Bioresource Technology 153 (2014) 95–100

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Bioresource Technology

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Hydroxylation and hydrolysis: Two main metabolic ways of spiramycin Iin anaerobic digestion

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.11.073

⇑ Corresponding author. Tel.: +86 21 64253021.E-mail address: juchu@ecust.edu.cn (J. Chu).

1 These authors contributed equally to this work.

Pei Zhu a,b,1, Daijie Chen a,1, Wenbin Liu a, Jianbin Zhang a, Lei Shao a, Ji-an Li a, Ju Chu c,⇑a State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, PR Chinab State Key Laboratory of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd., Shanghai 200436, PR Chinac State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China

h i g h l i g h t s

� Antibiotic like macrolide could bedegraded in anaerobic digestion.� Over 95% spiramycin was degraded in

32 day’s anaerobic digestion.� Hydroxylation and hydrolysis were

two metabolic ways of SPM I inanaerobic process.� Structural analysis confirmed that P-3

was a new compound.

g r a p h i c a l a b s t r a c t

O OHCH3

OH3CO

CH3

OO

N

CH3

CH3

CH3

O

OO

CH3

O

NCH3CH3

OCH3

OH

CH3OH

HO

O OHCH3

OH3CO

CH3

OO

N

CH3

CH3

CH3

OH

OH

a r t i c l e i n f o

Article history:Received 16 September 2013Received in revised form 20 November 2013Accepted 25 November 2013Available online 1 December 2013

Keywords:Anaerobic degradationSpiramycinHydroxylationHydrolysis

a b s t r a c t

The anaerobic degradation behaviors of five macrolides including spiramycin I, II, III, midecamycin andjosamycin by sludge were investigated. Within 32 days, 95% of spiramycin I, II or III was degraded, whilethe remove rate of midecamycin or josamycin was 75%. SPM I degradation was much higher in nutritionsupplementation than that just in sludge. The degradation products and processes of spiramycin I werefurther characterized. Three molecules, designated P-1, P-2 and P-3 according to their order of occur-rence, were obtained and purified. Structural determination was then performed by nuclear magneticresonance and MS/MS spectra, and data indicated that hydroxylation and hydrolysis were main reactionsduring the anaerobic digestion of spiramycin I. P-1 is the intermediate of hydroxylation, and P-2 is theintermediate of hydrolysis. P-3 is the final product of the both reaction. This study revealed a hydroxyl-ation and hydrolysis mechanism of macrolide in anaerobic digestion.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Antibiotics play a very important role in the treatments ofhuman and animal diseases and are widely used as animal growthpromoter in feed. Thousands of tons of antibiotics have been con-sumed in China each year; whereas, the resultant mass excretion ofundegraded antibiotics poses emerging environment issues. In-deed, the residual antibiotics have been detected in all tested soilsand rivers in China (Xu et al., 2007; Zheng et al., 2012; Zou et al.,2011). Of the administered antibiotics, only 10–70% will be

absorbed by the body, while the rest will be excreted, mostlythrough poultry manure and human excrement (Sarmah et al.,2006). Therefore municipal sewage and manure is one of the majorexposure routes that transport antibiotics or their metabolites intothe environment (Mohring et al., 2009; Motoyama et al., 2011).Another major exposure route of antibiotics is the plants and hos-pitals where a large amount of antibiotics-polluted liquid and solidis released to the environment.

Macrolides are a group of antibiotics widely used in humanand veterinary medicine. Meanwhile, they are also largely ex-creted into sewage with unchanged forms at extraction ratesgreater than 60% (Hirsch et al., 1999). It has been reported thatthe concentration of macrolides in raw sewage from Switzerlandvaries between 0.01 and 0.6 lg L�1 (Gobel et al., 2005a,b), andthe wastewater treatment plants (WWTPs) influent in the USA

96 P. Zhu et al. / Bioresource Technology 153 (2014) 95–100

contains macrolides at a concentration of 1.5 lg L�1 (Karthikeyanand Meyer, 2006). In individual cattle, chicken, and swine man-ures, the peak concentrations of tylosin reach 119, 35, and62 mg kg�1 (dry weight basis), respectively (Scott Teeter andMeyerhoff, 2003). Thus, substantial amounts of macrolides couldgo to rivers and soils through effluent discharge and manuring.Several investigations (Xu et al., 2007; Zheng et al., 2012; Zouet al., 2011) have demonstrated that residues of some macrolideshave been found in soils and adjacent environmental compart-ments such as surface and groundwater. Gullberg et al. (2011)suggested that the low antibiotic concentrations found in theenvironments were favorable for the enrichment and mainte-nance of resistance in bacterial population. Recent years, re-searches (Calza et al., 2009; Lange et al., 2006; Vione et al.,2009) have been focusing on the nature environmental degrada-tion ways on macrolides such as photolysis, ozonation and hydro-lysis. However, the effects of possible bioactivity of certainantibiotic degradations in the environment are still unknown.

