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Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity
international journal of hydrogen energy xxx (xxxx) xxx
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Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity Junfeng Chen, Yuewei Yang*, Yanyan Liu, Meizhen Tang, Renjun Wang, Hanwen Hu, Hongying Wang, Pingping Yang, Hanhan Xue, Xiao Zhang Department of Environmental Science, School of Life Sciences, Qufu Normal University, Qufu, 273165, PR China
highlights OTC
and
CTC
graphical abstract
inhibited
COD
removal efficiency and methane production. Antagonistic effect of CTC and OTC
acted
upon
methane
production. Antagonism
and
enhancement
acted on inhibition rate of COD removal efficiency. Bacterial diversity and similarities decreased with the increasing of CTC and OTC. Copy
number
of
16S
rRNA
increased with the increasing of CTC and OTC.
article info
abstract
Article history:
The treatment efficiency and bacterial diversity caused by chlortetracycline (CTC) and
Received 26 August 2019
oxytetracycline (OTC) in anaerobic digestion treatment of real piggery wastewater were
Received in revised form
studied by polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE)
12 January 2020
and real-time PCR. The COD removal efficiency and methane production were inhibited
Accepted 19 January 2020
with the increasing concentration of CTC and OTC. The antagonism of CTC and OTC on
Available online xxx
methane production was observed, the inhibition rate was expanded to 55.46% from 18.34%; antagonism and enhancement were observed on inhibition rate of COD removal
Keywords:
efficiency, and the inhibition rate was expanded to 24.84% from 4.16%. The bacterial di-
Microbiological effects
versity and similarities of each group were decreased with the increasing concentration of
PCR-DGGE
CTC and OTC. The identified species Thermotogae bacterium, Bacteroidetes bacterium, Chloro-
Anaerobic digestion treatment
flexi bacterium, Eubacterium, Mesophilic bacterium, etc. were beneficial for COD removal and
* Corresponding author. E-mail addresses: [email protected] (J. Chen), [email protected] (Y. Yang). https://doi.org/10.1016/j.ijhydene.2020.01.138 0360-3199/© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
2
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Real piggery wastewater
biodegradation of CTC and OTC. The quantitative fluorescence analysis was opposite to the
Oxytetracycline and
rule of bacterial diversity by real-time PCR.
chlortetracycline
© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction With the increasing consumption of pigs, the disorder discharge of piggery wastewater exceedingly influenced ecological environment and resident health. Piggery wastewater was originated from pig raising and excrement cleaning, which was featured by high concentration of organics (chemical oxygen demand, COD), ammonia nitrogen and total phosphorus (TP), therefore, piggery wastewater would lead to serious water pollution and eutrophication to a large extent [1]. Anaerobic digestion was a kind of basic and commonly used biological treatment for piggery wastewater, which was beneficial for wastewater pollution control and energy recovery, moreover, the process was also simple and easy to operate [2]. Oxytetracycline (OTC) and chlortetracycline (CTC) were key members of tetracyclines (TCs), which were important and commonly used broad spectrum antibiotics and have been diffusely used to prevent illness and advance the growth of pigs [3e5]. The massive abuse of OTC and CTC in pigs would lead to the accumulation of antibiotics in meat, which was harmful to human health. And also, the massive abuse of OTC and CTC in pigs would be discharged into the ecological environments in the form of the original drug, which would cause huge damages to the balances of ecological environments. Therefore, it is necessary to study on the treatment of antibiotics in piggery wastewater. Bacteria played a critical role in the biological treatment of wastewater. Ma et al. [6] studied the bacterial community compositions of coking wastewater treatment plants in steel industry, and demonstrated that Thiobacillus, Comamonas, Thauera, Azoarcus and Rhodoplanes were the dominant genera and played key role in treatment efficiency. Jena et al. [7] explored the anoxiceaerobic sequencing batch reactor system for 180 days, and obtained the removal efficiency of nitrate, phosphate and COD were 98%, 86%, and 72% respectively and analyzed that Proteobacteria, Alphaproteobacteria, Rhodobacterales, Rhodobacteraceae and Paracoccous were the prominent phylum, class, order, family and genus, respectively. These studies demonstrated that it was quite important to understand the bacterial species and diversity in grasping the mechanism of wastewater treatment. In our previous studies, we have discussed the effects of OTC on the anaerobic biological treatment of livestock wastewater [5], however, it was more complex and realistic in real wastewater. Therefore, it was meaningful and valuable to explore the effects on treatment efficiency and bacterial diversity by OTC and CTC in anaerobic digestion treatment of piggery wastewater. In this study, the COD removal efficiency, methane production, bacterial diversity and real-time PCR analysis were explored by OTC and CTC in anaerobic digestion treatment of
real piggery wastewater. The purposes of this study are to provide molecular biological basis and real piggery wastewater treatment results by CTC and OTC, which is of great significance for the removal of antibiotic contamination in real wastewater and the evaluation of antibiotic effects for the practical application.
Materials and methods Anaerobic digestion treatment of piggery wastewater There were 28 anaerobic digestion reactors used in this study (3 control groups and 25 experimental groups) and all reactors were settled under the temperature of 302 ± 2 K by a thermostat water bath and shaken regularly to ensure the sludge in suspension. There were three main parts: anaerobic digestion reactor, drainage method reactor and measuring cylinder. The anaerobic digestion reactor was 0.5 L, and the anaerobic granular activated sludge was 0.1 L, the real piggery wastewater was 0.4 L, and some certain concentration of OTC and CTC were added into the reactor (Table S1) according to previous studies [5]. The gas produced by anaerobic digestion was entered to 3% NaOH solution to absorb CO2, and the methane production was measured by drainage method (hydrogen production was very low and negligible). The pH of the anaerobic digestion treatment system was 7e8. The methane production was measured every day (across 24 h) until gas production ceased and COD concentration was measured every day (across 24 h), and the COD concentration was measured by the dichromate method (K2Cr2O7, CODCr) [5,8,9]. The anaerobic activated sludge was sampled at the end of all tests and stored at 253 K.
PCR-DGGE and quantitative fluorescence analysis The total DNA of anaerobic activated sludge was extracted by DNA kit. The bacterial universal 16S rRNA gene primer pairs 341F with a GC clamp and 517R were used in polymerase chain reaction (PCR) reaction mixture, and the PCR parameters were set as previous studies [4,10e12]. Denaturing gradient gel electrophoresis (DGGE) was operated by DCode™ Universal detection instrument (Bio-Rad), and the acrylamide concentration was 8%, and the denaturing gradient was 45%e65%, and the detailed parameters were set as previous study [5]. PCR-DGGE was used to illuminate bacterial diversity in anaerobic digestion treatment of real piggery wastewater by OTC and CTC. After DGGE, the distinct bands were excised under UV light, and DNA was extracted and purified with a quick gel extraction kit (Tian DZ, China). The purified DNA was re-amplified by primer pair 341F and517R, and the products
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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were ligated into the PMD18-T vector (Takara, Japan). The inserted fragments in the selected clones were sequenced by shotgun sequencing (Sangon, China). The quantitative fluorescence analysis (gene copy number) of 16S rRNA was determined by real-time PCR. Plasmid M13 ± was used to be sequenced and absolute quantitative plasmid number was XD2564-3. The real-time PCR was operated by Sybr Green qPCR Master Mix in fluorescence quantitative PCR instrument (ABI STEPONE PLUS) and cycling conditions were as previous study [4].
