ENVIRONMENTAL BIOTECHNOLOGY

Characterization of a novel melamine-degrading bacteriumisolated from a melamine-manufacturing factory in China

Han Wang & Chunnu Geng & Jiangwei Li & Anyi Hu &

Chang-Ping Yu

Received: 8 September 2013 /Revised: 24 October 2013 /Accepted: 28 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Melamine (2,4,6-triamino-1,3,5-triazine, C3H6N6),belonging to the s -triazine family, is an anthropogenic andversatile raw material for a large number of consumer prod-ucts and its extensive use has resulted in the contamination ofmelamine in the environment. A novel melamine-degradingbacterium strain CY1 was isolated from a melamine-manufacturing factory in China. The strain is phylogeneticallydifferent from the known melamine-degrading bacteria.Approximately, 94 % melamine (initial melamine concentra-tion 4.0 mM, initial cell OD 0.05) was degraded in 10 dayswithout the addition of additional carbon source. High-performance liquid chromatography showed the productionof degradation intermediates including ammeline, ammelide,cyanuric acid, biuret, and urea. Kinetic simulation analysisindicated that transformation of urea into ammonia was therate-limiting step for the degradation process. The melamine–cyanurate complex was formed due to self-assembly of mel-amine and cyanuric acid during the degradation. The trackingexperiment using CY1 cells and 13C3-melamine showed thatthe CY1 could mineralize s -triazine ring carbon to CO2. Thestrain CY1 could also catalyze partial transformation ofcyromazine, a cyclopropyl derivative of melamine, to6-(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, C3H6N6; CAS: 108-78-1), belonging to the s -triazine family, is an anthropogenicand versatile raw material for flame retardant, swimming poolbactericide, 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-manufacturersare situated in China. Melamine contamination has been de-tected in farmland soil samples, irrigation water samples, cropsamples 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 weredetected in the wastewater discharge of melamine-manufacturing factories, and demonstrated that melamine-manufacturing factories are important sources of melaminecontamination in the environment due to inappropriate treat-ment of their waste and wastewater. Although melamine hasbeen suggested to be of low toxicity to mammals (Bayneset al. 2008), the incident of melamine-tainted pet food andinfant formula made it an emerging contaminant (Hao andStone 2008). Researches have shown that the combination ofmelamine and cyanuric acid could induce nephrolithiasis(Chen et al. 2009). Melamine also caused rat neurotoxicity(Yang et al. 2010). Moreover, melamine is recalcitrant tobiodegradation due to its rigid and planar structure and inhib-itory to beneficial wastewater treatment microorganisms (Xuet al. 2013).

Several bacterial strains, including Pseudomonas sp. strainA (NRRL B-12227), Rhodococcus corallinus (NRRL B-15444R), Klebsiella terragena DRS-1 (ATCC 700372),Rhodococcus sp. strain Mel, and Nocardioides sp. strainATD6 were reported to degrade melamine (Cook and Hutter1981; 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 andHealth, Institute of UrbanEnvironment, Chinese Academy of Sciences, Xiamen 361021, Chinae-mail: cpyu@iue.ac.cn

Appl Microbiol BiotechnolDOI 10.1007/s00253-013-5363-2

1997; Takagi et al. 2012). In this study, a novel bacteriumCY1was isolated from the sludge of a melamine-manufacturingfactory, and it could completely degrade melamine. Melamineand its metabolites were monitored and their first-order kinet-ics was fitted to the dynamic data during the degradationprocess. We used 13C-labeled melamine to confirm that theisolate could degrade melamine to CO2. In addition, Ramanspectroscopy proved the formation of melamine–cyanuratecomplex (MC) during the melamine biodegradation process.To the best of our knowledge, this is the first attempt toobserve the formation of melamine–cyanurate complex by amelamine-degrading bacterium. This information will be use-ful to fill the knowledge gaps in microbial degradation ofmelamine.

