ENVIRONMENTAL BIOTECHNOLOGY
Characterization of a novel melamine-degrading bacterium isolated from a melamine-manufacturing factory in China
Han Wang &Chunnu Geng &Jiangwei Li &Anyi Hu &Chang-Ping Yu
Received:8September 2013/Revised:24October 2013/Accepted:28October 2013/Published online:3December 2013#Springer-Verlag Berlin Heidelberg 2013
Abstract Melamine (2,4,6-triamino-1,3,5-triazine,C 3H 6N 6),belonging to the s -triazine family,is an anthropogenic and versatile raw material for a large number of consumer prod-ucts and its extensive use has resulted in the contamination of melamine in the environment.A novel melamine-degrading bacterium strain CY1was isolated from a melamine-manufacturing factory in China.The strain is phylogenetically different from the known melamine-degrading bacteria.Approximately,94%melamine (initial melamine concentra-tion 4.0mM,initial cell OD 0.05)was degraded in 10days without the addition of additional carbon source.High-performance liquid chromatography showed the production of degradation intermediates including ammeline,ammelide,cyanuric acid,biuret,and urea.Kinetic simulation analysis indicated that transformation of urea into ammonia was the rate-limiting step for t
he degradation process.The melamine –cyanurate complex was formed due to self-assembly of mel-amine and cyanuric acid during the degradation.The tracking experiment using CY1cells and 13C 3-melamine showed that the CY1could mineralize s -triazine ring carbon to CO 2.The strain CY1could also catalyze partial transformation of cyromazine,a cyclopropyl derivative of melamine,to 6-(cyclopropylamino)-[1,3,5]triazine-2,4-diol.Keywords Melamine .Cyanuric acid .s -triazine .Cyromazine .Biodegradation
Introduction
Melamine (2,4,6-triamino-1,3,5-triazine,C 3H 6N 6;CAS:108-78-1),belonging to the s -triazine family,is an anthropogenic and versatile raw material for flame retardant,swimming pool bactericide,adhesive,paint,electrical molding,tableware,glass-reinforced substrates,and engineered wood products (Kandelbauer and Widsten 2009;Lund and Petersen 2006;Lv et al.2005).Nowadays,major melamine-manufacturers are situated in China.Melamine contamination has been de-tected in farmland soil samples,irrigation water samples,crop samples in China,and higher levels of melamine contamina-tion were observed in soil samples closer to melamine-manufacturing factories (Qin et al.2010).Qin et al.(2010)also indicated that highest melamine concentrations were detected in the wastewater discharge of melamine-manufacturing factories,and
demonstrated that melamine-manufacturing factories are important sources of melamine contamination in the environment due to inappropriate treat-ment of their waste and wastewater.Although melamine has been suggested to be of low toxicity to mammals (Baynes et al.2008),the incident of melamine-tainted pet food and infant formula made it an emerging contaminant (Hao and Stone 2008).Researches have shown that the combination of melamine and cyanuric acid could induce nephrolithiasis (Chen et al.2009).Melamine also caused rat neurotoxicity (Yang et al.2010).Moreover,melamine is recalcitrant to biodegradation due to its rigid and planar structure and inhib-itory to beneficial wastewater treatment microorganisms (Xu et al.2013).
Several bacterial strains,including Pseudomonas sp.strain A (NRRL B-12227),Rhodococcus corallinus (NRRL B-15444R),Klebsiella terragena DRS-1(ATCC 700372),Rhodococcus sp.strain Mel,and Nocardioides sp.strain ATD6were reported to degrade melamine (Cook and Hutter 1981;Cook and Hutter 1984;Dodge et al.2012;Shelton et al.
Electronic supplementary material The online version of this article (doi:10.1007/s00253-013-5363-2)contains supplementary material,which is available to authorized users.