Data statistics and analysis The Sig. analysis of methane production and COD removal efficiency experiments performed by SPPSS 17.0 (Sig. (2-tailed) test). The community abundance of each sample was presented using the index Ai compared to all the analyzed samples in this study. This index was calculated with the following equation: Aij ¼ lj/Lt. The phylotype profiles of the samples from every two samples were compared using Sorenson’s index as follows: CSAB ¼ 2LAB/(LA þ LB) 100; a pairwise similarity coefficient as previous study [5]. The plasmid fragment length was 2861 bp, concentration was 0.80 ng/mL and 2.60 108 copies/mL. The standard curves of plasmids were constructed by 10-fold gradient dilution and prepared by 90 mL dilution solution and 10 mL plasmid, and the quantitative fluorescence analysis was calculated as follows: a Plasmid concentration conversion formula (copies/mL) ¼ (mol number/mL) 6.02 1023 ¼ [quality (g)/molecular weight]/mL 6.02 1023 ¼ [quality (ng) 109/molecular weight]/mL 6.02 1023 ¼ concentration (ng/ uL) 6.02 1014/molecular weight b molecular weight ¼ (Carrier segment base pairs þ PCR product base pairs) 650 c Molecular weight of a double stranded DNA molecule (Dalton) ¼ Number of base pairs 650 d The average molecular weight of a base pair ¼ 650 Da
Results and discussion The effects on methane production by OTC and CTC The COD, ammonia nitrogen, total nitrogen and total phosphorus concentration of raw piggery wastewater were approximately 22,000 mg/L, 2250 mg/L, 2500 mg/L and 355 mg/ L, respectively. In this study, the raw piggery wastewater was diluted about 20 times to keep wastewater quality stable. The methane production, inhibitory rate of combined antibiotics, inhibitory rate of CTC, inhibitory rate of OTC, sum of inhibitory rates of OTC and CTC were summarized in Table 1. It could be seen that with the increased number of experimental groups designed, the curve of the whole inhibition rates was demonstrated as a fluctuating state. There was a turning point in each 5 groups of fluctuations, which was corresponded to the added concentration of the design, and the increase of each 5 points was close to that of the design. With the increase of CTC and OTC concentration, the inhibition rate of the system on cumulative methane production
3
was also increased. With the increase of CTC and OTC concentration, the inhibition rates of the system on cumulative methane production were also increased. The results showed that the inhibition rate of OTC (Experiment 1, 2, 3, 4 and 5) was increased faster than that of CTC (Experiment 1, 6, 11, 16 and 21). The combined effects of CTC and OTC were less than the sum of the two antibiotics used alone, which indicated that the inhibition of CTC and OTC on the volume of cumulative methane production was antagonistic, however, the inhibition rate of combined action on the volume of cumulative methane production was higher than that of CTC or OTC alone. The cumulative methane production volume data obtained from Table 1, and the significance analysis of cumulative methane production volume compared with control groups were analyzed in Table S2. It could be seen that the Sig. value of Experiment 1, 2, 6 and 11 was more than 0.05 from Table S2. It was indicated that the average value was equal in the probability of greater than 5%, but not equal in the probability of less than 95%. It was believed that the probability of equal mean was relatively large, indicating that the difference between the Experiment 1, 2, 6 and 11, and the blank control group was not significant, and no significant effects were achieved compared with the blank control group. The reasons for this were the relatively low concentrations of CTC and OTC. The Sig. value of the other 21 groups was less than 0.05, indicating that the average value was equal in the probability of less than 5%, but not in the probability of greater than 95%. It was believed that the probability of the average value being equal was relatively small, indicating that the difference was significant. The concentration of CTC and OTC in these groups was relatively high, and these results indicated that these groups had a significant inhibitory effect on methane production in anaerobic digestion treatment of real piggery wastewater under the influence of CTC and OTC.
The effects on COD removal efficiency by CTC and OTC The COD removal efficiency, inhibitory rate of combined antibiotics, inhibitory rate of CTC, inhibitory rate of OTC, sum of inhibitory rates of OTC and CTC were summarized in Table 2. It could be seen that with the increased number of experimental groups designed, the curve of the whole inhibition rates of COD removal efficiency were demonstrated as a fluctuating state. There was a turning point in each 5 groups of fluctuations, which was corresponded to the added concentration of the design, and the increase of each 5 points was close to that of the design. It could be seen Experiment 1, 2, 3, 4, 6, 7, 8, 9, 11, 12, 16 and 21, a total of 12 groups of the combined effect were less than the sum of the effects of two kinds of antibiotics when used alone in Table 2. It was demonstrated that two kinds of antibiotics inhibition of methane production volume were antagonism, through these groups can be seen in CTC or OTC in its lower concentration or both were lower cases could enhance the role. However, the inhibition rate of COD removal efficiency under the combined action was higher than that under the single action of CTC or OTC. The combined effects of 13 groups (5, 10, 13, 14, 15, 17, 18, 19, 20, 22, 23, 24 and 25) were greater than the sum of the two antibiotics
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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Table 1 e Effects of CTC, OTC and comparison of two antibiotics on methane production. Serial Methane number production (mL) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
374 348 319 295 271 359 335 303 278 254 340 315 281 256 236 318 298 260 241 220 299 279 238 222 204
Inhibitory rate of combined antibiotics (%) 18.34 24.02 30.35 35.59 40.83 21.61 26.86 33.84 39.30 44.54 25.76 31.22 38.65 44.10 48.47 30.56 34.93 43.23 47.38 51.97 34.72 39.08 48.03 51.53 55.46
Inhibitory Inhibitory Sum of inhibitory rate of CTC rate of OTC rates of OTC and (%) (%) CTC (%) 6.92 6.92 6.92 6.92 6.92 10.77 10.77 10.77 10.77 10.77 15.38 15.38 15.38 15.38 15.38 24.62 24.62 24.62 24.62 24.62 29.23 29.23 29.23 29.23 29.23
12.50 21.21 31.47 38.84 53.79 12.50 21.21 31.47 38.84 53.79 12.50 21.21 31.47 38.84 53.79 12.50 21.21 31.47 38.84 53.79 12.50 21.21 31.47 38.84 53.79
(Inhibitory rate of combined antibiotics)-(Sum of inhibitory rates of OTC and CTC) 1.08 4.11 8.04 10.17 19.88 1.66 5.12 8.40 10.31 20.02 2.12 5.37 8.20 10.12 20.70 6.56 10.9 12.86 16.08 26.44 7.01 11.36 12.67 16.54 27.56
19.42 28.13 38.39 45.76 60.71 23.27 31.98 42.24 49.61 64.56 27.88 36.59 46.85 54.22 69.17 37.12 45.83 56.09 63.46 78.41 41.73 50.44 60.7 68.07 83.02
Table 2 e Effects of CTC, OTC and comparison of two antibiotics on COD removal efficiency. Serial number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
COD removal efficiency (%) 79.74 78.37 76.52 76.05 70.03 78.65 77.76 75.98 70.23 68.45 77.89 76.73 72.90 69.41 65.03 77.14 74.13 70.85 66.00 63.19 76.80 72.83 69.48 63.60 60.38
Inhibitory rate of Inhibitory Inhibitory Sum of inhibitory combined rate of CTC rate of CTC rates of OTC and antibiotics (%) (%) (%) CTC (%) 4.16 5.56 7.60 8.11 14.54 5.33 6.28 8.18 14.31 16.22 6.14 7.38 11.47 15.19 19.87 6.94 10.15 13.66 18.85 21.84 7.30 11.54 15.12 21.40 24.84
3.78 3.78 3.78 3.78 3.78 5.76 5.76 5.76 5.76 5.76 5.85 5.85 5.85 5.85 5.85 6.89 6.89 6.89 6.89 6.89 7.94 7.94 7.94 7.94 7.94
1.65 2.94 5.46 8.70 10.27 1.65 2.94 5.46 8.7 10.27 1.65 2.94 5.46 8.70 10.27 1.65 2.94 5.46 8.70 10.27 1.65 2.94 5.46 8.70 10.27
5.43 6.72 9.24 12.48 14.05 7.41 8.70 11.22 14.46 16.03 7.50 8.79 11.31 14.55 16.12 8.54 9.83 12.35 15.59 17.16 9.59 10.88 13.40 16.64 18.21
(Inhibitory rate of combined antibiotics)-(Sum of inhibitory rates of OTC and CTC) 1.27 1.16 1.64 4.37 0.49 2.08 2.42 3.04 0.15 0.19 1.36 1.41 0.16 0.64 3.75 1.6 0.32 1.31 3.26 4.68 2.29 0.66 1.72 4.76 6.63
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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alone, indicating that the inhibitory effect of the two antibiotics on COD removal efficiency was enhanced. It could be seen from these groups that CTC or OTC had an enhanced effect when the concentration of CTC or OTC was relatively high or both are relatively high, antagonistic effect was usually observed when the increase was relatively small, and enhanced effect was usually observed when the increase was relatively large. The inhibitory rate of combined antibiotics on COD removal efficiency was obtained from Table 2, and the significance analysis of inhibitory rate of combined antibiotics on COD removal efficiency compared with control groups were analyzed in Table S3. It could be seen that the Sig. value of Comparision 4, 5, 9, 10, 13, 14, 15, 17, 18, 19, 20, 22, 23, 24 and 25 were less than 0.05. This means that the average probability of less than 5% was equal, while the probability of more than 95% was not equal. The results demonstrated that the difference was significant, so the average values between the two groups were not equal, which indicated that the 15 groups had significant inhibition effect on COD removal efficiency in anaerobic digestion system under the influence of CTC and OTC. However, these 15 groups were also generated at higher concentrations of either or both of CTC and OTC, and this was also consistent with the enhancement effect obtained in Table 2.
