ORIGINAL PAPER

Effect of oxytetracycline on biogas production and activemicrobial populations during batch anaerobic digestionof cow manure

Bahar Ince • Halil Coban • Gokhan Turker •

Emine Ertekin • Orhan Ince

Received: 7 April 2012 / Accepted: 6 August 2012 / Published online: 21 August 2012

� Springer-Verlag 2012

Abstract The aim of this study was to investigate the

effect of a common veterinary antibiotic in biogas plants.

20 mg/kg of oxytetracycline was intramuscularly injected

into a cow and its concentration in manure, which was

sampled daily during the following 20 days, was measured.

A total of 20 % of the injected oxytetracycline was detected

in manure. Collected manure samples on days 1, 2, 3, 5, 10,

15, and 20 were digested in triplicate serum bottles at 37 �C

for 30 days. Control serum bottles produced 255 ± 13 mL

biogas, whereas 50–60 % inhibitions were obtained for the

serum bottles operated with samples collected for the 5 days

after medication. Multivariate statistics used for the evalu-

ation of FISH results showed that Methanomicrobiales were

the main methanogenic group responsible for most of

the biogas production. Numbers of active Bacteria and

Methanomicrobiales were negatively correlated with the

presence of oxytetracycline, whereas Methanosarcinales and

Methanobacteriales were less affected.

Keywords Oxytetracycline � Fluorescent in situ

hybridization � Biogas � Anaerobic digestion � Redundancy

analysis (RDA)

Introduction

Antimicrobials, which are used to treat diseases, prevent

infections, as well as promote growth, make up about 70 %

of all used pharmaceuticals in veterinary medicine [1].

These compounds are not completely metabolized in the

body and are excreted via manure [2]. Oxytetracycline

(OTC) is a broad spectrum antibiotic which enters the

microbial cells and binds to ribosome to prevent binding of

aminoacyl tRNA, and is widely used to treat livestock

animals due to its low cost and low side effects [3]. Its

intense use generally results with its occurrence in manure

and further in biogas plants when manure is used as a

substrate [4]. Although there are several studies regarding

effects of OTC on biogas production, studies on the effects

of OTC on the microbiology of manure digesters are rather

limited [4–6]. Moreover, most of the studies are conducted

by adding antibiotics into the digester or using manure

from antibiotic-fed animals, and information on the effect

after intramuscular medication is almost absent despite

intramuscular injection being one of the main medication

methods. Therefore, the objective of this study is to

determine the effect of OTC excreted in manure from

intravascular medicated cows, both in terms of biogas

production and microbial groups involved in reactors.

Materials and methods

Chemicals

Oxytetracycline (Mw = 460, CAS no. 79-57-2) was sup-

plied by Acros Organics N.V (NJ, USA). HPLC grade

methanol, acetonitrile, oxalic acid, and citric acid were

obtained from Merck (NJ, USA). All other chemicals used

B. Ince � H. Coban � G. Turker � E. Ertekin (&)

Institute of Environmental Sciences, Bogazici University,

Bebek, 34342 Istanbul, Turkey

e-mail: emertekin@gmail.com

Present Address:H. Coban

UFZ - Helmholtz Centre for Environmental Research,

Department of Environmental Biotechnology,

Permoserstr. 15, 04318 Leipzig, Germany

O. Ince

Department of Environmental Engineering, Istanbul Technical

University, Maslak, 34469 Istanbul, Turkey

123

Bioprocess Biosyst Eng (2013) 36:541–546

DOI 10.1007/s00449-012-0809-y

in this study were of analytical grade. Double distilled

water used in this study was obtained using a Millipore

water purification system (Millipore Corporation, MA,

USA).

Animal medication and manure sampling

Manure samples of a Holstein race (3.5-year-old, 440 kg

body mass) dairy cow, which was kept in a barn belonging

to the Faculty of Veterinary Medicine, Istanbul University,

were used as the substrate for serum bottle tests. The

manure in the rectum was collected and stored at 4 �C until

future use as the control manure. The dairy cow was then

medicated once with Oxytetracycline injection solution

(20 mg OTC/kg animal weight) according to the standard

dosage in veterinary practice. Equal doses were injected to

the right and left body sides between the musculus semi-

tendinosus and musculus semimembranosus muscles.