Anaerobic digestion is an important way to reduce antibioticpollution by the interaction between anaerobic microorganismsand antibiotics. Increasing numbers of recent researches have fo-cused on the behaviors and kinetics of antibiotic degradation inanaerobic treatments of manure (Alvarez et al., 2010; Arikan,2008; Beneragama et al., 2013; Dreher et al., 2012), pharmaceuticalwastewater (Shimada et al., 2008; Sponza and Demirden, 2010)and municipal sewage (Gartiser et al., 2007; Le-Minh et al.,2010). The antibiotic concentrations have more or less inhibitiveeffects on methane production or methane percentage. Benera-gama demonstrated that cefazolin showed no inhibition for meth-ane production and oxytetracycline showed 70% inhibition(Beneragama et al., 2013). Dreher also found that chlortetracyclinedid not inhibit biogas production, however the methane percent-age was approximately 15% decreased (Dreher et al., 2012).Shimada demonstrated that tylosin can inhibit propionate- orbutyrate-oxidizing syntrophic and fermenting bacteria, and conse-quently lead to unfavorable effects on methanogenesis (Shimadaet al., 2008). The biochemical mechanism of antibiotics degrada-tion involved in anaerobic fermentation has not been elucidatedin detail, mainly because of failures in isolation and purificationof antibiotic metabolites to a detectable concentration fromcomplicated anaerobic digestion systems. Up to data only onemetabolite, formed by hydroxylation at the pyrimidine ring4-OH-sulfadiazine, has been identified and partly quantified bymass spectrometry (Mohring et al., 2009). The unknown metabo-lites discharged into the environment directly after anaerobictreatment may also bring potential negative effects.

Spiramycin is a 16-member macrolide mainly used to treatinfections of oropharynx, respiratory system and genito-urinary.It is a broad-spectrum antibiotic against most of Gram-positiveand Gram-negative cocci, but not against Enterobacteriaceae(Rubinstein and Keller, 1998). The basic structure of spiramycinis a lactone ring bearing 16 carbon atoms, and is modified withthree sugar radicals: mycarose, mycaminose and forosamine (Rich-ardson et al., 1990). The clinically used spiramycin is a mixture ofspiramycin I, spiramycin II (3-acetyl) and spiramycin III (3-propa-noyl). The criterion of the ratio of these components varies in dif-ferent countries. In France the major component is spiramycin I(>85%), while spiramycin II and III only account for <5% and 10%,respectively (Mourier and Brun, 1997). In contrast, the product inChina mainly contains spiramycin II and III (>35%, respectively)with moderate spiramycin I (<10%) (Liu et al., 1997). Diffusion testshave demonstrated that the relative microbial activities of spira-mycin II and III to spiramycin I are 57% and 72%, respectively(Liu et al., 1999). To date, seldom has been focused on the degrada-tion of spiramycin under anaerobic condition. Castiglioni et al.(2006) have investigated the removal rate of spiramycin. However,

the degradation pathways of spiramycin and the resultant metab-olites await investigation.

Our previous studies showed that after spiramycin-containingwaste was treated in the anaerobic digestion system, nearly nospiramycin could be detected by HPLC. This raised the questions:what and how spiramycin was changed to. To answer these,lab-scale anaerobic digestion tests were performed to verify thedegradation results of spiramycin I, II, III, midecamycin andjosamycin. Degradation products of spiramycin I were purifiedfor structure analysis and degradation processes assay.

2. Methods

2.1. Reagents

Spiramycin I (SPM I, 95%) and spiramycins (SPM I <10%, II and IIIabout 35%, respectively) were supported by Topfond (Henan, China).Spiramycin II (SPM II) and III (SPM III) were purified in lab with 93%purity. SPM I wastewater (COD 23,000 ± 3000 mg L�1, SPM I con-centration 65 ± 12 mg L�1) and sludge (TS, 24%) were also suppliedby Topfond. Midecamycin (MIM, 96%) and josamycin (JOM, 95%)were kindly supplied by NIFDC (Beijing, China). All other chemicalsand solvents were of analytical or HPLC grade. All the above fivemacrolides were prepared in ethanol at the test solution(1024 mg L�1) stored at �70 �C. Macroporous resin (NM100) wasa gift from Nano-Micro (Suzhou, China). The matrix of the resinwas polystyrene with a particle diameter of 40–100 lm.