test results of cumulative methane production volume and COD removal efficiency of previous statistical analysis.
Phylogenetic analysis 24 bands were recovered and sequenced in the total of 31 bands. The sequences were collected in Table 5, and phylogenetic trees of bacterial based on the results of BLAST of sequences were shown in Fig. 1 (neighbor-joining method). There were 9 bands (1, 2, 11, 13, 14, 15, 18, 19 and 20) clustered to be uncultured bacterium. Band 3 was Thermotogae bacterium, it was a genus of bacteria subordinate to the phylum Thermotogae, and it was a kind of thermophilic or superthermophilic bacteria. This might demonstrate that the concentration of CTC and OTC were suitable for the growth of Thermotogae bacterium [13]. Band 4 and 24 were Bacteroidetes bacterium, and it was a kind of Bacteroides and a special anaerobic bacterium with chemical and organic nutrition [14]. It also appeared alone in lane 6, which function was similar to that of lane 3. Band 5 (Chloroflexi bacterium) was a genus of phylum Chloroflexi. It was a kind of facultative anaerobic bacteria, which had strong resistance to CTC and OTC [15], and it disappeared in lane 10 when OTC and CTC were of very high concentrations. Eubacterium (band 6) was also resistant to CTC and OTC, and it would be impacted only when the two antibiotics were of high concentrations [16]. Mesophilic bacterium (band 7) was often found in the medium temperature anaerobic treatment system, and it was inhibited only under the condition of high concentration of two antibiotics. Clostridium sp. (band 8) was a hydrogen-producing bacterium [15], which existed in lanes 1 to 8 and disappeared in the latter two lanes, indicating the inhibitory effect of this bacterium at high concentrations of CTC. Lactobacillus fermentumis (band 9) was obvious in lane 7, which was also resistant to CTC and OTC. There were few studies on Uncultured Magnetobacterium sp. (band 10), however, it was also a kind of bacteria with strong resistance of antibiotics. The resistance of Proteobacterium (band 12) was also obvious [17]. Dehalococcoides sp. (band 16) was a kind of dehalogenated bacteria, which was widely existed in the whole system, and it was related with the biodegradation of two antibiotics [18]. Caldilineaceae bacterium (band 17) was widely existed in activated sludge, and it was less influenced by CTC and OTC. Enterobacteriaceae bacterium (band 21) was mainly influenced by CTC, and it was existed in low concentration and disappeared in high concentration [19]. Weissella confuse (band 22) was often found in anaerobic digestion, and was less influenced by CTC and OTC. The role of Spirochaetaceae bacterium (band 23) was similar to Weissella confuse [20]. These species: Thermotogae bacterium, Bacteroidetes bacterium, Chloroflexi bacterium, Eubacterium, Mesophilic bacterium, Lactobacillus fermentumi, Magnetobacterium sp, Proteobacterium, Dehalococcoides sp., Caldilineaceae bacterium,
PCR-DGGE analysis Experiment 1, 5, 6, 10, 12, 14, 17, 20, 21 and 25 were selected to extract the total DNA of anaerobic granular activated sludge, and the fragment length was normal (about 25 kb). The PCR amplification products were stained by 1% agarose electrophoresis with EB solution, and the length of PCR products were about 250 bp. The PCR products of selected 10 groups were applied for DGGE analysis. DGGE profile was screened by using the Gel Doc 2000 System (Quantity One V4.52, Bio-Rad), and 31 distinct bands were counted from the stained denaturing gradient gel (Fig. S1). The richness value (Rs) of bacterial community and comparability index (Cs) of bacterial population in different groups were shown in Tables 3 and 4. It could be seen from Table 3 that with the increasing concentration of OTC and CTC, the bands obtained was decreasing (from 30 to 20), and the same rule of Rs value (from 0.968 to 0.645). The Cs value compared with each two samples in Table 4 demonstrated that similarity was decreasing (from 91.5 of sample 1 and 3 to 55.6 of sample 10 and 5) with the increasing concentration of OTC and CTC. These indicated that the difference of bacterial community richness and similarity were not affected by the low concentration of CTC and OTC, but it had significant effects when the concentration of CTC and OTC were both high or one kind of antibiotics was much more, and these were consistent with the significant
Table 3 e Richness value of bacterial community. Sample Bands Rs
1
2
3
4
5
6
7
8
9
10
30 0.968
27 0.871
29 0.935
26 0.839
25 0.806
26 0.839
28 0.903
28 0.903
29 0.935
20 0.645
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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Table 4 e Comparability index of bacterial community in different groups. Sample 1 2 3 4 5 6 7 8 9 10
1
2
3
4
5
6
7
8
9
10
100 87.7 91.5 89.3 90.9 85.7 79.3 75.9 78.0 80.0
87.7 100 85.7 90.6 80.8 86.8 87.3 83.7 81.4 72.3
91.5 85.7 100 87.3 88.9 87.3 77.2 80.7 72.4 73.5
89.3 90.6 87.3 100 82.3 88.5 88.9 88.9 88.9 69.6
90.9 80.8 88.9 82.3 100 82.3 90.6 84.3 88.9 55.6
85.7 86.8 87.3 88.5 82.3 100 85.7 92.6 80.0 69.6
79.3 87.3 77.2 88.9 90.6 85.7 100 85.7 79.3 75.0
75.9 83.7 80.7 88.9 84.3 92.6 85.7 100 77.2 75.0
78.0 81.4 72.4 88.9 88.9 80.0 79.3 77.2 100 73.5
80.0 72.3 73.5 69.6 55.6 69.6 75.0 75.0 73.5 100
Table 5 e Results of sequences using BLAST. Band
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Length/bp
185 180 197 186 172 168 171 171 265 171 172 179 191 169 189 185 189 150 183 161 196 253 196 179
Closet blast match Request ID
Accession NO.
