ORIGINAL PAPER

J. L. Sanz · N. Rodrıguez · R. Amils

The action of antibiotics on the anaerobic digestion process

Received: 6 February 1996 / Received revision: 24 July 1996 / Accepted: 5 August 1996

Abstract Antibiotics can disturb the production ofbiogas during anaerobic digestion. This study shows asystematic approach to understanding how the differentbacterial populations involved in the final conversion oforganic matter into methane are inhibited by 15 anti-microbial agents with different specificities and modes ofaction. The results obtained show the following trends:(i) some inhibitors, such as the macrolide erythromycin,lack any inhibitory effect on biogas production; (ii) someantibiotics, with different specificities, have partial in-hibitory effects on anaerobic digestion and decreasemethane production by interfering with the activity ofpropionic-acid- and butyric-acid-degrading bacteria, (e.g.antibiotics that interfere with cell wall synthesis, RNApolymerase activity and protein synthesis, especially theaminoglycosides); (iii) the protein synthesis inhibitorschlortetracycline (IC50 40 mg l)1) and chloramphenicol(IC50 15–20 mg l)1) are very powerful inhibitors ofanaerobic digestion. The majority of the antibioticstested lacked activity against acetoclastic methanogens,being active only on the acetogenic bacteria. However,chloramphenicol and chlortetracycline could cause thecomplete inhibition of the acetoclastic methanogenicarchaea.

Introduction

Antibiotics can be found as contaminants in the waste-waters from pharmaceutical industries and in the solidand liquid wastes from livestock farms. Antibiotics have

several modes of action on prokaryotic systems: block-ing cell wall synthesis, inhibiting protein biosynthesis,interfering with the function of membranes, etc. Theeffect of common feed additives that interfere with cellmembranes (e.g. monensin, nonactin, lasalocid, salino-mycin and avoparcin) in anaerobic fermentation of an-imal manures has been widely studied (Varel andHashimoto 1981; Hilpert et al. 1984; Wildenauer et al.1984; Blotevogel and Jannsen 1988). There is, however,less information about the performance of anaerobicdigestion in the presence of antibiotics that inhibit pro-tein and cell wall biosynthesis. This is surprising in thelight of the fact that these antibiotics can be found notonly in manure (Hilpert et al. 1984; Poels et al. 1984) butalso in the wastewater of pharmaceutical industries(Glancer and Ban 1988; Struzeski 1975). Moreover, theconflicting results reported in the few published studiesavailable make it difficult to establish practical re-commendations (Hilpert at al. 1984; Poels et al. 1984;Camprubı et al. 1988).

At the end of the anaerobic food chain differentmicroorganisms are involved in bringing about miner-alization of the organic matter: acetogenic bacteria(responsible for converting volatile fatty acids intoacetic acid and H2), acetoclastic methanogenic archaea(using acetic acid to produce methane) and hydro-gentrophic methanogenic archaea (using hydrogen toproduce methane). The question arises of whether an-tibiotics with different specificities would affect thesepopulations similarly. Unfortunately, the informationon this subject is scarce. For this reason, in this studywe investigated the effects on anaerobic digestion of 15antibiotics, most of them commonly used as anti-microbial agents in livestock diets. The different groupsof antibiotics were chosen on the basis of their mode ofaction. The aim of the study was to investigate theeffect on biogas production and to determine possibledifferences in the antibiotics’ action on methanogenicand acetogenic bacteria during anaerobic digestion ofdefined substrates.

Appl Microbiol Biotechnol (1996) 46: 587–592 Springer-Verlag 1996

J. L. Sanz (&) · N. Rodrıguez · R. AmilsDepartamento de Biologıa Molecular,Universidad Autonoma de Madrid,Canto Blanco 28049 Madrid, Spain.Fax: 34 1 3978087e-mail: JLSANZ@CCUAM3.SDI.UAM.ES

Materials and methods

Chemicals

The chemicals used in this study were purchased from Merck(Darmstadt, Germany), except for the yeast extract, which wasobtained from Difco (Detroit, USA). The sources of the variousantibiotics were as follows: ampicillin, chloramphenicol, ery-thromycin, hygromycin B, kanamycin, novobiocin, rifampicin fromBoehringer (Mannheim, Germany); chlortetracycline, gentamicin,neomycin, penicillin G, spectinomycin, streptomycin, tylosin fromSigma (St. Louis, USA); doxycycline from Pfizer (Madrid, Spain).