Manure samples (around 1 kg) were collected from the

rectum daily for 20 days and used throughout experiments

after the OTC concentration was determined by HPLC.

Determination of extraction efficiencies for OTC

and method of HPLC analysis

Manure samples were extracted in triplicate by a method

modified from a previous study [7]. Briefly, 5 g of wet

manure was placed into 50 mL polycarbonate centrifuge

tubes with 0.5 g oxalic acid (C2O4H2�2H2O), 4 mL acetic

acid, and 7.5 mL of 90 % methanol, and shaken at 100 rpm

for 30 min. The tubes were further centrifuged at

11,000 rpm for 10 min. This procedure was repeated for 3

times and the supernatants were collected in 50 mL volu-

metric flasks. Collected supernatants (30 mL ± 5–10 %)

were diluted to a 50 mL volumetric curve with double

distilled water and centrifuged again at 14,000 rpm for

3 min and filtrated through 0.2 lm Millipore filters. The

extracts were kept in 2 mL amber vials at -20 �C until the

day of the HPLC analysis. The HPLC instrument (Schi-

madzu LC-10 AD) was equipped with an UV detector; (UV

VIS Detector, SPD 10-A), an autosampler; (SIL-10 AD), a

degasser (DGU-14A), and a system controller (SCL-10A).

The column used in this study was Inertsil ODS-3 HPLC

column (25 cm 9 4.6 mm). Degassing of the solvents was

achieved by sonication in a transonic ultrasonic bath

(ELMA D-78224, Singen/Htw) prior to use. The mobile

phase consisted of 75 % 0.1 M oxalic acid buffer and 25 %

Methanol: Acetonitrile (1:1.5) solution which was deliv-

ered isocratically at a flow rate of 1 mL/min. The total run

time was 30 min. The wavelength for the detection of

oxytetracycline was 357 nm at which the retention time

was 7.3 ± 0.1 min. The minimum detectable concentration

was 0.01 mg/L.

All results were analyzed by the system software; Class

VP Schimadzu Scientific Instruments Inc. In order to

determine extraction efficiencies, triplicate samples of non-

medicated manure were spiked with OTC in concentrations

given in Table 1 and incubated for 3 h and extracted as

described above. Recovery results shown in Table 1 were

calculated as means of triplicate samples at each

concentration.

Serum bottle tests

Manure samples collected after days 1, 2, 3, 4, 5, 10, 15, 20

of medication were used in the serum bottle tests. Exper-

iments were carried out batch-wise in 120 mL serum bot-

tles for 30 days with a working volume of 40 mL. Manure

samples were diluted with tap water to total solids (TS)

concentration of 5 % as a usual practice in commercial

farm operations [8]. Seed was obtained from a lab-scale

manure digester and added to the manure slurry at a ratio of

1:4 (Inoculum: Substrate). On operation days 0, 10, 20, and

30, biogas samples were collected from headspace, and

samples for biogas and molecular analyses were collected

every 10 days by sacrificing a set of serum bottles

appointed to each sampling time. The experiment set was

incubated in a temperature-controlled room at 37 ± 1 �C

for 30 days on an automatic shaker operated at 120 rpm.

All of the serum bottles were run in triplicate.

Total carbon, total nitrogen, Total Kjeldahl nitrogen

(TKN), total solids, and total volatile solids (TVS) mea-

surements were carried out according to the American

Public Health Association’s [9] guidelines. Characteristics

of manure samples are given in Table 2. Gas pressures

were measured using a manometer (HACH PM-9107) for

calculations of biogas production. Gas compositions were

determined using a Gas Chromatograph HP Agilent 6850

with a thermal conductivity detector and HP Plot Q Col-

umn (30 m, 530 lm). The carrier gas was Helium with an

inlet column flow of 5.4 mL/min.

