium for the degradation of DMA was designated and the strains were recognized by 16s rRNA amplification and sequencing tracked by sequence resemblance examines with BLAST program. In the present study whole genome analysis of Ochrobactrum intermedium has been discussed and genomic analysis of Agromyces indicus will be determined and discussed in the next communication.
2.5. Phylogenetic analysis The phylogeny at the chromosomal level has been analyzed for Ochrobactrum sp. Strain_AV using the final scaffold sequences against some of the closely related strains through neighbor-joining (NJ) algorithm of AAF phylogeny tool v2016104.1 (Fan et al., 2015) and the phylogeny plot was generated using MEGA (Kumar et al., 2016). 2.6. Data deposition and availability This Whole Genome Shotgun project has been placed at DDBJ/ENA/ GenBank under the accession SADZ00000000. The description defined in this paper is version SADZ01000000. 2.7. Biodegradation experiments
2.2. Genomic DNA extraction and microbial identification
The bacterial culture was prepared in 500 ml reagent bottles incubating in the minimal media at 30 °C on a horizontal shaker (150 rpm). The ingredients used for the minimal media are KH2PO4, K2HPO4, MgSO4.7H2O4, KCl, CaCl2, NaCl and FeSO4 (Liao et al., 2015). Five sets were arranged for the evaluation of biomass growth rate and DMA reduction rate which was operated in a 500 ml reagent bottles containing 200 ml of media: 10 mg/L DMA + Biomass 19.25 mg/L; (ii) 20 mg/L DMA + Biomass 19.25 mg/L; (iii) 30 mg/L DMA + Biomass 19.25 mg/L; (iv) 40 mg/L DMA + Biomass 19.25 mg/L; (v) 50 mg/L DMA + 19.25 mg/L.
Genomic DNA was extracted using the phenol-chloroform extraction method from the pure culture. The bacterial isolate was identified using 16S rRNA gene amplification and its subsequent Sanger sequencing. A similarity search against the NCBI database was performed for the 16S rRNA sequence using BLASTn (Altschul et al., 1990). 2.3. Whole genome sequencing The genomic libraries were prepared using the Illumina TruSeq Nano DNA library preparation kit (Illumina Inc USA) as per the Illumina protocol. The libraries were evaluated on the 4200 Tape Station system using High Sensitivity D5000 screen tape (Agilent Technologies, Santa Clara, CA, USA) to calculate the collection size. They were then measured on a Qubit 2.0 fluorometer by Qubitds DNA HS kit (Life Technologies, USA) consequent to the Illumina recommended procedure. A library was then introduced on Illumina Next Seq 500 platform (Illumina Inc USA) and 150 bp paired-end sequencing was performed by the Eurofins Genomics India Pvt. Ltd Bengaluru, India.
2.8. Analytical methods Bacteria were filtered from the minimal media with 0.22 μ membrane filter (Millex GV, Millipore, Ireland) before determining DMA and other derivatives. The filtrate was analyzed for DMA and ammonia in Ion chromatography (DIONEX- AS 500, Thermo Fisher, USA) equipped with the CS17 column. The flow rate of the gas in the instrument was sustained at 0.7 ml/min and suppressor temperature was adjusted at 30 °C. Methanesulfonic acid (6 mM) was prepared and used as an eluent to quantify DMA and ammonia. Bacterial growth was observed with UV-VIS spectrophotometer (Shimadzu, Japan) at OD600. An OD600 result obtained was changed to dry cell weight by a correlation acquired from a calibration curve. The curve was established by plotting dry weight of bacteria per liter against OD600 for the evaluation of growth and degradation rate. The total organic carbon was analyzed with TOC analyzer equipped with ASI-V autosampler (Shimadzu, Japan). The regular evaluation of pH was done with the help of the pH meter (Cyberscan, Eutech, USA) which was first calibrated with different pH standards. The bacterium mineralizing the DMA was isolated and amplified on a thermocycler (Bio-Rad T100, USA) which was further studied with the help of the Sanger sequencer and BLAST tool from NCBI.