2.2. Activation of sludge

The sludge was centrifuged and washed twice with distilledwater in order to remove any metabolites and 12.5 L of the pre-treated sludge was transferred to a 30 L jacketed and hermetic bio-reactor with a 25-L working volume. Then, 12.5 L medium whichcontaining 8 g L�1 starch, 5 g L�1 glucose, 4 g L�1 soybean meal,2 g L�1 NaCl, 3 g L�1 NH4NO3 and 1 g L�1 KH2PO4 (pH 7.2) wasadded into the reactor to activate the sludge at 37 �C for twoweeks. After that, 5 L activated sludge was withdrawn and samevolume of fresh medium was added simultaneously everyday forthree months with intermittent agitation (5 min twice a day)(Shimada et al., 2008).

2.3. Anaerobic degradation of five macrolides

In this study, 1 L hermetic jars with butyrate rubber septa wereconducted for degradation tests of the five macrolides in triplicate.Each jar that contains 0.4 L activated sludge and 0.4 L medium wasspiked with 160 mg of each of the five macrolides (final concentra-tion 200 mg L�1) (Table 1). The temperature was maintained at37 �C throughout the 32-day digestion. Every four days, 1 mL sam-ple from each fermentor was drawn and mixed with 1 mL ethanolfor shaking two hours and then centrifuged to remove cell massand other insoluble substances. Quantitative analysis was per-formed by HPLC (Waters, America; 2998 Photodiode Array Detec-tor) with a Welchrom C18 column, 5 lm, 4.6 � 250 mm (Welch,America). The mobile phase was a 63:37 (v/v) mixture of 50 mMammonium acetate water solution and acetonitrile. The columntemperature was 35 �C and the effluent was monitored at 232 nm.

2.4. Anaerobic degradation process of SPM I

SPM I was chosen to further explore the degradation process inanaerobic digestion. The digestion was conducted in two 1-L jarswith butyrate rubber septa. The activated sludge and medium usedwere same as above. In order to compare whether the nutrition af-

P. Zhu et al. / Bioresource Technology 153 (2014) 95–100 97

fects digestion, Jar No. 1 contained 0.4 L sludge and 0.4 L mediumand Jar No. 2 contained 0.4 L sludge and 0.4 L distilled water wereconducted. Each jar was spiked with 80 mg SPM I (final concentra-tion 100 mg L�1) (Table 1). The condition of digestion and themethods of sampling and analysis were same as above. Becauseof incomprehension of the degraded metabolites, the process wasdescribed only using the peak areas of HPLC as quantitative crite-rion rather than masses.

2.5. Anaerobic digestion process of SPM I wastewater

In order to understand the digestion process of SPM I in waste-water that is usually treated by anaerobic digestion further studywas conducted to imitate real anaerobic treatment of SPM I waste-water. Briefly, a 1-L jar with butyrate rubber septa containing 0.4 Lsludge and 0.4 L SPM I wastewater was maintained at 37 �C(Table 1). The mean concentration of SPM I in this wastewater is65 ± 12 mg L�1 and the digestion period was 20 days. The methodsof sampling and analysis were same as above.

2.6. Isolation and characterization of SPM I-degraded metabolites

P-1 and P-3 were isolated by microsphere resin (Nano-Micro,China) chromatography with specific degraded solution of SPM I.The resin (150 mL) was washed by distilled water and loaded ina glass column (diameter 2 cm). The degraded solution of SPM Iwas mixed with isometric ethanol and then isolated. The superna-tant was concentrated in a rotary vacuum concentrator (BUCHI,Switzerland) and poured onto the column at approximately1 mL min�1. After that, 600 mL 15% (v/v) ethanol water solutionwas used to wash the column at 2–3 mL min�1 to remove somewater-soluble impurities, and then 1.5 L 40% (v/v) ethanol watersolution was used to elute the SPM I-degradations at a averageflow rate of 3 mL min�1. Effluents were collected in each stepand analyzed by HPLC. The eluate with the purity of P-1 or P-3 over95% was pooled and vacuum concentrated. The enriched solutionswere freeze-dried (LABCONCO, America) to achieve the powderwhich was stored at �70 �C prior to further identification andcharacterization.