Determination of active microbial populations

Every 10 days of the operation, 5 mL of sample from the

serum bottles was transferred into sterile containers, diluted

with absolute ethanol (1:1, v/v) and fixed according to a

Table 1 Recovery rates of OTC in manure

Concentration in

manure (mg/kg)

Recovery

rate (%)

200 92 ± 0.1

20 85 ± 0.2

5 80 ± 0.1

0.5 86 ± 0.9

542 Bioprocess Biosyst Eng (2013) 36:541–546

123

protocol described previously [10]. Samples were taken

from the control serum bottle and serum bottles operated

with the 2nd, 10th, and 15th day manures as representative

to the level of biogas inhibition. Hybridization and visu-

alization of samples were carried out according to a pre-

vious study [11], except that 50–75 times dilution of the

fixed samples was spotted on Teflon-coated slides. Probes

used in this study were EUB338 [12], MB310, MG1200,

MS1414, and MSMX860 [13] for the detection of Bacteria,

Methanobacteriales, Methanomicrobiales, Methanosarcin-

aceae, and Methanosarcinales (complete acetoclastic

methanogens), respectively. For the detection of non-spe-

cific bindings, NON338 probe was used as the negative

control [14]. These probes were selected in reference to the

most frequently detected groups of methanogens in biogas

digesters. Results were reported as the total cell number

which gave a positive signal to the specified probe per mL.

Statistical analyses

Significant differences were determined at 0.05 level by

one sample t test by means of SPSS 11.5 (SPSS Inc., USA).

Redundancy analysis (RDA) was applied in order to

investigate the relation between microbial community

dynamics and environmental variables (Canoco 4.5,

Biometris, the Netherlands).

Results and discussion

Excretion of injected OTC in manure

Approximately 20 % of injected OTC was detected in the

manure samples during 20 days of collection (Table 3). The

total amount of OTC in the manure was calculated by mul-

tiplying the OTC concentration with the total manure pro-

duction which is 118 g manure/kg body weight [15] daily. A

manure sample which was collected on day 1 showed the

highest OTC concentration (10.38 ± 0.22 mg/kg). On the

4th day of collection, there was an increase in the OTC

concentration, but the level decreased after that rapidly until

the 13th day of collection (Fig. 1). Although studies on the

fate of OTC in manure after oral administration are plenty,

information on intramuscular medication has not been

reported. Our results indicated 80 % of OTC was either

absorbed and/or degraded to metabolites; theoretically, the

absorption rate of Tetracyclines can vary between 10 and

90 % [16]. According to similar studies reported in the lit-

erature, a total of 23 % of the oral-fed OTC was recovered

from the manure of beef calves [17], and about 10 mg/kg

OTC was detected in a fivefold diluted manure slurry of an

orally medicated calf [16]. Tetracyclines have been found in

Table 2 Characteristics of manure samples used in serum bottle tests

Manure

collection day

TS

(%)

TVS

(%)

TKN

(mg/kg)

C/Na

Day 0

C/Na

Day 30

0 (Control) 20 16 11,500 4.7 3.8

1 14 12 10,500 4.2 3.4

2 15 13 14,000 4.6 3.7

3 14 12 12,500 4.2 3.5

5 12 10 13,000 3.7 3.1

10 14 12 14,000 3.9 3.2

15 15 12 11,000 3.5 3.0

20 15 12 11,000 4.2 3.5

Table 3 Daily OTC concentra-

tion in manureManure

sampling the

day after

medication

Concentration

in manure

(mg/kg)

1 10.38

2 4.54

3 4.13

4 6.35

5 3.71

6 1.15

7 1.13

8 0.61

9 0.34

10 0.45

11 0.30

12 0.25

13 0.00

14 0.00

15 0.00

16 0.00

17 0.00

18 0.00

19 0.00

20 0.00

Fig. 1 Concentrations of OTC for 20 days of manure collection

Bioprocess Biosyst Eng (2013) 36:541–546 543

123

manure as low as 0.1 mg/kg [18]. All the differences

encountered in the literature can likely be due to adminis-

tration type of the drug, sampling and storage conditions, the

diets, general health of the animal, and type of animal.

