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    tracycline (OTC) on anaerobic digestion was tested.1 were compared with no-antibiotic control.ly affect (P > 0.05) methane production.OTC treatments showed a signicant reduction.

    lity from

    pectively, of the control values. The high CH4 production, optimal pH andVFA data during digestion indicated the process stability and treatment efciency.

    (Jjemba, 2002). Oxytetracycline (OTC) and cefazolin (CFZ) are themost commonly used antibiotics in Obihiro University farm, Hok-kaido, Japan. OTC is a broad spectrum antibiotic that is activeagainst variety of bacteria thus used in dairy cattle production pri-marily to treat or prevent diseases and to a lesser extent to increasemilk production or improve feed efciency. CFZ, a b-lactam antibi-otic, is also widely used to treat bacterial infections in cows (e.g.

    offers numerousction, lowvan Staikeas been re

    to affect anaerobic digestion efciencies, both at the laboand the eld scale. Masse et al. (2000) previously reported thracycline and penicillin reduced the methane production of psy-chrophilic anaerobic digestion (20 C) of swine manure slurry insequencing batch reactor by 25% and 35% respectively. Similarly,Alvarez et al. (2010) reported signicant inhibition of anaerobicdigestion of swine manure containing a combination of chlortetra-cycline (CTC) and OTC at concentrations of 10, 50 and 100 mg L1

    at 35 C, where maximum methane production decreased by 64%in manure containing 100 mg L1 of both CTC and OTC.

    Corresponding author. Tel.: +81 0155495515; fax: +81 0155495519.

    Bioresource Technology 133 (2013) 2330

    Contents lists available at

    T

    elsE-mail address: umetsu@obihiro.ac.jp (K. Umetsu).digestion, resulting in the excretion of potent parent and daughterproducts (Teeter and Meyerhoff, 2003). Between 17% and 76% ofantibiotics administered to animals are excreted via urine andfeces in an unaltered form or as metabolites of parent compounds

    reducing pollution from agricultural wastes andsignicant advantages, such as low sludge produrequirement, and possible energy recovery (1997). The presence of antibiotic compounds h0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.01.032energynburg,portedratoryat tet-Anaerobic digestionDairy manureCefazolinOxytetracyclineMethane production

    2013 Elsevier Ltd. All rights reserved.

    1. Introduction

    Many antibiotics are being used as veterinary medicines fortherapeutic and sub therapeutic treatments and as feed additivesto promote the growth of animals. Many of these compounds exhi-bit poor retention within the animal gut and intestine during the

    mastitis). The use of CFZ as a treatment to mastitis in cows leadsantibiotic residues in milk. This milk is not saleable and shouldbe withheld for a period recommended by the manufacturer anddisposed of. The presence of these compounds in manure andwaste milk warrants further consideration due to usage of thesematerials in anaerobic digestion which provides a method ofKeywords:OTC at concentrations of82.7%, 70.3% (P < 0.05) res" The effect of cefazolin (CFZ) and oxyte" Concentrations of 30, 60 and 90 mg L

    " CFZ concentrations did not signicant" Individual OTC and combined CFZ and" No noticeable effects on process stabi

    a r t i c l e i n f o

    Article history:Received 5 October 2012Received in revised form 31 December 2012Accepted 5 January 2013Available online 22 January 2013CFZ and OTC concentrations tested.

    a b s t r a c t

    The individual and combined effect of cefazolin (CFZ) and oxytetracycline (OTC) on thermophilic anaer-obic digestion (55 C) of dairy manure in batch digesters was investigated. Methane productions from theconcentrations tested (30, 60 and 90 mg L1) were compared with no-antibiotic control. CFZ concentra-tions showed no inhibition (P > 0.05) for methane production. The individual OTC and combined CFZ and

    30, 60 and 90 mg L1 represented 79.1%, 70.3%, 68.6% (P < 0.05) and 88.5%,h i g h l i g h t sThe combined effect of cefazolin and oxyproduction from thermophilic anaerobic

    Nilmini Beneragama, Suraju A. Lateef, Masahiro IwaDepartment of Environmental Hygiene, Obihiro University of Agriculture and Veterinary

    Bioresource

    journal homepage: www.rtracycline on biogasgestion of dairy manure

    i, Takaki Yamashiro, Kazutaka Umetsu dicine, Obihiro 080-8555, Japan

    SciVerse ScienceDirect

    echnology

    evier .com/locate /bior tech

  • 55 C. Digestate samples were taken before and after the experi-

    Methane production was observed from digesters of no-cefazo-1

    rcement to analyze for pH, TS (total solids), VS (volatile solids) degra-dation and volatile fatty acids (VFA).