P-2 was purified by C18 (DaYou, China) reversed phase chroma-tography with the help of the early elution (40%) of P-1 which justcontain P-2 and P-3. The packing (50 g) was filled in a glass column(diameter 1 cm) and washed with 200 mL 10% (v/v) and 90% (v/v)methanol respectively. The early elution of P-1 was concentratedin a rotary vacuum concentrator (BUCHI, Switzerland) and 5 mLwas poured onto the column at 0.5 mL min�1. 0.5 L of 70% (v/v)methanol was used to elute P-2 at the rate of 0.5 mL min�1. Efflu-ents were collected step by step and analyzed by HPLC. The eluatewith the purity of P-2 over 95% was pooled and vacuum concen-trated, stored at �70 �C for further identification.

Structural analysis of the metabolites of SPM I in anaerobicdigestion were characterized by mass spectra, 1D 1H, 13C, DEPTand 2D COSY, HMQC and GHMBC spectra. Determination of the ex-act mass was carried out by a Q-Tof micro mass spectrometer

Table 1Experimental design of the batch assays carried out in this work.

Assay Type of medium Slu

Five macrolidesa Defined mediumb 0.4SPM I with nutrition Defined medium 0.4SPM I without nutrition Deionized water 0.4SPM I wastewater SPM I wastewater 0.4

a Five macrolides: SPM I, II, III, MIM and JOM.b Defined medium: 8 g L�1 starch, 5 g L�1 glucose, 4 g L�1 soyb

(pH 7.2).

(Waters, America) using electrospray ionization (ESI). The NMRspectra were acquired on a Bruker AV400 (Bruker, Switzerland)spectrometer instrument (400 MHz).

2.7. Statistical analysis

All batch tests were repeated in triplicate. Mean values andstandard deviation were calculated and presented in tables andgraphs as coordinate pairs with corresponding error bars. Analysisof variance was performed by using the SPSS 18.0 statistical anal-ysis system (SPSS Inc., America), and a least significant differencetest was used to compare the means with a confidence intervalof 95%.

3. Results and discussion

3.1. Anaerobic degradation of five macrolides

All of the five macrolides were successfully degraded within the5-week-digestion with sludge (Table 2). No degradation was ob-served without sludge (data were not shown). The spiramycin I,II and III were nearly 95% eliminated after three weeks, while themidecamycin and josamycin experienced a minor (P < 0.05) elimi-nation by 75% during the 32 days. The degradation rate of spiramy-cin in this anaerobic process was opposite to the investigationconducted by Castiglioni et al. (2006). They found that the removerate (RR) of spiramycin in sewage treatment plants (STPs) of Italywas closed to zero both in winter and summer. The reason of thisenormous divergence may be the different microbial flora in sludgeand the different digestion condition (hydraulic retention time andthe initial macrolides concentration) used in our Lab. It is wellknown that bacteria can adapt their metabolism to degrade aro-matic compounds as an alternative carbon source (Tropel andvan der Meer, 2004). So the microorganisms in anaerobic digestioncan also consume antibiotics in a specific condition.

3.2. Anaerobic degradation process of SPM I

The anaerobic degradation process of SPM I were compared un-der the conditions with or without nutrient supplementation.Three major degraded metabolites were observed compared withthe ultraviolet spectroscopy of SPM I. They were designated as P-1, P-2 and P-3 according to chronological order of appearance.The retention time (tR) of P-1, P-2 and P-3 were 9.7, 7.8 and5.7 min, respectively.

Under the condition of nutrient supplementation, the t1/2 (timerequired to digest half substrate) of SPM I was approximately3 days and SPM I was eliminated completely on the ninth day.Meanwhile, P-1 was the main incipient metabolite in the first7 days, and it was then being eliminated in the following 6 days(Fig. 1a). The concentration of P-2 was low in the first 6 days andstarted to increase on the seventh day (Fig. 1a). P-3 was a third de-graded metabolite that largely appeared after the sixth day andwas kept accumulating in the rest of time course (Fig. 1a). This

dge (L) Medium (L) Macrolides (mg L�1)

0 0.40 2000 0.40 1000 0.40 1000 0.40 37

ean meal, 2 g L�1 NaCl, 3 g L�1 NH4NO3 and 1 g L�1 KH2PO4

Table 2Degradation of the five macrolides during a fermentation process of 32 days aspercentage of initial measured concentration.