H.Wang :C.Geng :J.Li :A.Hu :C.<P.Yu (*)
Key Laboratory of Urban Environment and Health,Institute of Urban Environment,Chinese Academy of Sciences,Xiamen 361021,China e-mail:cpyu@iue.ac
Appl Microbiol Biotechnol (2014)98:3287–3293DOI 10.1007/s00253-013-5363-2
1997;Takagi et al.2012).In this study,a novel bacterium CY1 was isolated from the sludge of a melamine-manufacturing factory,and it could completely degrade melamine.Melamine and its metabolites were monitored and their first-order kinet-ics was fitted to the dynamic data during the degradation process.We used13C-labeled melamine to confirm that the isolate could degrade melamine to CO2.In addition,Raman spectroscopy proved the formation of melamine–cyanurate complex(MC)during the melamine biodegradation process. To the best of our knowledge,this is the first attempt to observe the formation of melamine–cyanurate complex by a melamine-degrading bacterium.This information will be use-ful to fill the knowledge gaps in microbial degradation of melamine.
Materials and methods
Chemicals
Melamine and cyanuric acid were obtained from Alfa Aesar. Ammeline,ammelide,and cyromazine were purchased from Tokyo Chemical Industry.Biuret came from ChemService. Potassium allophanate was made from ethyl allophanate (Tokyo Chemical Industry)by hydrolysis according to the previous study(Whitney and Coopers1972).Urea and13C3-melamine were purchased from Sigma-Aldrich.Acetonitrile was purchased from Tedia Company.Perchloric acid and ammonia were obtained from Sinopharm Chemical Reagent Company.All chemicals were reagent grade,except that chromatographic grade chemicals were used in high-performance liquid chromatography(HPLC),Raman spec-trometry,ultraperformance liquid chromatography coupled to quadrupole-time of flight mass spectrometry(UPLC-Q-TOF),and stable isotope ratio mass spectrometry(SIRMS). Enrichment and isolation
The sewage sludge collected from a melamine-manufacturing factory was used as inoculum.The factory,located in the Sanming city of Fujian province in China,has manufactured melamine for more than a decade.The enrichment medium contained mineral salt medium(MSM),which was modified from the nitrate mineral salt medium(Yu et al.2007)by removing the nitrogen source and instead0.8mM melamine was used as the sole carbon and nitrogen source.
One hundred milliliters enrichment culture was placed in a 250-mL sterile flask and incubated at30°C
with shaking at 150rpm.Approximately every week,50mL cell suspension was discarded from the flask and then replaced with the equal amount of new enrichment medium.After1month of transfer, the enrichment culture was spread on R2A(Qingdao Hope Bio-Technology Co.,Ltd)agar plates for the isolation of melamine-degrading cultures.After numerous streaking,mor-phologically distinct colonies were selected and tested for their degradation capability toward melamine.Isolates show-ing degradation abilities were first identified by using16S rRNA gene sequences.The isolated melamine-degrading strain CY1is now deposited in the Deutsche Sammlung für Mikroorganismen und Zellkulturen under the number DSM 26006T.The16S rRNA gene sequence is deposited at GenBank with accession number JQ676982. Biodegradation assay
For the experiment of melamine degradation,1L MSM was put into a2-L flask.Melamine(4mM,∼500mg/L)was then added and used as the sole carbon and nitrogen source.The isolated strain CY1was pregrown in R2A broth,harvested by centrifugation,and washed and resuspended with MSM,be-fore inoculating into the medium containing melamine (OD600=0.05).Experiments were conducted in triplicates and autoclave-killed controls were also included.All experi-ments were incubated at30°C and150rpm.Time–course samples were collected and flash frozen in liquid nitrogen immediately and then stored at−40°C before analysis.