Effects of OTC on anaerobic digestion performance

Serum bottle tests were run for 30 days, during which a

total of 255 ± 13 mL biogas was obtained in the control

bottles. At the end of 30 days of digestion, inhibitions in

terms of biogas production compared to the control serum

bottles were 46, 58, 57, 51, and 21 % for the manures

collected on days 1, 2, 3, 5, and 10 after treatment,

respectively (Fig. 2). Serum bottles operated with manure

samples collected on days 15 and 20 were not significantly

different from the control bottles in terms of biogas pro-

duction (p \ 0.05). The methane percentages in the biogas

were 58 ± 5 for all serum bottles at all sampling times. In

this study, 1.0–3.3 mg/L OTC in the slurries (calculated by

dividing the OTC concentration of the daily manure sam-

ples by the dilution ratio) resulted in 50–60 % decreases in

biogas production. In a previous study, 27 % inhibition in

cumulative biogas production was reported in which OTC

was given to beef calves orally and found in the manure

slurry in a concentration of 3.1 mg/L [4]. In another study,

tetracycline was found to inhibit methane production by

25 % in a swine manure digester [5]. In this case, although

the highest concentration of OTC was detected after the

first day of medication, the most severe inhibition on bio-

gas productions was observed in serum bottles operated

with manure samples collected on days 2 and 3. In serum

bottles operated with manure samples collected after the

5th day of medication, the level of inhibition decreased.

Manure samples collected on days 15 and 20 caused almost

no inhibition compared to the control bottles. These out-

comes can beneficially be utilized in practice in order to

prevent possible decreases in biogas production; manures

collected for the first 5 days after medication should be

mixed with non-medicated manure and/or manures col-

lected after the 10th day of medication prior to transition to

biogas plants. However, in commercial barns, generally all

of the animals are medicated, instead of just the sick ones,

to prevent contamination of diseases. This can cause higher

than expected levels of OTC in biogas plants, especially

when manure is used as the sole substrate for biogas pro-

duction. This challenge could be overcome by using other

substrates such as agro-crops, bio solids, food wastes, etc.

for co-digestion.

Changes in active microbial populations in serum

bottles

Active microbial groups in serum bottles were character-

ized using fluorescent rRNA-targeted oligonucleotide

probes specific for phylogenetically defined groups of

methanogens and total Bacteria. The numbers of active

cells detected by the specified probes per mL are given in

Table 4. FISH results showed an increase in all samples

until the 20th day of digestion after which the number of

active cells decreased. Detected methanogens belonged to

the groups of Methanobacteriales, Methanomicrobiales,

and Methanosarcinales. The cells identified as Methano-

sarcinacea were almost equal to the number of cells iden-

tified as Methanosarcinales. Thereby, it could be assumed

that Methanosaetaceae group was nearly non-existent in

the serum bottles. The similar community structure of

manure digesters have been reported in various sources

[20–22].

In order to evaluate results, redundancy analysis (RDA)

was used as a statistical approach, which is known as the

most generally effective ordination method for ecological

community data, and was also used in similar studies

determining microbial community Dynamics in biogas

plants [19]. Figure 3 shows the RDA plot showing the

FISH results where Eigenvalue was 0.829. In the case of

cumulative percentage variance, 82.9 and 87.8 % of

the species–species and species-environment relations,

respectively, were explained.

RDA analysis showed that biogas production was sig-

nificantly negatively correlated with OTC concentration as

those arrows directed opposite to each other. Biogas pro-

duction was found in a positive correlation with total

Bacteria and order Methanomicrobiales in the serum bot-

tles. Orders Methanobacteriales and Methanosarcinales

explained less variance on biogas production. This indi-

cates that most of the methane production was accom-

plished through the hydrogenotrophic pathway by

Methanomicrobiales spp. The case has also been suggested

previously where methanogenesis through the syntrophic

association between hydrogenotrophic methanogens and

bacteria such as Clostridium spp. [22–24] was reported inFig. 2 Cumulative biogas production in serum bottles after 30 days

544 Bioprocess Biosyst Eng (2013) 36:541–546

123

manure digesters [25]. The stability of methane percent-

ages in the produced biogas from the serum bottles showed

that there was a cease in carbon dioxide production as well.