Biological assays

Batch

The toxicity assays were carried out according to Field et al. (1988)and Sierra et al. (1991). Batch assays were conducted in 250-mlglass serum bottles sealed with a butyl rubber septum and a screwcap. The medium was flushed with an 80% nitrogen and 20% car-bon dioxide mixture in order to remove the oxygen. The bottleswere incubated in a temperature-controlled room (30 ± 2 °C) andwere not shaken during the assay period. The methane productionwas monitored daily with modified Mariotte flasks by water dis-placement. These flasks were filled with a 3% NaOH solution toremove the CO2 contained in the biogas.

The assay substrate consisted of a mixture of volatile fatty acids(VFA) supplied at approximately 4 g chemical oxygen demand(COD)/l. The VFA stock solution was made up of acetate (C2),propionate (C3) and butyrate (C4), in a ratio of 100:100:100 g/kg(C2:C3:C4 24:34:41 on a COD basis), and was neutralized withNaOH.

All the assays contained essential inorganic nutrients. The fol-lowing macronutrients were added to give concentrations in themedia as follows (mg l)1): K2HPO4·3H2O 327, NH4Cl 280,MgSO4·7H2O 100, NaHCO3 400, CaCl2·2H2O 10, yeast extract100, and trace elements (in lg l)1): FeCl2·4H2O 2000, CoCl2·6H2O2000, MnCl2·4H2O 500, Na2SeO3·5H2O 164, NiCl2·6H2O 92, ZnCl250, H3BO3 50, (NH)6Mo7O24·4H2O 50, CuCl2·2H2O 38, EDTA1000, resazurin 200, and 36% HCl 0.001 ml.

Anaerobic granular sludge was obtained from the upflowanaerobic sludge blanket (UASB) reactor at the Latenstein wheatstarch factory (Nijmegen, The Netherlands). The sludge was storedat 4 °C before its use in the different experiments. In the assays,1.5 g l)1 volatile suspended solids (VSS) of granular sludge, rinsedfree of fines and debris, was used.

The assays were carried out in three consecutive feedings. In thefirst feeding, tap water, granular sludge, VFA as a substrate andnutrient solution were added to the serum flasks. No antibioticswere added at this stage. The aim of this feeding was to activate thesludge and to check the activity of the different systems. Only flaskswith similar methane production were selected for the assays. In thesecond feeding, the antibiotics were added together with the sub-strate. A third feeding was performed to evaluate the remainingactivity and recovery after exposure to the antibiotic. The super-natant was carefully decanted to avoid the loss of sludge and re-placed with new medium. No antibiotics were added in thereplacement medium. Each feeding was incubated for 2 weeks. Allthe experiments were carried out in duplicate and the resultscompared with those from controls, in triplicate, containing thesame medium without addition of antibiotic.

Reactor

The experiments were performed in UASB reactors with a workingvolume of 900 ml. The gas was passed through a 3% NaOH so-lution and measured using a Schlumberger gas meter.

The reactors were seeded with elutriated granular sludge (27 gVSS/reactor) from the UASB reactor at the Nedalco alcohol fac-

tory (Bergen op Zoom, The Netherlands). The sludge was stored at4 °C prior to the experiments.

Feed consisted of a mixture of VFA in a ratio of C2:C3:C4300:50:150 (on a COD basis). The loading rate reached 25 g CODl)1 day)1 in the steady state. The sludge loading rate was ap-proximately 1 g COD g VSS)1 day)1. The hydraulic retention timewas 12 h and the temperature was 30 °C throughout the experi-ment. The reactors were supplied with macronutrients in the ratioCOD:N:P:K of 1000:10:2:2 and trace elements as described above.KH2PO4/K2HPO4 was added to buffer the medium at pH 7–7.5.

Analytical methods

The COD (micro method with dichromate) was determined ac-cording to Standard Methods (1989) using a Hach COD reactorequipped with a DR/700 colorimeter. Total solids and volatilesuspended solids were determined according the Standard Methods(1989). The pH was measured with an Orion 420A pH meter im-mediately after a sample had been taken from the top of the reactorto avoid a rise in pH due to the loss of CO2.

Volatile fatty acids were analysed by gas chromatography usinga Shimadzu GC-8A equipped with a 2-m glass column packed withSupelcoport (100–200 mesh) coated with Fluorad. The temperatureof the column was 100 °C, that of the injection port 240 °C andthat of the flame ionization detector 240 °C. Nitrogen was used as acarrier gas and a device made in our laboratory was used to sa-turate the column with formic acid. The methane was analysed onthe same gas chromatograph with a 2-m glass column packed withPorapack Q. The temperatures of the column, the injector anddetector were 60 °C, 220 °C, 220 °C respectively.