The strain CY1was also tested for its degradation ability toward cyromazine(2-cyclopropylamino-4,6-diamino-s-tri-azine)which is a cyclopropyl derivative of melamine.The experimental procedures were similar to melamine degrada-tion except that the strain CY1was inoculated into the MSM containing0.75mM(125mg/L)cyromazine(OD600=0.5). HPLC analysis of melamine and degradation intermediates
Melamine,ammeline,ammelide,cyanuric acid,biuret, allophanate,and urea were determined by HPLC(Dionex Ultimate3000,USA)with UV detection at200nm.Hitachi lachrom C18column(4.6×150mm,5μm)was used as the analysis column.The mobile phase consisted of ion-pair re-agent and acetonitrile.Ion-pair reagent was prepared by adding12-mL12.7M perchloric acid into1L ultra-pure water and then adjusting the solution to pH2.5using ammonia solution.The change of acetonitrile concentration in the gra-dient elution was as follows:0.5–4%in0–5min,4–8%in5–9min,8–0.5%in9–17min,and0.5%in17–22min.The flow rate was0.7mL/min from0to5min,increased to1mL/ min from5to9min,further increased to1.5mL/min from9to 17min,then decreased to0.7mL/min from17to20min,and held0.7mL/min from20to22min.
Determination of ammonia,total organic carbon
Ammonia concentration was determined by flow injection analysis(LACHAT QC8500,USA)and TOC was determined
by TOC analyzer(SHIMADZU TOC-V CPH,Japan).The oven temperature of TOC analyzer was set at720°C. Fitting kinetic modeling
The concentration profiles of melamine and metabolites were modeled by first-order kinetics(see details in the supplemental material).In order to facilitate simulation,melamine and its metabolites were normalized into the N percentage in each bottle.The optimization was performed using the fmincon function of the MA TLAB.Degradation rate constants of mel-amine(K1),ammeline(K2),ammelide(K3),cyanuric acid (K4),biuret(K5),and urea(K6)were obtained after simulation.
Identifying unknown precipitate by scanning electron microscopy and Raman spectrometry
An unknown precipitate was observed during melamine deg-radation and was further collected through centrifugation for analysis.The collected precipitate was washed repeatedly with sterile deionized water.The MC was synthesized by mixing100mL4mM melamine and cyanuric acid together and the MC was precipitated.After treated by critical point drying,unknown precipitate and M
C were observed by field emission scanning electron microscopy(SEM;Hitachi S-4800,Japan)to capture their microstructure.Unknown pre-cipitate,MC,melamine,ammeline,ammelide,and cyanuric acid was also analyzed by confocal microscope Raman spec-trometer(Horiba Jobin Yvon S.A.S.LabRAM Aramis, France)at633nm diode laser source.The spectral signal ranged from0to2,000cm−1.The baseline was calibrated by silicon wafer at520.5cm−1Raman band.
Tracking melamine degradation using13C-melamine
13C
3
-melamine was used to confirm melamine degradation by the isolated bacterium CY1.Twenty-five milliliters sterile MSM containing0.8mM13C3-melamine was filled into a 120-mL serum bottle.Artificial air(nitrogen/oxygen,79/21, v/v)was used to purge the MSM to remove carbon dioxide. The vial was then inoculated with CY1and sealed using butyl rubber immediately.Unlabeled melamine of the same concen-tration was also used in another set of bottles for comparison. All treatments were incubated at30°C and150rpm.13C/12C ratio of the headspace CO2was determined by SIRMS at0,5, 11,24,44,and69h.Thermo trace gas chromatography(USA) was implemented for SIRMS with the chro
matographic col-umn Rt®-Q-BOND(Restek,30m×0.32mm ID)for carbon dioxide.The flow rates of carrier gas(He)were1.8mL/min and the temperature program was40°C for6min.13C value was determined by Thermo Delta V Advantage isotope ratio mass spectrometer(USA).Identifying the cyromazine metabolite by UPLC-Q-TOF After the degradation assay of cyromazine by CY1,for extracting metabolites,4mL ethyl acetate and2mL degrada-tion sample were placed in a20-mL vial.The vial was oscil-lated in a platform shaker at200rpm for12h.Two milliliters ethyl acetate was transferred to a new vial and was dried and redissolved in acetonitrile/water(50/50,v/v)and analyzed by UPLC-Q-TOF(Agilent,Ultimate3000/Acquity/micro TOF-Q II,USA).Waters ACQUITY UPLC BEH C18column (2.1×50mm,1.7μm)was used for UPLC analysis.A gradi-ent elution was performed using acetonitrile as the mobile phase A and water as the mobile phase B:0to0.5min(2% A,98%B),0.5to13min(linear gradient to40%A),16to 20min(linear gradient to2%A).The flow rate was0.2mL/ min.The mass spectrometry used electrospray ionization in positive mode.The range of m/z was70–1,000.Capillary and end plate offset voltages were4,500and−500V,respectively. Quadrupole ion energy and collision cell collision energy were2.0and8.0eV,respectively.