Day SPM I SPM II SPM III MIM JOM

0 100.0 ± 3.1 100.0 ± 2.0 100.0 ± 2.8 100.0 ± 3.6 100.0 ± 3.98 34.9 ± 10.9 53.8 ± 6.7 60.4 ± 7.2 54.8 ± 8.9 44.9 ± 7.7

16 13.7 ± 5.3 26.0 ± 6.0 26.5 ± 6.6 43.2 ± 7.1 36.4 ± 7.224 3.9 ± 1.4 8.3 ± 2.4 8.7 ± 3.0 33.1 ± 6.2 26.1 ± 5.632 1.5 ± 0.6 2.8 ± 0.6 3.5 ± 0.8 26.4 ± 3.6 22.1 ± 3.5

Fig. 1. Anaerobic digestion of SPM I at 37 �C using sludge with nutrition (a), sludgewithout nutrition (b) and sludge with wastewater (c). Peak area of HPLC (232 nm)was used to quantify the concentration of each compound.

98 P. Zhu et al. / Bioresource Technology 153 (2014) 95–100

changing pattern indicated that P-1 might be an intermediateproduct. Compared with P-1, the decreasing rate of P-2 was slowerin the following 12 days and still existed at the end of the forma-tion (Fig. 1a). However, the pattern was similar to P-1, suggestingthat P-2 might also be an intermediate product. P-3 is a third deg-radation that was largely appeared after the sixth day and wasaccumulating in the rest of the experiment (Fig. 1a). Therefore P-3 might be an end degraded metabolite of SPM I in this anaerobicdigestion as shown in Fig. S1.

Under the condition without nutrient supplementation, only P-1 was obviously increased, the biggest concentration of P-1 wasless (P < 0.05) 21% than that of with nutrition condition. P-2 andP-3 was maintained at a low level (Fig. 1b) and the concentrationof terminal product P-3 within nutrition supplementation washigher (P < 0.05) 5 times than that of without nutrition conditionin the nineteenth day. It is obvious that the degradation rate ofSPM I was increased with nutrition supplementation. There is asignificant difference (P < 0.05) under the condition without acti-

vated sludge, SPM I was relatively stable within 19-days (Fig. 1),demonstrating the straightforward interaction between SPM Iand the anaerobic microorganisms which was affected by nutri-tion. Although few related anaerobic microorganisms has been re-ported that can degrade SPM I, researchers (Liu et al., 2012) foundthat Microcystis aeruginosa a kind of cyanobacteria strain whichshowed 12.5%-32.9% degradation of SPM I in 7-day exposure.Physical–chemical processes are also effective for SPM I degrada-tion, such as acid catalysis (Shi et al., 2004) and illumination (Calzaet al., 2009).

In the anaerobic treatment of wastewater containing 37 mg L�1

SPM I, SPM I was completely dispelled in 4 days, and P-1 and P-2were produced in the time course and vanished with the increasingof P-3 (Fig. 1c). This profile was consistent with that of pure SPM Iin nutrient supplementation condition.

3.3. Isolation and characterization of SPM I-degraded metabolites

Three SPM I degraded metabolites P-1, P-2 and P-3 wereisolated and purified with the purities above 95%, and the structureidentification was as follows.

The mass spectrum of P-1 showed a molecular ion at m/z 845and a doubly charged ion at m/z 423, indicating that the molecularweight (M.W.) of P-1 is 844 amu. The pseudo-molecular ion wasjust 2 amu higher than that of SPM I, indicating P-1 was a producthydrogenated from SPM I. However, there were four positions thatcan be hydrogenated in SPM I. The NMR was further conducted toconfirm the exact position. Comparing the 13C NMR spectrum(DMSO-d6) of SPM I with P-1, the C-19 signal (d 203.6) of SPM Iwas against with that (d 59.2) of P-1, without change in other C sin-gles. This observation was further confirmed by the disappearanceof the 19-H single (d 9.7). It is concluded that P-1 was a metabolitethat contains an alcohol function instead of the aldehyde functionin the C-19 position of SPM I (Fig. S1). Previously, it has been re-ported that the product with a hydroxyl in the C-19 position wasbe named spiramycin IV (Chepkwony et al., 2001; González et al.,1999).

P-2 gave a molecular ion at m/z 526 indicating that the M.W. ofP-2 is 525 amu. This pseudo-molecular ion is just hydrolysis ofmycaminose–mycarose sugar from SPM I. Because P-2 was notmuch available for NMR, the proposed structure was supportedby MS2 spectra analysis. MS2 spectrum showed an intense ion at349 that is concerned loss of forosamine and a hydrone. The otherpeaks at m/z 331, 317 and 299 were exactly the same to the re-search about the fate of spiramycin in river water (Calza et al.,2009), suggesting that P-2 (Fig. S1) is a metabolite of demycami-nose–mycarose SPM I.