Results

The outline of our batch experiment, using a VFAmixture as substrate, enabled us to determine the effectof the different antibiotics at two levels: first, on thevarious microbial populations, that is, acetogenic bac-teria (which convert propionate and butyrate into acet-ate) as well as methanogenic archaea (which convertacetate into methane) and, second, on the final step ofanaerobic digestion determined as a whole by the me-thane produced. The results for C2, C3 and C4 removedafter exposure of the anaerobic sludge to antibiotics areshown in Table 1. The global effect of the different an-tibiotics tested on the methane production are sum-marized in Table 2.

DNA-dependent RNA polymerase inhibitors

Rifampicin, the only inhibitor of this mode of action tes-ted, needed 2–3 days to affect biogas production. How-ever, a high concentration of this antibiotic (200 mg l)1)was able to inhibit anaerobic digestion, affecting the de-grading activity of C3 and, especially, of C4 (Table 1).

Cell-wall inhibitors (b-lactamic antibiotics)

These inhibitors caused a 30%–40% reduction in biogasproduction at a concentration of 10 mg l)1. At higherconcentrations, up to 500 mg l)1 (Fig. 1A), increased

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inhibition was not observed. The fact that there was noobservable acetic acid accumulation indicates that thedecrease in biogas production must be related to theinhibitory effect of these antibiotics on the propionic-acid- and butyric-acid-consuming bacteria. The presenceof b-lactamic antibiotics (10–500 mg l-1) caused a de-crease in the activity of acetogenic bacteria of around55%, compared with the control assays (Table 1).

Inhibitors of protein synthesis

Four types of protein synthesis inhibitors were tested inthis study.

Aminoglycosides

Most of the aminoglycoside compounds tested had onlya slight effect on biogas production. These inhibitors didnot act against acetate-consuming methanogenic bac-teria (Table 1).

Streptomycin showed no effect on the sludge duringthe first few days. At concentrations higher than50 mg l)1, the activity of the C3- and C4-degradingbacteria was low. In these conditions, a 35%–40% in-hibition of biogas production was observed (Fig. 1B).Kanamycin, gentamicin and spectinomycin did not dis-play any significant effect on anaerobic digestion(Fig. 1B). Neomycin showed some inhibition, rangingfrom 17% to 40% at 10–50 mg l)1 antibiotic. Higherconcentrations did not cause higher inhibition. Neomy-cin affected C2-, C3- and, especially, C4-degrading bac-teria.

It is remarkable that hygromycin B, an aminoglyco-side antibiotic with a differential mode of action andspecificity (Vazquez 1979), is the only antibiotic of thisgroup for which a linear relationship exists between theconcentration of the tested antibiotic and the inhibitoryreaction (IC50 = 220 mg l)1) (Table 2 and Fig. 1B).

Tetracyclines

Chlortetracycline was found to be a powerful inhibitorof anaerobic digestion, with an IC50 of 40 mg l)1 (Table 2and Fig. 1C). The acetoclastic methanogenic bacteriawere not affected up to 25 mg l)1 but, at antibioticconcentrations higher than 200 mg l)1, C2 was notconsumed. C4 removal was affected even at low con-centrations of chlortetracycline, and above 100 mg l)1

the C4-consuming acetogenic bacteria died.Doxycycline was considerably less active; it seemed to

affect only the C4-degrading bacteria significantly. Theresponse of the sludge to the antibiotic was directlyproportional, between 10 mg l)1 and 100 mg l)1 doxy-

Table 1 Levels of volatile fatty acids (VFA), expressed as percen-tages of VFA removed, after exposure of the anaerobic sludge tothe antibiotics. The values have been compared to the unexposedcontrols. Higher levels of VFA removed signify lower inhibitoryactivity of the antibiotic. ) VFA removed ≤ 10% (similar values tothose of the initial levels), + 10% < VFA removed < 50%, ++ 50%< VFA removed < 90%, +++ VFA removed ≥ 90% (similar valuesto those of the levels found in the unexposed controls). ND notdetermined

Antibiotic Extent of VFA removal

C2 C3 C4

Rifampicin +++ ++ +b-Lactamic

Aminoglycosides +++ ++ ++Streptomycin +++ + +Kanamycin + gentamicin +spectinomycin

+++ ++ ++

Neomycin ++ ++ +Hygromycin B ND ND ND

TetracyclinesChlortetracycline ) + )Doxycycline +++ +++ )