Species name
Similariy%
95D1Y41U01R 95D9DFAN01R 95DD0XGV01R 95DMRJ9D01R 95DTXRMS01R 95DYJBH501R 95E5RG7U015 95E9XDF901R 95EGM20W01R 95EMT0K101R 95ETTW2C01R 95F03BX201R 95F6FU4K01R 95FJ3C2D01R 95FRBG8A01R 95FY3W3601R 95G1NANW01R 95G9MY0W01R 95GCXMPZ01R 95GGZNVE015 95GMX7W4014 95P8VCBZ01R 95PDEHBZ01R 95PG7CUZ01R
KF247577.1 HE966326.1| JX473505.1 HQ003601.1 CU924009.1 U81649.2 KF668160.1 KC215464.1 AM117157.1 JN806323.1 FJ645699.1 GQ242550.1 AJ249105.1 FJ645699.1 HE984826.1 GU556343.1 GU472730.1 KF171508.1 AB364749.1 KF232138.1 EU572076.1 AM117131.1 JF946844.1 CU926305.1
Uncultured bacterium Uncultured bacterium Thermotogae bacterium Bacteroidetes bacterium Chloroflexi bacterium Eubacterium Mesophilic bacterium Clostridium sp. Lactobacillus fermentum Uncultured Magnetobacterium sp. Uncultured bacterium Proteobacterium Uncultured bacterium Uncultured bacterium Uncultured bacterium Dehalococcoides sp. Caldilineaceae bacterium Uncultured bacterium Uncultured bacterium Uncultured bacterium Enterobacteriaceae bacterium Weissella confusa Spirochaetaceae bacterium Bacteroidetes bacterium
95% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 98% 99% 99% 99% 99% 95% 97% 99% 99% 99% 99% 99%
Enterobacteriaceae bacterium, Weissella confuse were proved to be beneficial for the COD removal and biodegradation of CTC and OTC [21e23].
Real-time PCR analysis The absolute quantitative standard curve results were shown in Fig. 2. In the PCR reaction process, the cyclic number was taken as the horizontal coordinate, and the real-time fluorescence intensity in the reaction process was taken as the vertical coordinate for the amplification curve to synthesize the gradient dilution sample amplification curve of bacterial target gene 16S rRNA (Fig. 2a). When the PCR product was heated, the double chain amplification products were gradually dissolved with the increase of temperature, which lead to the decrease of fluorescence intensity; when the products reached a certain temperature, a large number of products
would be dislinked and the fluorescence would dropped sharply; the PCR specificity could be identified to obtain the fusion curve, and the fusion curve of the target gene 16S rRNA was shown in Fig. 2b. Standard products were diluted to different concentrations and used as templates for PCR reactions. The standard curve was drawn by taking the logarithm value of copy number of standard substance as the abscissa, the measured CT value as the ordinate, and then the standard curve results of 16S rRNA of target gene were obtained (Fig. 2c). The slope of standard curve was 3.096, the amplification efficiency was E ¼ 101/slope-1 ¼ 101/ 3.096 e1 ¼ 110.40%, the correlation coefficient was R2 ¼ 0.99, and Y-inter was 32.43. The mean copies of 10 samples were shown in Table 6 according to Fig. 2. The copy number of sample 8 (CTC: 2 mg/L, OTC: 80 mg/L) was 1.75 107copies/mL, which was the maximum number among these 10 samples; however, the
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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Fig. 1 e Phylogenetic trees of bacterial based on the results of BLAST of sequences. copy number of sample 1 (CTC: 0.4 mg/L, OTC: 10 mg/L) was 5.61 106copies/mL, which was the minimum number among these 10 samples. The mean copy number of samples showed an increasing trend, which demonstrated that with the
increase of antibiotics, the dominant species were more and more obvious. This was opposite to the bacterial diversity, and which might be beneficial for the treatment of piggery wastewater to enhance the number of dominant bacteria [24e27].
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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Fig. 2 e (a) The amplification plot map (Cycle - DRn); (b) Map of melt curve (Temperature e Derivative reporter); (c) Standard curve of 16S rRNA (Quantity - Ct).
Table 6 e The mean copy number of samples. Sample
16S rRNA (Ct value)
1 2 3 4 5 6 7 8 9 10
11.54 10.69 10.63 10.72 10.72 10.34 10.76 10.01 10.68 10.28
16S rRNA (average copies number) 5.61 1.06 1.10 1.03 1.03 1.37 1.00 1.75 1.06 1.43
106 107 107 107 107 107 107 107 107 107
Conclusions The COD removal efficiency and methane production were inhibited with the increasing concentration of OTC and CTC. The antagonistic effect of CTC and OTC on methane production was observed; antagonism and enhancement were observed on inhibition rate of COD removal efficiency. The bacterial diversity and similarities of each group were
decreased with the increasing concentration of CTC and OTC. The identified species Thermotogae bacterium, Bacteroidetes bacterium, Chloroflexi bacterium, Eubacterium, Mesophilic bacterium, etc. were beneficial for the treatment of real piggery wastewater. The copy number of 16S rRNA increased with the increasing concentration of antibiotics, and was opposite to the law of bacterial diversity.
Acknowledgement The authors gratefully thank the financial support provided by Shandong Provincial Natural Science Foundation (ZR2019BB040), the National Natural Science Fund of China (NO. 31901188, NO. 31700433, and NO. 31672314) and Shandong Provincial Agricultural Fine Species Project (2019LZGC020).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijhydene.2020.01.138.
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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references
[1] Wang S, Ma X, Wang Y, Du G, Tay J-H, Li J. Piggery wastewater treatment by aerobic granular sludge: granulation process and antibiotics and antibiotic-resistant bacteria removal and transport. Bioresour Technol 2019;273:350e7. [2] Mao C, Feng Y, Wang X, Ren G. Review on research achievements of biogas from anaerobic digestion. Renew Sustain Energy Rev 2015;45:540e55. [3] Chen J, Hu Y, Huang W, Liu Y, Tang M, Zhang L, Sun J. Biodegradation of oxytetracycline and electricity generation in microbial fuel cell with in situ dual graphene modified bioelectrode. Bioresour Technol 2018;270:482e8. [4] Chen J, Yang Y, Liu Y, Tang M, Wang R, Tian Y, Jia C. Bacterial community shift and antibiotics resistant genes analysis in response to biodegradation of oxytetracycline in dual graphene modified bioelectrode microbial fuel cell. Bioresour Technol 2019;276:236e43. [5] Yan Z, Chen J, Liu Y, Shao J, Shu P, Wen S. Effects oxytetracycline on bacterial diversity in livestock wastewater. Environ Eng Sci 2017;34(4):265e71. [6] Ma Q, Qu Y, Shen W, Zhang Z, Wang J, Liu Z, Li D, Li H, Zhou J. Bacterial community compositions of coking wastewater treatment plants in steel industry revealed by Illumina highthroughput sequencing. Bioresour Technol 2015;179:436e43. [7] Jena J, Kumar R, Saifuddin M, Dixit A, Das T. Anoxiceaerobic SBR system for nitrate, phosphate and COD removal from high-strength wastewater and diversity study of microbial communities. Biochem Eng J 2016;105:80e9. [8] Chen J, Hu Y, Huang W, Zhang L. Enhanced electricity generation for biocathode microbial fuel cell by in situ microbial-induced reduction of graphene oxide and polarity reversion. Int J Hydrogen Energy 2017;42(17):12574e82. [9] Chen J, Yang Y, Liu Y, Tang M, Wang R, Zhang C, Jiang J, Jia C. Bacterial community shift in response to a deep municipal tail wastewater treatment system. Bioresour Technol 2019;281:195e201. [10] Chen J, Hu Y, Zhang L, Huang W, Sun J. Bacterial community shift and improved performance induced by in situ preparing dual graphene modified bioelectrode in microbial fuel cell. Bioresour Technol 2017;238:273e80. [11] Chen J, Liu S, Yan J, Wen J, Hu Y, Zhang W. Intensive removal efficiency and mechanisms of carbon and ammonium in municipal wastewater treatment plant tail water by ozone oyster shells fix-bed bioreactor membrane bioreactor combined system. Ecol Eng 2017;101:75e83. [12] Chen J, Zhang L, Hu Y, Huang W, Niu Z, Sun J. Bacterial community shift and incurred performance in response to in situ microbial self-assembly graphene and polarity reversion in microbial fuel cell. Bioresour Technol 2017;241:220e7. [13] Cheng DL, Ngo HH, Guo WS, Chang SW, Nguyen DD, Kumar SM, Du B, Wei Q, Wei D. Problematic effects of antibiotics on anaerobic treatment of swine wastewater. Bioresour Technol 2018;263:642e53.