2.4. De novo assembly and annotation A total of 6,872,010 reads were generated after sequencing which resulted in ∼2.06 Gb of sequenced data. The pre-processing i.e. ambiguity filtration and quality filtration, of the raw reads obtained from Illumina sequencing, were performed using Trimmomatic v0.35 (Bolger et al., 2014). The filtered reads obtained with a cut-off value for PHRED quality score of 20 were selected for de novo assembly which was carried out using SPAdes genome assembler (Nurk et al., 2013). A total of 1,152 scaffolds generated after assembly was then subjected to Contiguator, which is a finishing tool for bacterial genomes (Galardini et al., 2011). By applying prodigal from the assembled scaffolds genes were predicted, with default parameters, which resulted in the prediction of 4,062 genes (Hyatt et al., 2010). Functional annotation was accomplished by BLASTx program, which is a part of the NCBI-BLAST2.3.0 + standalone tool. Blast 2 GO platform was used to evaluate the gene ontology (GO) annotations (Conesa et al., 2005). GO assignments were applied to categorize the utilities of the predicted genes and a WEGO (Web Gene Ontology Annotation) plot was generated. Predicted genes were assigned to COG categories and KOs by
2.9. Theory and calculation The bacterial growth rate and DMA removal rate was evaluated in the present study by correlating the log of the bacterial culture (lnb) and time (t) by applying the Monod equation (Metcalf and Eddy, 2003)
μ = (Inb2 − Inb1)/(t2 − t1)
(1)
where, b1 and b2 are the bacterial concentrations (mg/L) at time t1 and t2 (h), respectively. The constraints of growth kinetics μ varies with (S) was observed 323
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hence, a double-reciprocal plot i.e. Lineweaver berk's plot was applied between the inverse of μ and the inverse of (S) which brings an association between μmax and KS as shown below (Metcalf and Eddy, 2003)
1/ μ = Ks / μmax (S ) + 1/ μmax
Table 2 Summary of KEGG pathway analysis for Ochrobactrum sp. Strain AV.
(2)
Here, μmax (per hour) is the maximum specific growth rate and Ks (mg/ L) is the half saturation constant for DMA. From 1st equation growth rate possibly be transformed and applied for determining the specific removal rate of DMA (VDMA), simultaneously resulted in the media is shown below:
V DMA = −(InDMA2 − InDMA1 )/(t2 − t1)C
(3)
where, DMA1 and DMA2 are the DMA concentrations at time t1 and t2, correspondingly, C is the initial microbial concentration in both situations. The specific removal rate (V) evaluated by equation (3) which contrasts with the initial DMA concentration. Therefore, values of maximum specific removal rate, Vmax (per hour) and half saturation constant, KS (mg/L) for DMA was estimated by applying Lineweaver berk's plot was drawn between the inverse of (V) and the inverse of (S), which brings an association between Vmax and KS as shown below: (4)
1/ V = K S /(Vmax . S ) + 1/ Vmax
3. Results and discussion 3.1. Microbial genomic analysis High quality of bacterial strain was enriched and developed from activated sludge sampled from Indian Oil Corporation Limited (IOCL), Assam. After regular maintenance of 120 days, DMA oxidizing bacterial strain was cultured and isolated. The isolate designated as IR-26 was gram-positive, rod-shaped cell bacteria. Further, the isolated colony was sequenced through 16s rRNA Sanger sequencing and found to be Agromyces and Ochrobactrum sp. The isolated strain has been submitted with accession number MF156974 and MG696218 in the NCBI database. The reported DMA degrading cultures comprised of genera Arthrobacter, Mycobacterium, Hyphomicrobium, Bacillus, Methylobacterium, Paracoccus, Pseudomonas, Microccocus, and Methylophilus (Rappert and Muller, 2005). Information on isolates of the alkyl amine degrading microbial community was quite limited. Till date, Agromyces and Ochrobactrum sp. has not been reported in the degradation of DMA and as per the author's best knowledge, the present study was first to report this new culture. The 16S rRNA study showed that the genome affiliated to Ochrobactrum species. The assembly of the draft genome sequence consisted of 1,152 scaffolds amounting to 4,958,020 bp with 78 bp and 666,518 bp as the minimum and maximum size of the scaffold, respectively (4,303 bp average size of the scaffolds). The scaffold N50 value was 383,647 bp. The genome features are summarized in Table 1. A total of 4,062 protein-coding genes were determined from the contigs with an average size of genes as 924 bases, and 90 and 8655 bases as a minimum and maximum size of genes, respectively. The distribution of genes into KEGG pathways is presented in Table 2. The
Pathway Metabolism
Number of genes
Amino acid metabolism Carbohydrate metabolism Metabolism of cofactors and vitamins Energy metabolism Nucleotide metabolism Metabolism of other amino acids Lipid metabolism Xenobiotics biodegradation and metabolism Biosynthesis of other secondary metabolites Metabolism of terpenoids and polyketides Glycan biosynthesis and metabolism Enzyme families Genetic information processing Transcription Translation Folding, sorting and degradation Replication and repair Environmental information processing Membrane transport Signal transduction Cellular processes Transport and catabolism Cell growth and death Cellular community - prokaryotes Cell motility Organismal systems Environmental adaptation
234 228 164 153 88 70 70 49 36 31 30 1 4 81 39 51 261 94 12 35 125 34 3