    2.3. Analytical methods

    Total gas productions were monitored every other day. Wet gasmeter was used to measure the volume of produced gas. All gasmeasurements were expressed at 0 C and a pressure of one atmo-sphere. Prior to measure the volume of produced gas, gas compo-sition of each gas bag was determined using gas chromatograph(GC) (Shimadzu GC-14A) equipped with a thermal conductivitydetector (stainless column and Porapak Q packing). The opera-However, there is lack of information on the individual andcombined effect of OTC and CFZ on the anaerobic digestion processof dairy manure at thermophilic temperature (55 C). It is thereforeof considerable importance to determine whether amending dairymanure with OTC and CFZ (individual and combined) would ad-versely impact process efciencies and stability of batch digestersused for anaerobic treatment of dairy manure. The manure thatwas directly spiked with environmentally relevant OTC and CFZdoses was used in this study instead of using manure collectedfrom cows administered these antibiotics. Direct OTC and CFZspiked manure enable one to proportionally control the antibioticdoses within each digester.

    2. Methods

    2.1. Materials

    Cow manure and digested slurry were obtained from receptionpit and digester of biogas plant at Obihiro University, Obihiro, Hok-kaido, Japan respectively. Cow manure, discharged to the pit, wasobtained from a herd of lactating Holstein cows and collected dailyfrom concrete oor of free stall barn. The straw (bedding material)presented in manure was separated manually. Digested slurry, theinoculum, is produced from digested cow manure in a digesteroperated at thermophilic temperature (55 C).

    Oxytetracycline (OTC) and cefazolin (CFZ) were obtained fromZigma chemicals.

    2.2. Experimental design and procedure

    Laboratory-scale anaerobic degradation experiments were per-formed to evaluate the impact of individual OTC and CFZ and com-bined OTC and CFZ presence in manure on biogas production fromthermophilic anaerobic digestion of dairy manure. Three concen-trations; 30, 60, 90 mg L1 of OTC, CFZ and OTC + CFZ were tested.OTC and CFZ were combined each 50% as to form the same concen-trations (15 + 15, 30 + 30, and 45 + 45 mg L1). The control and thethree treatments in each group were tested in triplicate in 1 Lbatch digesters with active volume of 700 mL at 55 C in a thermo-statically controlled water bath for 16 days. For the control andeach treatment, digested slurry (S) and manure (M) were com-bined as to make the ratio slurry 50% and manure 50%. The com-bined contents were thoroughly mixed with a hand mixerseparately. OTC, CFZ and combined OTC and CFZ were addedaccordingly to manure and slurry mixture to obtain the desiredconcentrations of them. 700 mL of each mixture was added to eachdigester in triplicate. Digesters were ushed with argon gas priorto sealing. Gas bags were xed to each digester to collect theevolved biogas and the digesters were placed in a water bath at

    24 N. Beneragama et al. / Bioresoutional temperatures of injector port, column and the detector were220, 150 and 220 C, respectively. Argon was the carrier gas at aow rate of 50 mL min1.lin control (SM) and cefazolin spiked at concentration of 30 mg L(SM(C30)) with no lag phase and other two digesters with cefazolinconcentration of 60 mg L1 (SM(C60)) and 90 mg L1 (SM(C90))with a lag phase of about one day. SM and SM(C30) digestersreached peak methane production (38.49 and 35.71 mL/gVS addedrespectively) by the fourth day of digestion while SM(C60) andSM(C90) reached their peak (36.82 and 35.03 mL/gVS addedrespectively) by sixth day of digestion due to the initial lag phase.Methanogens are responsible for producing methane in an anaero-bic digestion process. As the anaerobic digestion process advancesand the methanogen populations become more established, theproduction of methane begins to increase. However, cefazolin con-centrations of 60 and 90 mg L1 have exerted some pressure onmethanogens at the beginning so that methanogens had to under-go some acclimatization period and as a result, a lag phase could beobserved in those two digesters. After 16 days, the mean cumula-tive total biogas production values for digesters SM, (SM(C30)),(SM(C60)) and (SM(C90)) were 10.544 0.329, 10.5 0.497,10.693 0.088 and 11.065 0.574 L respectively.

    The methane productivity of SM, (SM(C30)), (SM(C60)) and(SM(C90)) in terms of volatile solids added (mL/g VS added) were145.026 3.171, 148.142 7.051, 150.482 3.079 and159.253 5.952 mL respectively. The no-antimicrobial controltreatment (SM) showed no signicant difference (P > 0.05) inTS and VS were measured according to the Standard Methods(Standard Methods, 2005). The pH was measured using HoribaD-55 pH meter. Slurry samples were analyzed for VFA (acetic, pro-pionic, butyric and formic acids) with a high performance liquidchromatograph (HPLC, Shimadzu LC-10AD) with Shim-Pack SCR-102H column. The analytical procedure used in the current studyfor analyzing VFA using HPLC was described in detail by Kimuraet al. (1

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