Materials and methods

Chemicals

Melamine and cyanuric acid were obtained from Alfa Aesar.Ammeline, ammelide, and cyromazine were purchased fromTokyo Chemical Industry. Biuret came from ChemService.Potassium allophanate was made from ethyl allophanate(Tokyo Chemical Industry) by hydrolysis according to theprevious study (Whitney and Coopers 1972). Urea and 13C3-melamine were purchased from Sigma-Aldrich. Acetonitrilewas purchased from Tedia Company. Perchloric acid andammonia were obtained from Sinopharm Chemical ReagentCompany. All chemicals were reagent grade, except thatchromatographic grade chemicals were used in high-performance liquid chromatography (HPLC), Raman spec-trometry, ultraperformance liquid chromatography coupledto 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-manufacturingfactory was used as inoculum. The factory, located in theSanming city of Fujian province in China, has manufacturedmelamine for more than a decade. The enrichment mediumcontained mineral salt medium (MSM), which was modifiedfrom the nitrate mineral salt medium (Yu et al. 2007) byremoving the nitrogen source and instead 0.8 mM melaminewas used as the sole carbon and nitrogen source.

One hundred milliliters enrichment culture was placed in a250-mL sterile flask and incubated at 30 °C with shaking at150 rpm. Approximately every week, 50 mL cell suspensionwas discarded from the flask and then replaced with the equalamount of new enrichment medium. After 1 month of transfer,the enrichment culture was spread on R2A (Qingdao HopeBio-Technology Co., Ltd) agar plates for the isolation of

melamine-degrading cultures. After numerous streaking, mor-phologically distinct colonies were selected and tested fortheir degradation capability toward melamine. Isolates show-ing degradation abilities were first identified by using 16SrRNA gene sequences. The isolated melamine-degradingstrain CY1 is now deposited in the Deutsche Sammlung fürMikroorganismen und Zellkulturen under the number DSM26006T. The 16S rRNA gene sequence is deposited atGenBank with accession number JQ676982.

Biodegradation assay

For the experiment of melamine degradation, 1 L MSM wasput into a 2-L flask. Melamine (4 mM, ∼500 mg/L) was thenadded and used as the sole carbon and nitrogen source. Theisolated strain CY1 was pregrown in R2A broth, harvested bycentrifugation, and washed and resuspended with MSM, be-fore inoculating into the medium containing melamine(OD600=0.05). Experiments were conducted in triplicatesand autoclave-killed controls were also included. All experi-ments were incubated at 30 °C and 150 rpm. Time–coursesamples were collected and flash frozen in liquid nitrogenimmediately and then stored at −40 °C before analysis.

The strain CY1 was also tested for its degradation abilitytoward cyromazine (2-cyclopropylamino-4,6-diamino-s -tri-azine) which is a cyclopropyl derivative of melamine. Theexperimental procedures were similar to melamine degrada-tion except that the strain CY1 was inoculated into the MSMcontaining 0.75 mM (125 mg/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 (DionexUltimate 3000, USA) with UV detection at 200 nm. Hitachilachrom C18 column (4.6×150 mm, 5 μm) was used as theanalysis column. The mobile phase consisted of ion-pair re-agent and acetonitrile. Ion-pair reagent was prepared byadding 12-mL 12.7M perchloric acid into 1 L ultra-pure waterand then adjusting the solution to pH 2.5 using ammoniasolution. The change of acetonitrile concentration in the gra-dient elution was as follows: 0.5–4 % in 0–5 min, 4–8 % in 5–9 min, 8–0.5 % in 9–17 min, and 0.5 % in 17–22 min. Theflow rate was 0.7 mL/min from 0 to 5 min, increased to 1 mL/min from 5 to 9min, further increased to 1.5 mL/min from 9 to17 min, then decreased to 0.7 mL/min from 17 to 20 min, andheld 0.7 mL/min from 20 to 22 min.

Determination of ammonia, total organic carbon

Ammonia concentration was determined by flow injectionanalysis (LACHAT QC8500, USA) and TOC was determined

Appl Microbiol Biotechnol

by TOC analyzer (SHIMADZU TOC-V CPH, Japan). Theoven temperature of TOC analyzer was set at 720 °C.

Fitting kinetic modeling

The concentration profiles of melamine and metabolites weremodeled by first-order kinetics (see details in the supplementalmaterial). In order to facilitate simulation, melamine and itsmetabolites were normalized into the N percentage in eachbottle. The optimization was performed using the fminconfunction of the MATLAB. Degradation rate constants of mel-amine (K1), ammeline (K2), ammelide (K3), cyanuric acid(K 4), biuret (K 5), and urea (K 6) were obtained aftersimulation.