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[14] Huang X, Liu C, Li K, Su J, Zhu G, Liu L. Performance of vertical up-flow constructed wetlands on swine wastewater containing tetracyclines and tet genes. Water Res 2015;70:109e17. [15] Li H, Li B, Zhang Z, Zhu C, Tian Y, Ye J. Evolution of microbial communities during electrokinetic treatment of antibioticpolluted soil. Ecotox Environ Safe 2018;148:842e50. [16] Xu J, Xu Y, Wang H, Guo C, Qiu H, He Y, Zhang Y, Li X, Meng W. Occurrence of antibiotics and antibiotic resistance genes in a sewage treatment plant and its effluent-receiving river. Chemosphere 2015;119:1379e85. [17] Liu S, Chen J, Yan J, Hu Y, Zhou D. Conversion mechanisms of carbon, nitrogen, and phosphorus in ozone-fixed-bed and membrane bioreactors for deep treatment of municipal tail water. Environ Eng Sci 2017;34(8):562e8. [18] Xiong W, Sun Y, Zhang T, Ding X, Li Y, Wang M, Zeng Z. Antibiotics, antibiotic resistance genes, and bacterial community composition in fresh water aquaculture environment in China. Microb Ecol 2015;70(2):425e32. [19] Qu F, Sun Z, Liu D, Zhao X, You J. Direct and indirect fluorescent detection of tetracyclines using dually emitting carbon dots. Microchim Acta 2016;183(9):2547e53. [20] Yin F, Dong H, Ji C, Tao X, Chen Y. Effects of anaerobic digestion on chlortetracycline and oxytetracycline degradation efficiency for swine manure. Waste Manag 2016;56:540e6. [21] Zhang G, Feng S, Jiao Y, Lee DJ, Xin Y, Sun H. Cathodic reducing bacteria of dual-chambered microbial fuel cell. Int J Hydrogen Energy 2017;42(45):27607e17. [22] Hasany M, Mardanpour MM, Yaghmaei S. Biocatalysts in microbial electrolysis cells: a review. Int J Hydrogen Energy 2016;41(3):1477e93. [23] Shen R, Liu Z, He Y, Zhang Y, Lu J, Zhu Z, Si B, Zhang C, Xing XH. Microbial electrolysis cell to treat hydrothermal liquefied wastewater from cornstalk and recover hydrogen: degradation of organic compounds and characterization of microbial community. Int J Hydrogen Energy 2016;41(7):4132e42. [24] Sun J, Xu W, Yang P, Li N, Yuan Y, Zhang H, Ning X, Zhang Y, Chang K, Peng Y, Chen K. Enhancing the performance of photo-bioelectrochemical fuel cell using graphene oxide/ cobalt/polypyrrole composite modified photo-biocathode in the presence of antibiotic. Int J Hydrogen Energy 2019;44(3):1919e29. [25] Yu Y, Ndayisenga F, Yu Z, Zhao M, Lay CH, Zhou D. Cosubstrate strategy for improved power production and chlorophenol degradation in a microbial fuel cell. Int J Hydrogen Energy 2019;44(36):20312e22. [26] Yang G, Yin Y, Wang J. Microbial community diversity during fermentative hydrogen production inoculating various pretreated cultures. Int J Hydrogen Energy 2019;44(26):13147e56. [27] Sharma P, Melkania U. Effect of bioaugmentation on hydrogen production from organic fraction of municipal solid waste. Int J Hydrogen Energy 2018;43(15):7290e8.
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
Available online at www.sciencedirect.com
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Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity Junfeng Chen, Yuewei Yang*, Yanyan Liu, Meizhen Tang, Renjun Wang, Hanwen Hu, Hongying Wang, Pingping Yang, Hanhan Xue, Xiao Zhang Department of Environmental Science, School of Life Sciences, Qufu Normal University, Qufu, 273165, PR China
highlights OTC
and
CTC
graphical abstract
inhibited
COD
removal efficiency and methane production. Antagonistic effect of CTC and OTC
acted
upon
methane
production. Antagonism
and
enhancement
acted on inhibition rate of COD removal efficiency. Bacterial diversity and similarities decreased with the increasing of CTC and OTC. Copy
number
of
16S
rRNA
increased with the increasing of CTC and OTC.
article info
abstract
Article history:
The treatment efficiency and bacterial diversity caused by chlortetracycline (CTC) and
Received 26 August 2019
oxytetracycline (OTC) in anaerobic digestion treatment of real piggery wastewater were
Received in revised form
studied by polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE)
12 January 2020
and real-time PCR. The COD removal efficiency and methane production were inhibited
Accepted 19 January 2020
with the increasing concentration of CTC and OTC. The antagonism of CTC and OTC on
Available online xxx
methane production was observed, the inhibition rate was expanded to 55.46% from 18.34%; antagonism and enhancement were observed on inhibition rate of COD removal
Keywords:
efficiency, and the inhibition rate was expanded to 24.84% from 4.16%. The bacterial di-
Microbiological effects
versity and similarities of each group were decreased with the increasing concentration of
PCR-DGGE
CTC and OTC. The identified species Thermotogae bacterium, Bacteroidetes bacterium, Chloro-
Anaerobic digestion treatment
flexi bacterium, Eubacterium, Mesophilic bacterium, etc. were beneficial for COD removal and
* Corresponding author. E-mail addresses: [email protected] (J. Chen), [email protected] (Y. Yang). https://doi.org/10.1016/j.ijhydene.2020.01.138 0360-3199/© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
2
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Real piggery wastewater
biodegradation of CTC and OTC. The quantitative fluorescence analysis was opposite to the
Oxytetracycline and
rule of bacterial diversity by real-time PCR.
chlortetracycline
© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction With the increasing consumption of pigs, the disorder discharge of piggery wastewater exceedingly influenced ecological environment and resident health. Piggery wastewater was originated from pig raising and excrement cleaning, which was featured by high concentration of organics (chemical oxygen demand, COD), ammonia nitrogen and total phosphorus (TP), therefore, piggery wastewater would lead to serious water pollution and eutrophication to a large extent [1]. Anaerobic digestion was a kind of basic and commonly used biological treatment for piggery wastewater, which was beneficial for wastewater pollution control and energy recovery, moreover, the process was also simple and easy to operate [2]. Oxytetracycline (OTC) and chlortetracycline (CTC) were key members of tetracyclines (TCs), which were important and commonly used broad spectrum antibiotics and have been diffusely used to prevent illness and advance the growth of pigs [3e5]. The massive abuse of OTC and CTC in pigs would lead to the accumulation of antibiotics in meat, which was harmful to human health. And also, the massive abuse of OTC and CTC in pigs would be discharged into the ecological environments in the form of the original drug, which would cause huge damages to the balances of ecological environments. Therefore, it is necessary to study on the treatment of antibiotics in piggery wastewater. Bacteria played a critical role in the biological treatment of wastewater. Ma et al. [6] studied the bacterial community compositions of coking wastewater treatment plants in steel industry, and demonstrated that Thiobacillus, Comamonas, Thauera, Azoarcus and Rhodoplanes were the dominant genera and played key role in treatment efficiency. Jena et al. [7] explored the anoxiceaerobic sequencing batch reactor system for 180 days, and obtained the removal efficiency of nitrate, phosphate and COD were 98%, 86%, and 72% respectively and analyzed that Proteobacteria, Alphaproteobacteria, Rhodobacterales, Rhodobacteraceae and Paracoccous were the prominent phylum, class, order, family and genus, respectively. These studies demonstrated that it was quite important to understand the bacterial species and diversity in grasping the mechanism of wastewater treatment. In our previous studies, we have discussed the effects of OTC on the anaerobic biological treatment of livestock wastewater [5], however, it was more complex and realistic in real wastewater. Therefore, it was meaningful and valuable to explore the effects on treatment efficiency and bacterial diversity by OTC and CTC in anaerobic digestion treatment of piggery wastewater. In this study, the COD removal efficiency, methane production, bacterial diversity and real-time PCR analysis were explored by OTC and CTC in anaerobic digestion treatment of
real piggery wastewater. The purposes of this study are to provide molecular biological basis and real piggery wastewater treatment results by CTC and OTC, which is of great significance for the removal of antibiotic contamination in real wastewater and the evaluation of antibiotic effects for the practical application.