Results
The phylogenetic relationship of the melamine-degrading bacterium CY1
A nearly full-length16S rRNA gene sequence(1,436bp)of strain CY1was determined.Phylogenetic analysis indicated that strain CY1was considered to be a novel species withinβ-proteobacteria and its16S rRNA gene sequence had95.5% similarity to the most closely related species Alicycliphilus denitrificans K601T.In addition,it is phylogenetically differ-ent from the previously known melamine-degrading bacteria (Fig.1).The16S rRNA gene sequence of the strain shared 93.3%similarity to the Acidovorax citrulli(previously known as Pseudomonas sp.strain A NRRL B-12227), 77.7%similarity with Raoultella terragena DRS-1(previ-ously known as Klebsiella terragena DRS-1ATCC700372), 73.3%similarity with Norcadioides sp.ATD6,63.7%sim-ilarity with Micrococcus sp.MF-1,73.1%similarity with Rhodococcus corallines NRRL B-15444R and73.6%simi-larity with Rhodococcus sp.strain Mel.
Melamine biodegradation
Previous studies reported that melamine could be biodegraded by adding other carbon ,lactate,glycerol,and sodium acetate(Cook and Hutter1981;Dodge et al.2012; Shelton et al.1997;Takagi et al.2012).In this study,we found that CY1cells could mineralize melamine without additional carbon source.In the initial24h,approximately64%of melamine was degraded.Meanwhile,ammeline increased to
1.2mM in10h(Fig.2a).The result indicated that CY1 immediately deaminized melamine in the initial stage. Ammeline,ammelide,and cyanuric acid concentration in-creased and then decreased gradually,but biuret and urea accumulated continuously.
The ammonia concentration increased continuously to 12.7mM(Fig.2b).Nitrogen content of each compound was calculated based on HPLC and flow injection analysis of target compounds at each sampling time.Total nitrogen amount did not change significantly and was around24mM during incubation time,suggesting negligible amount of ni-trogen was assimilated by CY1.Simultaneously,the pH in-creased from7.3to8.9during degradation(Fig.2b).The significant correlation between the pH and ammonia content was observed during degradation(r2=0.935,p<0.01),sug-gesting that ammonia release resulted in the pH increase during melamine biodegradation.Seventy-two percent TOC removal was observed at the end of incubation although almost94%melamine was degraded(Fig.2c). Verification of s-triazine ring mineralization
In the tracking experiment using13C3-melamine,the percent-age of13C/12C of the headspace carbon dioxide rapidly in-creased to about80%in24h.Yet,in unlabeled melamine the percentage of13C/12C of the headspace carbon dioxide remained around1%(Fig.3).Above results directly reflected the mineralization of s-triazine ring.
Modeling of melamine degradation
The dynamic change of melamine and its metabolites in the aqueous phase was simulated by the first-order kinetics,with K1=0.032,K2=0.024,K3=0.046,K4=0.021,K5=0.02,and K6=0.003h−1(see Fig.S1in the supplemental material).The gradual accumulation of urea was observed to reach28%of total N from initial melamine.It was supported by the K6of 0.003h−1,indicating that urea degradation into ammonia was the rate-limiting step of the degradation process.The mass balance indicated that only18%of ammonia was transformed from urea,while82%was from the deamination of melamine.
Unknown precipitate identification
An unknown white precipitate appeared within24h during melamine degradation.The precipitate disappeared gradually with time afterwards.SEM photographs showed the crystal-line structure of the unknown precipitate(Fig.4).We specu-lated that MC,ammeline,or ammelide might be candidates of the precipitate due to their lower solubility in neutral solution.