genome contained genes involved in basic metabolic pathways, such as pyruvate metabolism, glycolysis and TCA cycle, and was concluded to carry out aerobic respiration. Majority of the genes were observed to be associated with amino acid and carbohydrate metabolism. The GO assignments were used to classify the functions of the predicted genes and a WEGO (Web Gene Ontology Annotation) plot was generated, which suggested the pathways to majorly belong to the cellular component, molecular functions and biological processes (Fig. 1). The phylogenetic analysis for Ochrbactrum sp. Strain AV at the chromosomal level suggested the strain to be closest to Ochrobactrum sp. Strain UBA 7634 (Fig. 2). This suggests that the strain AV could be a novel member of the genus Ochrobactrum. Circular representation of Ochrobactrum strain_AV genome by CGView server (Fig. 3) demonstrates high proportion regions of the genome being similar to Ochrobactrum sp. UBA7634, Ochrobactrum intermedium, and Ochrabacterum A44. The figure also enunciates that there are large regions which are unique to only Ochrobactrum strain AV and not found in other Ochrobactrum sp. UBA7634, Ochrobactrum intermedium and Ochrobactrum Ochrobactrum intermedium strain_AV
Table 1 Genome features of Ochrobactrum intermedium strain_AV. Attribute
Value
NCBI accession number Genome size Total no. of scaffolds Shortest scaffold Longest scaffold N50 average length Total no. of protein-coding genes
SADZ00000000 4,958,020 bp 1,152 78 bp 666,518 bp 383,647 bp 4,303 bp 4,062
Fig. 1. Graphical representation of GO terms via WEGO plot Ochrobactrum intermedium strain_AV. 324
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Fig. 2. Phylogeny of the isolated bacteria among Ochrobactrum species.
shown to utilize nitrogen from methylamine and not the carbon for its energy and growth. More such microorganism needs to be explored for finding a high metabolic oxidation rate of DMA and other alkyl amines. In the present study, Agromyces sp. and Ochrabacterum sp. has been reported for the first time for the biodegradation of DMA as per the author's best knowledge.
A44. Phylogenetic analysis of Ochrobactrum strain AV with other Ochrobactrum species shows similar trends as obtained by the CG View result. Different microorganisms of genera Pseudomonas, Arthobacter and Paracoccus have been reported degrading TMA and DMA both in water and air (Chang et al., 2004). Recently Dziewit et al. (2015) have reported Paracoccus aminophilus JCM 7686 which was proficient to consume different kinds of carbon composites as the only energy source. The gene responsible for oxidizing DMA and other alkyl amines were identified. Chen et al. (2010) have also documented the degradation of methylamine by nonmethylotrophs (eg. Agrobacterium tumefaciens) has
3.2. Kinetics study of DMA Biological degradation of DMA by different microbial culture has been documented (Sharp et al., 2010; Weidhaas et al., 2012; Liao et al.,
Fig. 3. Graphical comparison Ochrobactrum Intermedium strain_AV (SADZ00000000) genome with Ochrobactrum intermedium LMG 3301, Ochrobactrum UB7634 and Ochrabactrum A44 retrireved from NCBI database using CGView server using default parameters. 325
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different previous reports. The degradation kinetics of DMA was analyzed, seeded with biomass concentrations 19 mg/L. The results of the microbial growth are shown in Fig. 4a. Agromyces sp. and Ochrobactrum sp. initiate its growth in 1.5 h of the study. DMA as a carbon substrate dependent growth periods includes lag phase, log, stationary were observed during the incubation period of 45 h. The microbial growth was determined very slow though high degradation capacity was observed. Hence, it can be inferred that the bacterial culture possesses a high metabolism which could consume DMA as a sole energy source. Various literature has reported the degradation kinetics by different microbial culture on alkyl amine especially TMA but very less literature on biodegradation kinetics of DMA was found. The growth rate of Methylophilus methylotrophus and Methylobacterium sp. was determined for TMA degradation (Roseiro et al., 1999). The growth kinetics parameters were shown by Agromyces sp. and Ochrobactrum sp. was calculated by applying Eqs (1) and (2) and represented in Fig. 4b and c. The μmax of the reaction was calculated using Eq. (3) was 0.013 per hour and the saturation constant (Ks) of the system has been calculated 0.63 mg/L. The kinetics studies have shown and demonstrated a very slow growth of microbial culture and growth rate slow down with a rise in the concentration which was a standard observation in the kinetic modeling. The inhibition effect on the growth was negligible which shows the ability of the microbial culture to degrade the higher DMA concentration. No inhibition effect on growth was noticed during the incubation period which established the capability of these bacteria in degrading TMA at much higher concentration. Biodegradation study of DMA was evaluated by analyzing the degradation of different concentration of DMA (Fig. 5a). Rapid biodegradation of DMA was observed when enriched with Agromyces and Ochrobactrum sp. A DMA concentration of 10 mg/L has been completely oxidized in 20 h of study. Liao et al. (2015) has reported DMA degradation from drinking water biofilter and has shown the degradation of 10 mg/L in 7 days supplementing with an additional carbon source. The present study has shown a high removal rate of DMA and high metabolic rate of bacteria with DMA as an only nitrogen and carbon source to the isolated degraders. The DMA biodegradation mechanism
Fig. 4. (a) Bacterial growth curve (b) specific growth (c) maximum growth rate is evaluated by lineweaver-berk plot.