Materials and methods Anaerobic digestion treatment of piggery wastewater There were 28 anaerobic digestion reactors used in this study (3 control groups and 25 experimental groups) and all reactors were settled under the temperature of 302 ± 2 K by a thermostat water bath and shaken regularly to ensure the sludge in suspension. There were three main parts: anaerobic digestion reactor, drainage method reactor and measuring cylinder. The anaerobic digestion reactor was 0.5 L, and the anaerobic granular activated sludge was 0.1 L, the real piggery wastewater was 0.4 L, and some certain concentration of OTC and CTC were added into the reactor (Table S1) according to previous studies [5]. The gas produced by anaerobic digestion was entered to 3% NaOH solution to absorb CO2, and the methane production was measured by drainage method (hydrogen production was very low and negligible). The pH of the anaerobic digestion treatment system was 7e8. The methane production was measured every day (across 24 h) until gas production ceased and COD concentration was measured every day (across 24 h), and the COD concentration was measured by the dichromate method (K2Cr2O7, CODCr) [5,8,9]. The anaerobic activated sludge was sampled at the end of all tests and stored at 253 K.
PCR-DGGE and quantitative fluorescence analysis The total DNA of anaerobic activated sludge was extracted by DNA kit. The bacterial universal 16S rRNA gene primer pairs 341F with a GC clamp and 517R were used in polymerase chain reaction (PCR) reaction mixture, and the PCR parameters were set as previous studies [4,10e12]. Denaturing gradient gel electrophoresis (DGGE) was operated by DCode™ Universal detection instrument (Bio-Rad), and the acrylamide concentration was 8%, and the denaturing gradient was 45%e65%, and the detailed parameters were set as previous study [5]. PCR-DGGE was used to illuminate bacterial diversity in anaerobic digestion treatment of real piggery wastewater by OTC and CTC. After DGGE, the distinct bands were excised under UV light, and DNA was extracted and purified with a quick gel extraction kit (Tian DZ, China). The purified DNA was re-amplified by primer pair 341F and517R, and the products
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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were ligated into the PMD18-T vector (Takara, Japan). The inserted fragments in the selected clones were sequenced by shotgun sequencing (Sangon, China). The quantitative fluorescence analysis (gene copy number) of 16S rRNA was determined by real-time PCR. Plasmid M13 ± was used to be sequenced and absolute quantitative plasmid number was XD2564-3. The real-time PCR was operated by Sybr Green qPCR Master Mix in fluorescence quantitative PCR instrument (ABI STEPONE PLUS) and cycling conditions were as previous study [4].
Data statistics and analysis The Sig. analysis of methane production and COD removal efficiency experiments performed by SPPSS 17.0 (Sig. (2-tailed) test). The community abundance of each sample was presented using the index Ai compared to all the analyzed samples in this study. This index was calculated with the following equation: Aij ¼ lj/Lt. The phylotype profiles of the samples from every two samples were compared using Sorenson’s index as follows: CSAB ¼ 2LAB/(LA þ LB) 100; a pairwise similarity coefficient as previous study [5]. The plasmid fragment length was 2861 bp, concentration was 0.80 ng/mL and 2.60 108 copies/mL. The standard curves of plasmids were constructed by 10-fold gradient dilution and prepared by 90 mL dilution solution and 10 mL plasmid, and the quantitative fluorescence analysis was calculated as follows: a Plasmid concentration conversion formula (copies/mL) ¼ (mol number/mL) 6.02 1023 ¼ [quality (g)/molecular weight]/mL 6.02 1023 ¼ [quality (ng) 109/molecular weight]/mL 6.02 1023 ¼ concentration (ng/ uL) 6.02 1014/molecular weight b molecular weight ¼ (Carrier segment base pairs þ PCR product base pairs) 650 c Molecular weight of a double stranded DNA molecule (Dalton) ¼ Number of base pairs 650 d The average molecular weight of a base pair ¼ 650 Da
Results and discussion The effects on methane production by OTC and CTC The COD, ammonia nitrogen, total nitrogen and total phosphorus concentration of raw piggery wastewater were approximately 22,000 mg/L, 2250 mg/L, 2500 mg/L and 355 mg/ L, respectively. In this study, the raw piggery wastewater was diluted about 20 times to keep wastewater quality stable. The methane production, inhibitory rate of combined antibiotics, inhibitory rate of CTC, inhibitory rate of OTC, sum of inhibitory rates of OTC and CTC were summarized in Table 1. It could be seen that with the increased number of experimental groups designed, the curve of the whole inhibition rates was demonstrated as a fluctuating state. There was a turning point in each 5 groups of fluctuations, which was corresponded to the added concentration of the design, and the increase of each 5 points was close to that of the design. With the increase of CTC and OTC concentration, the inhibition rate of the system on cumulative methane production
3
was also increased. With the increase of CTC and OTC concentration, the inhibition rates of the system on cumulative methane production were also increased. The results showed that the inhibition rate of OTC (Experiment 1, 2, 3, 4 and 5) was increased faster than that of CTC (Experiment 1, 6, 11, 16 and 21). The combined effects of CTC and OTC were less than the sum of the two antibiotics used alone, which indicated that the inhibition of CTC and OTC on the volume of cumulative methane production was antagonistic, however, the inhibition rate of combined action on the volume of cumulative methane production was higher than that of CTC or OTC alone. The cumulative methane production volume data obtained from Table 1, and the significance analysis of cumulative methane production volume compared with control groups were analyzed in Table S2. It could be seen that the Sig. value of Experiment 1, 2, 6 and 11 was more than 0.05 from Table S2. It was indicated that the average value was equal in the probability of greater than 5%, but not equal in the probability of less than 95%. It was believed that the probability of equal mean was relatively large, indicating that the difference between the Experiment 1, 2, 6 and 11, and the blank control group was not significant, and no significant effects were achieved compared with the blank control group. The reasons for this were the relatively low concentrations of CTC and OTC. The Sig. value of the other 21 groups was less than 0.05, indicating that the average value was equal in the probability of less than 5%, but not in the probability of greater than 95%. It was believed that the probability of the average value being equal was relatively small, indicating that the difference was significant. The concentration of CTC and OTC in these groups was relatively high, and these results indicated that these groups had a significant inhibitory effect on methane production in anaerobic digestion treatment of real piggery wastewater under the influence of CTC and OTC.