Identifying unknown precipitate by scanning electronmicroscopy and Raman spectrometry

An unknown precipitate was observed during melamine deg-radation and was further collected through centrifugation foranalysis. The collected precipitate was washed repeatedlywith sterile deionized water. The MC was synthesized bymixing 100 mL 4 mM melamine and cyanuric acid togetherand the MC was precipitated. After treated by critical pointdrying, unknown precipitate and MC were observed by fieldemission scanning electron microscopy (SEM; Hitachi S-4800, Japan) to capture their microstructure. Unknown pre-cipitate, MC, melamine, ammeline, ammelide, and cyanuricacid was also analyzed by confocal microscope Raman spec-trometer (Horiba Jobin Yvon S.A.S. LabRAM Aramis,France) at 633 nm diode laser source. The spectral signalranged from 0 to 2,000 cm−1. The baseline was calibrated bysilicon wafer at 520.5 cm−1 Raman band.

Tracking melamine degradation using 13C-melamine

13C3-melamine was used to confirm melamine degradation bythe isolated bacterium CY1. Twenty-five milliliters sterileMSM containing 0.8 mM 13C3-melamine was filled into a120-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 CY1 and sealed using butylrubber immediately. Unlabeled melamine of the same concen-tration was also used in another set of bottles for comparison.All treatments were incubated at 30 °C and 150 rpm. 13C/12Cratio of the headspace CO2 was determined by SIRMS at 0, 5,11, 24, 44, and 69 h. Thermo trace gas chromatography (USA)was implemented for SIRMS with the chromatographic col-umn Rt®-Q-BOND (Restek, 30 m×0.32 mm ID) for carbondioxide. The flow rates of carrier gas (He) were 1.8 mL/minand the temperature program was 40 °C for 6 min. 13C valuewas determined by Thermo Delta VAdvantage isotope ratiomass spectrometer (USA).

Identifying the cyromazine metabolite by UPLC-Q-TOF

After the degradation assay of cyromazine by CY1, forextracting metabolites, 4 mL ethyl acetate and 2 mL degrada-tion sample were placed in a 20-mL vial. The vial was oscil-lated in a platform shaker at 200 rpm for 12 h. Two millilitersethyl acetate was transferred to a new vial and was dried andredissolved in acetonitrile/water (50/50, v /v ) and analyzed byUPLC-Q-TOF (Agilent, Ultimate 3000/Acquity/micro TOF-Q II, USA). Waters ACQUITY UPLC BEH C18 column(2.1×50 mm, 1.7 μm) was used for UPLC analysis. A gradi-ent elution was performed using acetonitrile as the mobilephase A and water as the mobile phase B: 0 to 0.5 min (2 %A, 98 % B), 0.5 to 13 min (linear gradient to 40 % A), 16 to20 min (linear gradient to 2 % A). The flow rate was 0.2 mL/min. The mass spectrometry used electrospray ionization inpositive mode. The range ofm /z was 70–1,000. Capillary andend plate offset voltages were 4,500 and −500 V, respectively.Quadrupole ion energy and collision cell collision energywere 2.0 and 8.0 eV, respectively.

Results

The phylogenetic relationship of the melamine-degradingbacterium CY1

A nearly full-length 16S rRNA gene sequence (1,436 bp) ofstrain CY1 was determined. Phylogenetic analysis indicatedthat strain CY1 was considered to be a novel species withinβ-proteobacteria and its 16S rRNA gene sequence had 95.5 %similarity to the most closely related species Alicycliphilusdenitrificans K601T. In addition, it is phylogenetically differ-ent from the previously known melamine-degrading bacteria(Fig. 1). The 16S rRNA gene sequence of the strain shared93.3 % similarity to the Acidovorax citrulli (previouslyknown as Pseudomonas sp. strain A NRRL B-12227),77.7 % similarity with Raoultella terragena DRS-1 (previ-ously known as Klebsiella terragena DRS-1 ATCC 700372),73.3 % similarity with Norcadioides sp. ATD6, 63.7 % sim-ilarity with Micrococcus sp. MF-1, 73.1 % similarity withRhodococcus corallines NRRL B-15444R and 73.6 % simi-larity with Rhodococcus sp. strain Mel.