2015). Moreover, DMA is among the transitional product in the TMA breakdown (Ho et al., 2008). Biological degradation of different alkyl amines by isolated inoculum has been well described in different studies (Rappert and Muller, 2005). Recently Oyarzun et al. (2019) has shown the degradation of alkyl amine from the fishmeal industry and DMA with a removal efficiency of 83% was reported. Different reports specified that biotreatment reactors inoculated with various microbial cultures could efficiently treat alkyl amine in gaseous form (Chang et al., 2004; Ho et al., 2008; Yin and Xu, 2012). However, there has been very less study on biodegradation kinetics of DMA or other aliphatic amines. Survey of the literature reveals as first report on the evaluation of the degradation rate of DMA by the A. indicus and O. intermedium. In the present study, the rapid degradation of DMA was established by the enriched culture. This result proposed that the degradation kinetics of DMA has been highly potent when enriched with Agromyces and Ochrobactrum sp. treatment when compared with
Fig. 5. DMA degradation is evaluated with the activity of microbial culture. (A) DMA degradation and carbon consumption (B) specific degradation of DMA and halflife of DMA (C) maximum degradation rate is evaluated by lineweaver-berk plot. 326
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mainly depends upon the intake of carbon present in the DMA by the biomass which degraded DMA into MMA which finally mineralizes to ammonia. The bacterial culture contains Agromyces and Ochrobactrum sp. has shown a normal sigmoidal growth phase in the study. Growth kinetics was calculated by applying equations (1) and (2) to the observation obtained from the proliferation of bacteria in the presence of DMA (Fig. 5). The maximum growth rate has been calculated by applying equation (3) which has been observed around 0.013 per hour. The growth rate was very less and it confirms that the bacterial species Agromyces and Ochrobactrum sp. grows very slowly in presence of DMA which was the only energy source. The saturation constant Ks of the bacterial growth was 0.6 mg/L which was quite high and show the low affinity of the bacteria. Similarly, the degradation rate has been evaluated of different concentration of DMA and 100% removal efficiency was observed in all setup. Specific degradation rate (VDMA) for different concentration of DMA has shown a declining pattern with the increase in the concentration and half-life (T-half) of DMA has also shown an integrated pattern with the escalation of DMA (Fig. 5b). From the study, the maximum degradation rate has been evaluated with the help of equation (4), the Lineweaver berk's plot has shown maximum degradation rate in higher concentration. The Vmax of degradation study was observed around 0.11per hour and a saturation constant of DMA (Km) degradation was 1.96 mg/L. The Vmax in the study was quite low but Km has shown a good affinity of DMA (Fig. 5c). The degradation rate has been checked, compare and evaluated using different models presented in Table 3. Biodegradation of DMA has been described in various literature (Webster et al., 2013; Liao et al., 2015). Rappert and Muller (2005) have shown different bacterial species capable to degrade DMA. Recently, Santawee et al. (2019) has also shown the rapid degradation of TMA when inoculated with Bacillus Thuringiensis. Also, Lobo et al. (1999) have reported the biological degradation kinetics of TMA with the help of Aminobacter aminovorans in which the μmax reported was 0.08 g/L/h which were quite higher when we compare it to the present study and the Vmax reported was 1.79 mM/h. There were no reports which have elaborated the DMA biodegradation kinetics so far as per the author's best knowledge. Subsequently, Ho et al. (2008) have calculated Vmax of DMA along with other alkyl amines with the application of Paracoccus sp. CP2 resulted in Vm = 0.9g-N Kg-1 and Km = 64 ppm which has shown a slow rate of degradation when compared to the degradation with Agromyces and Ochrobactrum sp. The saturation constant (Km) in Ho et al. study was very high which depicts a very low affinity of Paracoccus with the substrate. DMA was a limited energy source for the biomass to nurture. DMA offers a carbon and nitrogen source to the bacteria. The nitrogen was a vital component to the bacteria to proliferate (helps in replication). Hence, the nitrogen consumption by the bacteria from the DMA to the ammonia has been estimated in varied concentration (Table 4). The nitrogen content present in the initial study phase of TMA and final degradation product ammonia has been calculated and the difference among them has been assumed to be taken up by the bacteria. It has been observed that Agromyces sp. and Ochrobactrum sp. was consuming nitrogen source for its growth and metabolic activity. Thus, the isolated biomass was capable of DMA degradation. As per best of the author's knowledge, this is the first study to evaluate the biodegradation kinetic parameters of
Table 4 Nitrogen utilization by the isolated degraders from DMA degradation to ammonia formation. S.No
DMA concentration (mg/L)
Initial N content in DMA (mg/L)
Final N content in ammonia (mg/L) (PPM)
N utilized by the bacteria (mg/L)(P
1. 2. 3. 4. 5.