The effects on COD removal efficiency by CTC and OTC The COD removal efficiency, inhibitory rate of combined antibiotics, inhibitory rate of CTC, inhibitory rate of OTC, sum of inhibitory rates of OTC and CTC were summarized in Table 2. It could be seen that with the increased number of experimental groups designed, the curve of the whole inhibition rates of COD removal efficiency were demonstrated as a fluctuating state. There was a turning point in each 5 groups of fluctuations, which was corresponded to the added concentration of the design, and the increase of each 5 points was close to that of the design. It could be seen Experiment 1, 2, 3, 4, 6, 7, 8, 9, 11, 12, 16 and 21, a total of 12 groups of the combined effect were less than the sum of the effects of two kinds of antibiotics when used alone in Table 2. It was demonstrated that two kinds of antibiotics inhibition of methane production volume were antagonism, through these groups can be seen in CTC or OTC in its lower concentration or both were lower cases could enhance the role. However, the inhibition rate of COD removal efficiency under the combined action was higher than that under the single action of CTC or OTC. The combined effects of 13 groups (5, 10, 13, 14, 15, 17, 18, 19, 20, 22, 23, 24 and 25) were greater than the sum of the two antibiotics
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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Table 1 e Effects of CTC, OTC and comparison of two antibiotics on methane production. Serial Methane number production (mL) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
374 348 319 295 271 359 335 303 278 254 340 315 281 256 236 318 298 260 241 220 299 279 238 222 204
Inhibitory rate of combined antibiotics (%) 18.34 24.02 30.35 35.59 40.83 21.61 26.86 33.84 39.30 44.54 25.76 31.22 38.65 44.10 48.47 30.56 34.93 43.23 47.38 51.97 34.72 39.08 48.03 51.53 55.46
Inhibitory Inhibitory Sum of inhibitory rate of CTC rate of OTC rates of OTC and (%) (%) CTC (%) 6.92 6.92 6.92 6.92 6.92 10.77 10.77 10.77 10.77 10.77 15.38 15.38 15.38 15.38 15.38 24.62 24.62 24.62 24.62 24.62 29.23 29.23 29.23 29.23 29.23
12.50 21.21 31.47 38.84 53.79 12.50 21.21 31.47 38.84 53.79 12.50 21.21 31.47 38.84 53.79 12.50 21.21 31.47 38.84 53.79 12.50 21.21 31.47 38.84 53.79
(Inhibitory rate of combined antibiotics)-(Sum of inhibitory rates of OTC and CTC) 1.08 4.11 8.04 10.17 19.88 1.66 5.12 8.40 10.31 20.02 2.12 5.37 8.20 10.12 20.70 6.56 10.9 12.86 16.08 26.44 7.01 11.36 12.67 16.54 27.56
19.42 28.13 38.39 45.76 60.71 23.27 31.98 42.24 49.61 64.56 27.88 36.59 46.85 54.22 69.17 37.12 45.83 56.09 63.46 78.41 41.73 50.44 60.7 68.07 83.02
Table 2 e Effects of CTC, OTC and comparison of two antibiotics on COD removal efficiency. Serial number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
COD removal efficiency (%) 79.74 78.37 76.52 76.05 70.03 78.65 77.76 75.98 70.23 68.45 77.89 76.73 72.90 69.41 65.03 77.14 74.13 70.85 66.00 63.19 76.80 72.83 69.48 63.60 60.38
Inhibitory rate of Inhibitory Inhibitory Sum of inhibitory combined rate of CTC rate of CTC rates of OTC and antibiotics (%) (%) (%) CTC (%) 4.16 5.56 7.60 8.11 14.54 5.33 6.28 8.18 14.31 16.22 6.14 7.38 11.47 15.19 19.87 6.94 10.15 13.66 18.85 21.84 7.30 11.54 15.12 21.40 24.84
3.78 3.78 3.78 3.78 3.78 5.76 5.76 5.76 5.76 5.76 5.85 5.85 5.85 5.85 5.85 6.89 6.89 6.89 6.89 6.89 7.94 7.94 7.94 7.94 7.94
1.65 2.94 5.46 8.70 10.27 1.65 2.94 5.46 8.7 10.27 1.65 2.94 5.46 8.70 10.27 1.65 2.94 5.46 8.70 10.27 1.65 2.94 5.46 8.70 10.27
5.43 6.72 9.24 12.48 14.05 7.41 8.70 11.22 14.46 16.03 7.50 8.79 11.31 14.55 16.12 8.54 9.83 12.35 15.59 17.16 9.59 10.88 13.40 16.64 18.21
(Inhibitory rate of combined antibiotics)-(Sum of inhibitory rates of OTC and CTC) 1.27 1.16 1.64 4.37 0.49 2.08 2.42 3.04 0.15 0.19 1.36 1.41 0.16 0.64 3.75 1.6 0.32 1.31 3.26 4.68 2.29 0.66 1.72 4.76 6.63
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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alone, indicating that the inhibitory effect of the two antibiotics on COD removal efficiency was enhanced. It could be seen from these groups that CTC or OTC had an enhanced effect when the concentration of CTC or OTC was relatively high or both are relatively high, antagonistic effect was usually observed when the increase was relatively small, and enhanced effect was usually observed when the increase was relatively large. The inhibitory rate of combined antibiotics on COD removal efficiency was obtained from Table 2, and the significance analysis of inhibitory rate of combined antibiotics on COD removal efficiency compared with control groups were analyzed in Table S3. It could be seen that the Sig. value of Comparision 4, 5, 9, 10, 13, 14, 15, 17, 18, 19, 20, 22, 23, 24 and 25 were less than 0.05. This means that the average probability of less than 5% was equal, while the probability of more than 95% was not equal. The results demonstrated that the difference was significant, so the average values between the two groups were not equal, which indicated that the 15 groups had significant inhibition effect on COD removal efficiency in anaerobic digestion system under the influence of CTC and OTC. However, these 15 groups were also generated at higher concentrations of either or both of CTC and OTC, and this was also consistent with the enhancement effect obtained in Table 2.
test results of cumulative methane production volume and COD removal efficiency of previous statistical analysis.
Phylogenetic analysis 24 bands were recovered and sequenced in the total of 31 bands. The sequences were collected in Table 5, and phylogenetic trees of bacterial based on the results of BLAST of sequences were shown in Fig. 1 (neighbor-joining method). There were 9 bands (1, 2, 11, 13, 14, 15, 18, 19 and 20) clustered to be uncultured bacterium. Band 3 was Thermotogae bacterium, it was a genus of bacteria subordinate to the phylum Thermotogae, and it was a kind of thermophilic or superthermophilic bacteria. This might demonstrate that the concentration of CTC and OTC were suitable for the growth of Thermotogae bacterium [13]. Band 4 and 24 were Bacteroidetes bacterium, and it was a kind of Bacteroides and a special anaerobic bacterium with chemical and organic nutrition [14]. It also appeared alone in lane 6, which function was similar to that of lane 3. Band 5 (Chloroflexi bacterium) was a genus of phylum Chloroflexi. It was a kind of facultative anaerobic bacteria, which had strong resistance to CTC and OTC [15], and it disappeared in lane 10 when OTC and CTC were of very high concentrations. Eubacterium (band 6) was also resistant to CTC and OTC, and it would be impacted only when the two antibiotics were of high concentrations [16]. Mesophilic bacterium (band 7) was often found in the medium temperature anaerobic treatment system, and it was inhibited only under the condition of high concentration of two antibiotics. Clostridium sp. (band 8) was a hydrogen-producing bacterium [15], which existed in lanes 1 to 8 and disappeared in the latter two lanes, indicating the inhibitory effect of this bacterium at high concentrations of CTC. Lactobacillus fermentumis (band 9) was obvious in lane 7, which was also resistant to CTC and OTC. There were few studies on Uncultured Magnetobacterium sp. (band 10), however, it was also a kind of bacteria with strong resistance of antibiotics. The resistance of Proteobacterium (band 12) was also obvious [17]. Dehalococcoides sp. (band 16) was a kind of dehalogenated bacteria, which was widely existed in the whole system, and it was related with the biodegradation of two antibiotics [18]. Caldilineaceae bacterium (band 17) was widely existed in activated sludge, and it was less influenced by CTC and OTC. Enterobacteriaceae bacterium (band 21) was mainly influenced by CTC, and it was existed in low concentration and disappeared in high concentration [19]. Weissella confuse (band 22) was often found in anaerobic digestion, and was less influenced by CTC and OTC. The role of Spirochaetaceae bacterium (band 23) was similar to Weissella confuse [20]. These species: Thermotogae bacterium, Bacteroidetes bacterium, Chloroflexi bacterium, Eubacterium, Mesophilic bacterium, Lactobacillus fermentumi, Magnetobacterium sp, Proteobacterium, Dehalococcoides sp., Caldilineaceae bacterium,