Melamine biodegradation

Previous studies reported that melamine could be biodegradedby adding other carbon sources, e.g., lactate, glycerol, andsodium acetate (Cook and Hutter 1981; Dodge et al. 2012;Shelton et al. 1997; Takagi et al. 2012). In this study, we foundthat CY1 cells could mineralize melamine without additionalcarbon source. In the initial 24 h, approximately 64 % ofmelamine was degraded. Meanwhile, ammeline increased to

Appl Microbiol Biotechnol

1.2 mM in 10 h (Fig. 2a). The result indicated that CY1immediately deaminized melamine in the initial stage.Ammeline, ammelide, and cyanuric acid concentration in-creased and then decreased gradually, but biuret and ureaaccumulated continuously.

The ammonia concentration increased continuously to12.7 mM (Fig. 2b). Nitrogen content of each compound wascalculated based on HPLC and flow injection analysis oftarget compounds at each sampling time. Total nitrogenamount did not change significantly and was around 24 mMduring incubation time, suggesting negligible amount of ni-trogen was assimilated by CY1. Simultaneously, the pH in-creased from 7.3 to 8.9 during degradation (Fig. 2b). Thesignificant correlation between the pH and ammonia contentwas observed during degradation (r2=0.935, p <0.01), sug-gesting that ammonia release resulted in the pH increaseduring melamine biodegradation. Seventy-two percent TOCremoval was observed at the end of incubation althoughalmost 94 % melamine was degraded (Fig. 2c).

Verification of s-triazine ring mineralization

In the tracking experiment using 13C3-melamine, the percent-age of 13C/12C of the headspace carbon dioxide rapidly in-creased to about 80 % in 24 h. Yet, in unlabeled melamine the

percentage of 13C/12C of the headspace carbon dioxideremained around 1 % (Fig. 3). Above results directly reflectedthe mineralization of s -triazine ring.

Modeling of melamine degradation

The dynamic change of melamine and its metabolites in theaqueous phase was simulated by the first-order kinetics, withK1=0.032, K2=0.024, K3=0.046, K4=0.021, K5=0.02, andK6=0.003 h

−1 (see Fig. S1 in the supplemental material). Thegradual accumulation of urea was observed to reach 28 % oftotal N from initial melamine. It was supported by the K6 of0.003 h−1, indicating that urea degradation into ammonia wasthe rate-limiting step of the degradation process. The massbalance indicated that only 18% of ammonia was transformedfrom urea, while 82%was from the deamination ofmelamine.

Unknown precipitate identification

An unknown white precipitate appeared within 24 h duringmelamine degradation. The precipitate disappeared graduallywith 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 ofthe precipitate due to their lower solubility in neutral solution.

Acidovorax citrulli NRRL B-12227 (AF078761)Acidovorax cattleyae NCPPB 961T (AF078762)

Acidovorax oryzae FC-143T (DQ360414)Acidovorax avenae subsp. avenae ATCC 19860T (AF078759)

Acidovorax konjaci ATCC 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 NA10BT (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 nitrativorans 23310T (AJ251577)Comamonas denitrificans 123T (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)

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Fig. 1 The phylogenetic tree ofknown melamine-degradingbacteria. Bootstrap values above50% (expressed as percentages of1000 replications) are shown atbranch points

Appl Microbiol Biotechnol

For identification, melamine, ammeline, ammelide, cyanuricacid, MC, and unknown precipitate were analyzed by Ramanspectroscopy. The spectrum of unknown precipitate matchedwell with MC, which had noticeable Raman peaks of 75, 99,154, 409, 528, 594, 678, 694, and 1,739 cm−1 (Fig. 5). Asimilar spectrum of MC was also obtained by the previous

study (He et al. 2008). Therefore, the unknown precipitate wasidentified as MC.