Acidovorax citrulli NRRL B-12227(AF078761)
Acidovorax cattleyae NCPPB 961T(AF078762)
degrade
Acidovorax oryzae FC-143T(DQ360414)
Acidovorax avenae subsp. avenae ATCC 19860T(AF078759)
Acidovorax konjaci A TCC 33996T(AF078760)
Acidovorax valerianellae CFBP 4730T(AJ431731)
Acidovorax temperans CCUG 11779T(AF078766)
Acidovorax soli BL21T(FJ599672)
Acidovorax defluvii BSB411T(Y18616)
Acidovorax delafieldii ATCC 17505T(AF078764)
Acidovorax facilis CCUG 2113T(AF078765)
Acidovorax caeni R-24608T(AM084006)
Pseudacidovorax intermedius CC21T(EF469609)
Simplicispira metamorpha DSM 1837T(Y18618)
Simplicispira limi EMB325T(DQ372987)
Strain CY1T(JQ676982)
Alicycliphilus denitrificans K601T(NR_025510)
Diaphorobacter nitroreducens NA10B T(AB064317)
Diaphorobacter oryzae RF3T(EU342381)
Variovorax dokdonensis DS-43T(DQ178978)
Ottowia pentelensis RB3-7T(EU518930)
Comamonas granuli Ko03T(AB187586)
Delftia lacustris DSM 21246T(EU888308)
Delftia tsuruhatensis T7T(AB075017)
Hydrogenophaga temperata TR7-01T(AB166886)
Comamonas composti CC-YY287T(EF015884)
Comamonas odontotermitis Dant 3-8T(DQ453128)
Comamonas koreensis KCTC 12005T(AF275377)
Comamonas zonglianii BF-3T(GQ245981)
Comamonas nitrativorans23310T(AJ251577)
Comamonas denitrificans123T(AF233877)
Raoultella terragena DRS-1(Y17658)
Norcadioides sp. ATD6(AB638612)
Micrococcus sp. MF-1(AB213661)
Rhodococcus corallines NRRLB-15444R(JN201861)
Rhodococcus sp.strain Mel(JN201860)
100
100
100
99
62
53
94
86
80
64
60
54
51
52
51
100
100
97 0.02Actinobacteria
Fig.1The phylogenetic tree of known melamine-degrading bacteria.Bootstrap values above 50%(expressed as percentages of 1000replications)are shown at branch points
For identification,melamine,ammeline,ammelide,cyanuric acid,MC,and unknown precipitate were analyzed by Raman spectroscopy.The spectrum of unknown precipitate matched well with MC,which
had noticeable Raman peaks of 75,99,154,409,528,594,678,694,and 1,739cm −1(Fig.5).A similar spectrum of MC was also obtained by the previous
study (He et al.2008).Therefore,the unknown precipitate was identified as MC.
Degradation of cyromazine by strain CY1
Our result indicated that cyromazine could be degraded by strain CY1(Fig.S2in the supplemental material).HPLC analysis showed that two unknown intermediates were formed during cyromazine degradation.Cyromazine and one un-known intermediate disappeared gradually but the other un-known compound accumulated and was not degraded.This result suggested that strain CY1could only transform cyromazine to an intermediate but could not completely min-eralize cyromazine.Considering the deamination ability of strain CY1,we hypothesized that strain CY1could catalyze consecutive hydrolysis of the two amino substituents of cyromazine,and 6-(cyclopropylamino)-[1,3,5]triazine-2,4-di-ol (molecular weight of 168)was formed as the final product.The sample after incubation was further analyzed by
UPLC-
Fig.4The SEM photograph of unknown precipitate
13
C /12
C %
Time (h)
Fig.3
13
C/12C proportion changes in vial headspace CO 2
C o n c e n t r a t i o n (m M )
Time (h)
Time (h)
N H 3-N (m M )
p H
C o n c e n t r a t i o n (m g /L )
Time (h)
a
b
c Fig.2The changes of melamine an
d intermediates (a ),NH 3–N,pH (b ),TOC (c )during melamin
e degradation
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