MacrolidesTylosin +++ +/++ +/++Erythromycin +++ +++ +++

Chloramphenicol ) ) )

Table 2 The concentration of thevarious antibiotics evaluated in thisstudy that resulted in an 80%, a 50%and a 20% production of methane.In all cases activity recovery wasnot observed after the toxiccompound was removed. ) Noinhibition at the highest concentra-tion tested (500–1000 mg l)1)

Antibiotic Concentration (mg l)1) inhibiting CH4 production by:

Mode of action 20% 50% 80%

Rifampicin RNA polymerase 100 >250 –Ampicillin Cell wall 10 – –Novobiocin Cell wall 10 – –Penicillin Cell wall 10 – –Gentamicin Protein synthesis 35 – –Hygromycin B Protein synthesis 64 210 >300Kanamycin Protein synthesis 100 – –Neomycin Protein synthesis 20 >500 –Spectinomycin Protein synthesis >20 – –Streptomycin Protein synthesis 18 – –Chlortetracycline Protein synthesis 5 40 152Doxycycline Protein synthesis 8 – –Tylosin Protein synthesis 15 – –Erythromycin Protein synthesis – – –Chloramphenicol Protein synthesis 11 26 41

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cycline, resulting in a 25%–45% reduction in methaneproduction. There was no further reduction at higherantibiotic concentrations (Fig. 1C).

Macrolide antibiotics

Tylosin was able to inhibit methane production (35% in-hibition) at the lowest concentration tested (25 mg l)1),although at higher concentrations, up to 250 mg l)1, onlya faint increase in the inhibitory effect (up to 45% inhibi-tion) was observed (Fig. 1D). This was due to the specificinhibition of C3- and C4-degrading bacteria, while no ef-fects were observed on acetoclastic methanogens. Theacetogenic bacteria showed 40%–50% of the activity thatthe control showed when exposed to tylosin (Table 1).

The other macrolide tested, erythromycin, had noinfluence on methane production even at 250 mg l-1,which was the maximum concentration tested (Fig. 1D).

Chloramphenicol

This antibiotic inhibited anaerobic digestion at ratherlow concentrations, with an IC50 of 15–20 mg l)1

(Table 2). At concentrations of 25 mg l)1 there was 90%inhibition, and complete inhibition was observed at50 mg l)1 (Fig. 1E).

Effect of chloramphenicol on the performanceof a UASB reactor

Working with UASB reactors produced similar results.After two 900-ml UASB reactors had run for 50 days,steady-state conditions were reached. The antibiotic was

Fig. 1A–E Inhibitory effect on biogas production caused byincreasing concentrations of antibiotics. Experiments were carriedout as described in Materials and methods. Different inhibitors havebeen clustered according their structure

Fig. 2 Effect of chloramphenicol on the performance of an upflowanaerobic sludge blanket reactor. After 2 months of operation,chloramphenicol at 40 mg/l was added to the reactor, and removed 2months later. The efficiency was calculated on the basis of chemicaloxygen demand (COD) removal and methane produced. OLR loadingrate

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added to one of them at a concentration of 40 mg l)1.The other was left as a control. After 4–5 days of op-eration, the performance was dramatically affected. TheCOD removal and methane production efficiencydropped from 80% to zero. The antibiotic was added tothe influent feed solution of the reactor for 2 monthslonger, but no adaptation to the chloramphenicol wasobserved. No recovery was observed for 1 month afterremoval of the antibiotic (Fig. 2).

Discussion

The inhibitory effect on methane production producedby different antibiotics can be classified into differentgroups according to the inhibition curves generated.

Some antibiotics do not interfere with methane pro-duction; erythromycin is the only antibiotic of this classstudied here. This result is consistent with the absence ofany inhibitory effect reported for erythromycin in thedigestion of manure slurry (Camprubı et al. 1988; Poelset al. 1984).

A wide group of antibiotics (ampicillin, novobiocin,penicillin, kanamycin, gentamicin, spectinomycin,streptomycin, tylosin and tetracycline) partially inhibitmethane production by granular sludge fed with VFAmixture. At low concentrations of antibiotics, inhibitionis produced, which then increases at higher concentra-tions until a plateau is reached (between 25% and 45%).Higher concentrations of antibiotic do not produce anyadditional inhibitory effect. These results are probablydue to the fact that the inhibitors specifically affect thedifferent populations involved in the final stages ofanaerobic digestion. The non-utilization of acetate, andthe partial degradation of propionate and butyrate seemto confirm this hypothesis (Table 1).