Degradation of cyromazine by strain CY1

Our result indicated that cyromazine could be degraded bystrain CY1 (Fig. S2 in the supplemental material). HPLCanalysis showed that two unknown intermediates were formedduring cyromazine degradation. Cyromazine and one un-known intermediate disappeared gradually but the other un-known compound accumulated and was not degraded. Thisresult suggested that strain CY1 could only transformcyromazine to an intermediate but could not completely min-eralize cyromazine. Considering the deamination ability ofstrain CY1, we hypothesized that strain CY1 could catalyzeconsecutive hydrolysis of the two amino substituents ofcyromazine, 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. 4 The SEM photograph of unknown precipitate

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Q-TOF. Strong signals for [M+H]+ at m /z 169 was observedin the selected ion chromatogram of the 10-day incubationsample, and the signal of m /z 169 was not observed in thebeginning of incubation (Fig. 6). The result of UPLC-Q-TOFsupported the proposed conversion of cyromazine to6-(cyclopropylamino)-[1,3,5]triazine-2,4-diol by strain CY1.

Discussion

As a pesticide and a cyclopropyl derivative of melamine,cyromazine is used to control fly in crop production and animalfeed by inhibiting inset growth. Previous study has indicated thatcyromazine could be dealkylated tomelamine by photochemicalreactions or by plant biotransformation systems (Lim et al. 1990;

Sancho et al. 2005). Our result indicated that microbial transfor-mation could convert cyromazine to a dead end product of6-(cyclopropylamino)-[1,3,5]triazine-2,4-diol from consecutivehydrolysis of the two amino substituents of cyromazine.Previous study has developed analytical method for simulta-neous determination of cyromazine and melamine in the envi-ronment since melamine is an important intermediate duringcyromazine degradation (Yokley et al. 2000). However, ourresult suggested that when examining the environmental fateof cyromazine, possible formation of 6-(cyclopropylamino)-[1,3,5]triazine-2,4-diol should also be considered.

The insolubleMC is infamous in causing the “kidney stone”or acute renal failure in children and pets in the incident ofmelamine-tainted milk products and pet food (Chen et al.2009). It is surprising to see the occurrence of MC during themicrobial degradation of melamine. We firstly detected theabnormal increasing of the medium OD due to the formationof the insoluble co-crystals during melamine degradation. TheOD value did not match the bacterial protein content duringmelamine biodegradation (data not shown). We observed theappearance of precipitate roughly after 24 h incubation duringmelamine degradation and meanwhile, cyanuric acid concen-tration almost reached its peak. The self-assembly of melamineand cyanuric acid generated a macromolecule which wassustained by noncovalent multiple hydrogen bonds. The disap-pearance of aggregates was likely resulted from the successiveconsumption of dissociative melamine and cyanuric acid in themedium. Previous studies did not report the observation of theformation of MC during melamine biodegradation. This wasmost likely due to their addition of extra carbon sources in themedium and the higher cell density would likely conceal thephenomenon of self-assembly of melamine and cyanuric acidin the medium. Our findings ofMC formation during microbial

120.9591

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Fig. 6 Mass spectra of cyromazine (m /z 167) (a) and 6-(cyclopropylamino)-[1,3,5]triazine-2,4-diol (m /z 169) (b) from the selected ion chromatogramsof biodegradation of cyromazine by strain CY1

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degradation melamine also provided supporting evidence for arecent proposal that melamine-induced kidney stones could bemediated by the gut microbiota (Zheng et al. 2013).

Acknowledgments We thank Professor Oliver Hao for his valuablecomments. This work was supported by the Science and TechnologyInnovation and Collaboration Team Project of the Chinese Academy ofSciences, Technology Foundation for Selected Overseas Chinese Scholarof MOHRSS, China, the Hundred Talents Program of the Chinese Acad-emy of Sciences, Science, Technology Planning Project of Xiamen,China (3502Z20120012), and the CAS/SAFEA International PartnershipProgram for Creative Research Teams (KZCX2-YW-T08).