10 20 30 40 50
3.10 6.21 9.31 12.4 15.5
1 2 2.93 3.68 4.61
2.1 4.21 6.38 8.72 10.9
DMA by Agromyces and Ochrobactrum sp. 3.3. TOC reduction, ammonia formation, and change in pH The reduction of TOC of different concentration inoculated with Agromyces and Ochrobactrum sp. has been presented in Fig. 5a. The reduction in carbon concentration from the initial period towards the completion of the study has shown the depletion of DMA by the bacterial inoculum. The higher concentration of DMA has high TOC value and provided more carbon to the bacterium which has given maximum bacterial growth in the study. Liao et al. (2015) have reported the TOC reduction of DMA by a microbial consortium. Although the degradation activity of the isolated degraders was far way slower when compared to the present study. DMA metabolizes into ammonia and formaldehyde under aerobic condition and follows the same degradation pathway of trimethylamine (TMA) (Kim et al., 2001). TMA could be primarily dissolved to DMA and to methylamine (MA). MA finally dissolved to ammonia under a specific environment (Ho et al., 2008). In the present study, arise in ammonia was noticed in the presence of A. indicus and O. intermedium during the incubation, endorsing that DMA was finally mineralized to ammonia. The increase in ammonia has similarly been described in the earlier studies in the degradation of alkyl amine (Ding et al., 2007; Liao et al., 2015).Ammonia formation was observed with the biodegradation of different concentration of DMA. Ammonia as the end product of DMA removal was calculated (Fig. 6) and the higher concentration of 50 mg/L of DMA has resulted in the high concentration of ammonia i.e. 5.5 mg/L. Ho et al. (2008) subsequently degraded ammonia formed by the degradation of alkylamine by employing Arthobacter sp. (ammonia oxidizer) alongside Paracoccus sp. (amine oxidizer) results finally to nitrite and nitrate as a residual in a bioreactor system. However, ammonia was in ion form in the residual left after the amine degradation and hence it was not toxic but eradicating it alongside with alkyl amine is a needful approach. The pH of the biodegradation study was presented in Fig. 7. The value of pH has shown a reduction with the reduction of DMA or TOC in the sample. DMA was having a higher pKa value and hence, a high concentration of DMA will have high pH. Therefore, with the degradation of DMA when inoculated with Agromyces and Ochrobactrum has resulted in the reduction of pH. The value of all the different concentration of DMA has come close to neutral pH which helps in ammonia nitrogen present in the residual to be stable which was present in the ionic form. 4. Conclusion
Table 3 Comparative model study for maximum degradation rate (Vmax) and saturation constant (Km). Vmax (per h)
The present study comprises of oxidation for DMA with investigating the kinetic parameter and specifies the ideal condition for establishing the biological process. Genome information could be suitable for determining the potential of Ochrobactrum sp. and validated its application for the degradation of DMA and other alkyl amines. Slow bacterial growth with a high metabolic activity was observed. Monod model was successfully applied in evaluating the kinetic parameters in the present study. The observation of TOC and ammonia has further validated the degradation capacity of the isolated degraders on DMA.
Km (mg/L)
Model 3.24 Hanes Plot Lineweaver-Burk Plot Eadie-Hofstee Plot Results
0 .1083 0.1144 0.1148
1.96 1.92
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Fig. 7. Effect on pH on the degradation of DMA with different concentration.
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