This can be explained by the simultaneous inhibitory effect

of OTC on both bacteria and methanogens.

As seen, total bacteria and Methanomicrobiales were

negatively correlated with OTC. Consequently, it can be

said that effect of OTC was most viable on Methanomi-

crobiales, whereas Methanosarcinales and Methanobacte-

riales groups were comparatively less affected. However, it

should be kept in mind that this population structure was

reserved for batch systems, meaning that a washout of the

most susceptible groups to oxytetracycline would have

occured if the system was operated in a continuous manner.

Future studies focusing on the microbiology in earlier

stages of digesters exposed to tetracyclines would be

enlightening on these matters.

Conclusion

In this study, a total 10 % of the administrated OTC was

excreted in the manure. A discharge pattern could be

monitored daily. Around 1–3.3 mg/L OTC caused a

50–60 % decrease in biogas production in which the

methane percentage was stable. The collection time of

manure was identified as an important factor upon trans-

ferring manure to the biogas plants. Redundancy analysis

made for the evaluation of microbial dynamics with envi-

ronmental parameters provided sensible results and

revealed that a hydrogentrophic group, Methanomicrobi-

ales, was mostly associated with biogas production and was

the most affected group from the OTC. However, bacterial

groups that mediate earlier stages should also be examined

in future studies in order to reveal the effects on syntrophic

associations.

Acknowledgments This study was financially supported by the

Scientific and Technical Research Council of Turkey (TUBITAK,

Project No: 109Y275). The authors are thankful to the Pharmacology

and Toxicology Department of Istanbul University, Faculty of

Veterinary Medicine for animal medication and manure sampling.

Table 4 Numbers of active microbial cells in serum bottles (cells/ml)

Manure sampling

day

Digestion

day

Eubmix MB310 MG1200 MSMS1414 MSMX

Control 0 2.69 9 107 2.47 9 107 2.98 9 107 2.33 9 107 2.52 9 107

Control 10 6.83 9 107 4.64 9 106 8.14 9 107 2.31 9 106 2.51 9 106

Control 20 9.22 9 107 5.51 9 107 9.52 9 107 3.81 9 107 3.98 9 107

Control 30 6.67 9 106 4.90 9 106 5.98 9 106 3.65 9 106 2.98 9 106

2 0 3.41 9 107 1.01 9 107 1.23 9 107 9.07 9 106 8.22 9 106

2 10 3.37 9 107 8.79 9 106 1.63 9 107 1.70 9 107 2.06 9 107

2 20 3.98 9 107 1.29 9 107 2.39 9 107 1.76 9 107 2.67 9 107

2 30 2.31 9 106 1.36 9 106 8.38 9 106 4.31 9 106 3.85 9 106

10 0 3.90 9 107 6.63 9 106 1.23 9 107 1.03 9 107 1.08 9 107

10 10 3.83 9 107 1.54 9 107 4.36 9 107 1.08 9 107 1.46 9 107

10 20 6.23 9 107 2.00 9 107 4.17 9 107 2.84 9 107 2.94 9 107

10 30 6.22 9 106 3.73 9 106 1.12 9 107 6.96 9 106 7.46 9 106

15 0 3.30 9 107 1.58 9 107 5.80 9 106 1.94 9 107 1.46 9 107

15 10 6.01 9 107 1.18 9 107 5.15 9 107 1.22 9 107 1.30 9 107

15 20 8.62 9 107 2.09 9 107 1.19 9 108 4.37 9 107 5.12 9 107

15 30 8.85 9 106 2.97 9 106 5.06 9 106 2.92 9 106 2.85 9 106

Fig. 3 RDA analysis of FISH results with environmental parameters

Bioprocess Biosyst Eng (2013) 36:541–546 545

123

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