As deduced above, P-3 is the ultimate metabolite of SPM I inanaerobic digestion. The m/z of the protonated form of P-3 is528, and thus P-3 has a M.W. of 527, which is just 2 amu largerthan P-2. When comparing M.V. between P-3 and P-1, we founda mycaminose–mycarose is off from P-1, resulting in P-3. This phe-nomenon was also seen between SPM I and P-2. To further confirmthe structure of P-3, 1D 1H, 13C, DEPT and 2D COSY, HMQC andGHMBC spectra were recorded. The assignments of the 1H and

Table 3NMR spectroscopic datas of P-3.

Position dHa (J) dC

a, mult. HMBC (H ? C#)

1 171.9, qC2 2.42, dd (14.7, 11.0)

2.20–2.25, m39.5, CH2 1

3 3.44–3.47, m 67.9, CH 14 2.73, dd (8.8, 1.2) 85.1, CH5 3.62, dd (8.8, 1.5) 71.7, CH6 2.19–2.25, m 40.3, CH7 1.38–1.46, m

0.80–0.84, m30.3, CH2 18

8 1.96–2.00, m 31.8, CH9 4.10, dd (9.6, 4.0) 78.1, CH 11,17

10 5.57, dd (15.1, 9.4) 128.4, CH11 6.13, dd (15.1, 10.6) 133.6, CH 912 6.00, dd (15.0, 10.6) 132.6, CH13 5.48, ddd (15.0, 11.3, 4.0) 130.7, CH14 2.49–2.51, m

2.20–2.25, m40.4, CH2 13,16

15 4.92–4.97, m 68.5, CH16 1.21, d (6.7) 20.3, CH3 1417 0.87, d (6.7) 15.4, CH3

18 1.50–1.53, m 32.1, CH2

19 3.44–3.47, m 59.2, CH2

20 3.40, s 60.7, CH3 410 4.41, dd (8.9, 1.5) 99.1, CH 920 1.71–1.80, m

1.40–1.46, m31.2, CH2

30 1.71–1.80, m1.40–1.46, m

17.9, CH2

40 2.19–2.25, m 64.8, CH50 3.42–3.45, m 72.9, CH60 1.12, d (6.7) 19.1, CH3

70/80 2.10, s 40.6, CH3 40

a Recorded in DMSO-d6 and obtained at 400 and 100 MHz for 1H and 13C NMR,respectively.

P. Zhu et al. / Bioresource Technology 153 (2014) 95–100 99

13C spectra of P-3 are shown in Table 3. After blasting against theSciFinder database, we found that P-3 has not been reported so far.

Above all, SPM I can be eliminated in anaerobic conditions.Three degraded metabolites were isolated and characterized. P-1and P-2 are mesostates that were hydroxylated or demycami-nose–mycarose metabolites of SPM I, respectively, while P-3 isdemycaminose–mycarose metabolite of P-1 or hydroxylatedmetabolite of P-2 (Fig. S1). The hydrolytic position (mycaminose–mycarose) of SPM I in anaerobic condition is against with thehydrolysis of mycrose or forosamine in acid catalyzed degradation(Shi et al., 2004). As demonstrated above, forosamine a sugar rad-ical still existed at C-9 of SPM I during anaerobic digestion whichmaybe hydrolyzed by photolysis in the environmental. This studyreports hydrogenation or hydroxylation of SPM I in anaerobicdigestion for the first time.

4. Conclusion

The comprehensive technology based on anaerobic digestionplays an important role in treatment of municipal sewage, pharma-ceutical wastewater and manure. This report showed that macro-lides especially spiramycin could be effectively degraded byanaerobic digestion. Two intermediate metabolites and a terminalmetabolite were obtained and characterized, suggesting that theremight exist two main routes of SPM I degradation, the hydrogena-tion of the aldehyde group and the hydrolysis of mycaminose–mycarose.

Acknowledgements

We thank Prof. Ping Xu, Shanghai Jiao Tong University, for hisexcellent writing assistance. We are grateful to Dr. Zhijun Yang,Shanghai Jiao Tong University, for his excellent analysis technology

help. This work was supported in part by grants from the Ministryof Science and Technology of China (Grant No. 2012ZX09201101-008).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2013.11.073.

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