References

Baynes RE, Smith G, Mason SE, Barrett E, Barlow BM, Riviere JE(2008) Pharmacokinetics of melamine in pigs following intravenousadministration. Food Chem Toxicol 46:1196–1200

Chen KC, Liao CW, Cheng FP, Chou CC, Chang SC, Wu JH, Zen JM,Chen YT, Liao JW (2009) Evaluation of subchronic toxicity of petfood contaminated with melamine and cyanuric acid in rats. ToxicolPathol 37:959–968

Cook AM, Hutter R (1981) s-Triazines as nitrogen sources for bacteria. JAgric Food Chem 29:1135–1143

Cook AM, Hutter R (1984) Deet hylsimazine bacterial dechlorination,deamination, and complete degradation. J Agric Food Chem 32:581–585

Dodge AG, Wackett LP, Sadowsky MJ (2012) Plasmid localization andorganization of melamine degradation genes in Rhodococcus sp.strain Mel. Appl Environ Microbiol 78:1397–1403

Hao X, Stone R (2008) Chinese probe unmasks high-tech adulterationwith melamine. Science 322:1310–1311

He L, Liu Y, Lin M, Awika J, Ledoux DR, Li H, Mustapha A(2008) A new approach to measure melamine, cyanuric acid,and melamine cyanurate using surface enhanced Raman spec-troscopy coupled with gold nanosubstrates. Sens Instrum FoodQual 2:66–71

Kandelbauer A, Widsten P (2009) Antibacterial melamine resin surfacesfor wood-based furniture and flooring. Prog Org Coat 65:305–313

Lim L, Scherer SJ, Shuler KD, Toth JP (1990) Disposition of cyromazinein plants under environmental conditions. J Agric Food Chem 38:860–864

Lund KH, Petersen JH (2006) Migration of formaldehyde and melaminemonomers from kitchen- and tableware made of melamine plastic.Food Addit Contam 23:948–955

Lv P, Wang Z, Hu K, Fan W (2005) Flammability and thermal degrada-tion of flame retarded polypropylene composites containing mela-mine phosphate and pentaerythritol derivatives. Polym Degrad Stab90:523–534

Qin Y, Lv X, Li J, Qi G, Diao Q, Liu G, XueM,Wang J, Tong J, Zhang L,Zhang K (2010) Assessment of melamine contamination in crop,soil and water in China and risks of melamine accumulation inanimal tissues and products. Environ Int 36:446–452

Sancho JV, Ibáñez M, Grimalt S, Pozo ÓJ, Hernández F (2005) Residuedetermination of cyromazine and its metabolite melamine in chardsamples by ion-pair liquid chromatography coupled to electrospraytandem mass spectrometry. Anal Chim Acta 530:237–243

Shelton DR, Karns JS, Mccarty GW, Durhum DR (1997) Metabolism ofmelamine by Klebsiella terragena . Appl Environ Microbiol 63:2832–2835

Takagi K, Fujii K, Yamazaki K, Harada N, Iwasaki A (2012)Biodegradation of melamine and its hydroxy derivatives by a bac-terial consortium containing a novel Nocardioides species. ApplMicrobiol Biotechnol 94:1647–1656

Whitney PA, Coopers TG (1972) Urea carboxylase and allophana hydro-lase: two components of a multienzyme complex in Saccharomycescerevisiae . J Biol Chem 247:1349–1353

Xu S, Zhang Y, Sims A, Bernards M, Hu Z (2013) Fate and toxicity ofmelamine in activated sludge treatment systems after a long-termsludge adaptation. Water Res 47:2307–2314

Yang JJ, Tian YT, Yang Z, Zhang T (2010) Effect of melamine onpotassium currents in rat hippocampal CA1 neurons. Toxicol InVitro 24:397–403

Yokley RA, Mayer LC, Rezaaiyan R, Manuli ME, Cheung MW (2000)Analytical method for the determination of cyromazine and mela-mine residues in soil using LC-UV and GC-MSD. J Agric FoodChem 48:3352–3358

Yu CP, Roh H, Chu KH (2007) 17β-Estradiol-degrading bacteria isolatedfrom activated sludge. Environ Sci Technol 41:486–492

Zheng X, Zhao A, Xie G, Chi Y, Zhao L, Li H, Wang C, Bao Y, Jia W,Luther M, Su M, Nicholson JK, Jia W (2013) Melamine-inducedrenal toxicity is mediated by the gut microbiota. Sci Transl Med 5:172ra22

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