References discussing the effect of these antibiotics inanaerobic digestion processes are very scarce. The onlyinhibitor widely studied is tylosine (Camprubı et al.1988; Hansen 1981; Hilpert et al. 1984; Poels et al. 1984).Although our inhibitory data agree with those Hilpertdescribed for municipal sludge treatment, the resultsreported in these papers often contradict one another.Studies with pure cultures show that, in general, me-thanogens tolerate high dosages of the aminoglycosidesincluded in this group (Hilpert et al. 1981; Pecher andBock 1981; Weisburg and Tanner 1982) while the degreeof inhibition caused by tylosin varies greatly dependingon the organisms tested (Hummel et al. 1985; Sanz,Hummel, Amils and Bock unpublished results).

A third type of inhibition is observed for neomycinand hygromycin B. Increasing concentrations of anti-biotics produce a steady increase of inhibition, althoughat the highest concentrations used only partial inhibitionis obtained (50%).

This behaviour could be the result of the differentsensitivities of the methanogens present in the granularsludge to these inhibitors, given that, in pure cultures,

Methanobacteriales are only slightly affected, while theMethanococcales and, especially, Methanosarcina bar-keri show a high sensitivity to neomycin and hygromycinB (Pecher and Bock 1981; Weisburg and Tanner 1982).It is well documented that, in granular sludge developedin UASB reactors, the predominant populations ofmethanogenic archaea are members of the Methano-sarcina and Methanosaeta (former Methanotrix) genera(Hulshoff Pol 1989; Alfenaar 1994).

A biphasic inhibition is observed for chlortetracycline(Fig. 1C). The pattern of utilization of the substrateindicates a selective effect of the antibiotic on the dif-ferent microbial populations (Table 1). The bacteria thatdegrade C4 compounds are quickly and drastically af-fected, while higher concentrations of antibiotic areneeded (greater than 20 mg l)1) to act on the methano-genic acetoclastic archaea.

Finally chloramphenicol strongly inhibits methaneproduction at low concentrations (IC50=25 mg l)1),acting on all the microorganisms involved in the process.Because of the pharmacological interest of this anti-biotic, its effect on anaerobic digestion has been widelystudied. Nevertheless, available reports are not con-clusive about its action. Some authors have found noinhibitory effect on the digestion of piggery wastes (Poelset al. 1984; Strauch and Winterhalder 1985), while oth-ers have reported strong inhibitions (Camprubı et al.1988). In pure cultures chloramphenicol inhibits thegrowth of all methanogens tested at rather low con-centrations (Hilpert et al. 1981; Pecher and Bock 1981)with even greater effect than that produced in bacteria.

We studied the inhibition caused by chloramphenicolin a UASB reactor, in order to ascertain its effects ac-curately and determine its long-term action. The lack ofdegradation of acetate, propionate and butyratestrongly suggests that the antibiotic affects both aceto-gens and acetotrophic populations. Moreover, eventhough we did not observe any significant deteriorationof the granules, the microorganisms must have beenseriously damaged because there was no evidence ofacclimatization or recovery of the reactor after removalof the inhibitor during the period studied. These resultsagree with those obtained with batch experiments andconfirm the high inhibitory capacity of chloramphenicol.

Our results indicate that most of the antibioticsnormally used in livestock farms will not drastically af-fect biogas production when used at recommendedconcentrations. Only chloramphenicol and chlorte-tracycline inhibit methanogenesis strongly, probablybecause they are potent inhibitors of archaea. Otherantibiotics, such as doxycycline, tylosin or some ami-noglycosides, produce a partial inhibition at high con-centrations, probably because of their inhibitory effecton the acetogenic bacteria. Although these antibioticsseem to have no effect on methanogens they could causeserious problems in methane production, since duringthe degradation of the organic matter a lot of potentialCH4 is locked up in acetogenic substrates. The failure toproduce gas or the decrease in gas production in the

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digesters appears to result from the incorporation ofantibiotics into the manure, so our results strongly ad-vise avoiding the use of these inhibitors. They can bereplaced by other antibiotics, like erythromycin, whichhave slighter effect or even no effect on anaerobic di-gestion.

Acknowledgements This work was supported by grants from theComunidad Autonoma de Madrid (C140/90) and the ComisionInterministerial de Ciencia y Tecnologıa (BIO91-1153-C02-02 andPB92-0129). We are also grateful to the Fundacion N. GarcıaGomez for providing part of the laboratory equipment. We wish tothank Dr. J. A. Field for his encouragement and valuable discus-sions.

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