PCR-DGGE analysis Experiment 1, 5, 6, 10, 12, 14, 17, 20, 21 and 25 were selected to extract the total DNA of anaerobic granular activated sludge, and the fragment length was normal (about 25 kb). The PCR amplification products were stained by 1% agarose electrophoresis with EB solution, and the length of PCR products were about 250 bp. The PCR products of selected 10 groups were applied for DGGE analysis. DGGE profile was screened by using the Gel Doc 2000 System (Quantity One V4.52, Bio-Rad), and 31 distinct bands were counted from the stained denaturing gradient gel (Fig. S1). The richness value (Rs) of bacterial community and comparability index (Cs) of bacterial population in different groups were shown in Tables 3 and 4. It could be seen from Table 3 that with the increasing concentration of OTC and CTC, the bands obtained was decreasing (from 30 to 20), and the same rule of Rs value (from 0.968 to 0.645). The Cs value compared with each two samples in Table 4 demonstrated that similarity was decreasing (from 91.5 of sample 1 and 3 to 55.6 of sample 10 and 5) with the increasing concentration of OTC and CTC. These indicated that the difference of bacterial community richness and similarity were not affected by the low concentration of CTC and OTC, but it had significant effects when the concentration of CTC and OTC were both high or one kind of antibiotics was much more, and these were consistent with the significant
Table 3 e Richness value of bacterial community. Sample Bands Rs
1
2
3
4
5
6
7
8
9
10
30 0.968
27 0.871
29 0.935
26 0.839
25 0.806
26 0.839
28 0.903
28 0.903
29 0.935
20 0.645
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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Table 4 e Comparability index of bacterial community in different groups. Sample 1 2 3 4 5 6 7 8 9 10
1
2
3
4
5
6
7
8
9
10
100 87.7 91.5 89.3 90.9 85.7 79.3 75.9 78.0 80.0
87.7 100 85.7 90.6 80.8 86.8 87.3 83.7 81.4 72.3
91.5 85.7 100 87.3 88.9 87.3 77.2 80.7 72.4 73.5
89.3 90.6 87.3 100 82.3 88.5 88.9 88.9 88.9 69.6
90.9 80.8 88.9 82.3 100 82.3 90.6 84.3 88.9 55.6
85.7 86.8 87.3 88.5 82.3 100 85.7 92.6 80.0 69.6
79.3 87.3 77.2 88.9 90.6 85.7 100 85.7 79.3 75.0
75.9 83.7 80.7 88.9 84.3 92.6 85.7 100 77.2 75.0
78.0 81.4 72.4 88.9 88.9 80.0 79.3 77.2 100 73.5
80.0 72.3 73.5 69.6 55.6 69.6 75.0 75.0 73.5 100
Table 5 e Results of sequences using BLAST. Band
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Length/bp
185 180 197 186 172 168 171 171 265 171 172 179 191 169 189 185 189 150 183 161 196 253 196 179
Closet blast match Request ID
Accession NO.
Species name
Similariy%
95D1Y41U01R 95D9DFAN01R 95DD0XGV01R 95DMRJ9D01R 95DTXRMS01R 95DYJBH501R 95E5RG7U015 95E9XDF901R 95EGM20W01R 95EMT0K101R 95ETTW2C01R 95F03BX201R 95F6FU4K01R 95FJ3C2D01R 95FRBG8A01R 95FY3W3601R 95G1NANW01R 95G9MY0W01R 95GCXMPZ01R 95GGZNVE015 95GMX7W4014 95P8VCBZ01R 95PDEHBZ01R 95PG7CUZ01R
KF247577.1 HE966326.1| JX473505.1 HQ003601.1 CU924009.1 U81649.2 KF668160.1 KC215464.1 AM117157.1 JN806323.1 FJ645699.1 GQ242550.1 AJ249105.1 FJ645699.1 HE984826.1 GU556343.1 GU472730.1 KF171508.1 AB364749.1 KF232138.1 EU572076.1 AM117131.1 JF946844.1 CU926305.1
Uncultured bacterium Uncultured bacterium Thermotogae bacterium Bacteroidetes bacterium Chloroflexi bacterium Eubacterium Mesophilic bacterium Clostridium sp. Lactobacillus fermentum Uncultured Magnetobacterium sp. Uncultured bacterium Proteobacterium Uncultured bacterium Uncultured bacterium Uncultured bacterium Dehalococcoides sp. Caldilineaceae bacterium Uncultured bacterium Uncultured bacterium Uncultured bacterium Enterobacteriaceae bacterium Weissella confusa Spirochaetaceae bacterium Bacteroidetes bacterium
95% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 98% 99% 99% 99% 99% 95% 97% 99% 99% 99% 99% 99%
Enterobacteriaceae bacterium, Weissella confuse were proved to be beneficial for the COD removal and biodegradation of CTC and OTC [21e23].
Real-time PCR analysis The absolute quantitative standard curve results were shown in Fig. 2. In the PCR reaction process, the cyclic number was taken as the horizontal coordinate, and the real-time fluorescence intensity in the reaction process was taken as the vertical coordinate for the amplification curve to synthesize the gradient dilution sample amplification curve of bacterial target gene 16S rRNA (Fig. 2a). When the PCR product was heated, the double chain amplification products were gradually dissolved with the increase of temperature, which lead to the decrease of fluorescence intensity; when the products reached a certain temperature, a large number of products
would be dislinked and the fluorescence would dropped sharply; the PCR specificity could be identified to obtain the fusion curve, and the fusion curve of the target gene 16S rRNA was shown in Fig. 2b. Standard products were diluted to different concentrations and used as templates for PCR reactions. The standard curve was drawn by taking the logarithm value of copy number of standard substance as the abscissa, the measured CT value as the ordinate, and then the standard curve results of 16S rRNA of target gene were obtained (Fig. 2c). The slope of standard curve was 3.096, the amplification efficiency was E ¼ 101/slope-1 ¼ 101/ 3.096 e1 ¼ 110.40%, the correlation coefficient was R2 ¼ 0.99, and Y-inter was 32.43. The mean copies of 10 samples were shown in Table 6 according to Fig. 2. The copy number of sample 8 (CTC: 2 mg/L, OTC: 80 mg/L) was 1.75 107copies/mL, which was the maximum number among these 10 samples; however, the
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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Fig. 1 e Phylogenetic trees of bacterial based on the results of BLAST of sequences. copy number of sample 1 (CTC: 0.4 mg/L, OTC: 10 mg/L) was 5.61 106copies/mL, which was the minimum number among these 10 samples. The mean copy number of samples showed an increasing trend, which demonstrated that with the
increase of antibiotics, the dominant species were more and more obvious. This was opposite to the bacterial diversity, and which might be beneficial for the treatment of piggery wastewater to enhance the number of dominant bacteria [24e27].
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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Fig. 2 e (a) The amplification plot map (Cycle - DRn); (b) Map of melt curve (Temperature e Derivative reporter); (c) Standard curve of 16S rRNA (Quantity - Ct).
Table 6 e The mean copy number of samples. Sample
16S rRNA (Ct value)
1 2 3 4 5 6 7 8 9 10
11.54 10.69 10.63 10.72 10.72 10.34 10.76 10.01 10.68 10.28
16S rRNA (average copies number) 5.61 1.06 1.10 1.03 1.03 1.37 1.00 1.75 1.06 1.43
106 107 107 107 107 107 107 107 107 107
Conclusions The COD removal efficiency and methane production were inhibited with the increasing concentration of OTC and CTC. The antagonistic effect of CTC and OTC on methane production was observed; antagonism and enhancement were observed on inhibition rate of COD removal efficiency. The bacterial diversity and similarities of each group were
decreased with the increasing concentration of CTC and OTC. The identified species Thermotogae bacterium, Bacteroidetes bacterium, Chloroflexi bacterium, Eubacterium, Mesophilic bacterium, etc. were beneficial for the treatment of real piggery wastewater. The copy number of 16S rRNA increased with the increasing concentration of antibiotics, and was opposite to the law of bacterial diversity.
Acknowledgement The authors gratefully thank the financial support provided by Shandong Provincial Natural Science Foundation (ZR2019BB040), the National Natural Science Fund of China (NO. 31901188, NO. 31700433, and NO. 31672314) and Shandong Provincial Agricultural Fine Species Project (2019LZGC020).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijhydene.2020.01.138.
Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138
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Please cite this article as: Chen J et al., Effects caused by chlortetracycline and oxytetracycline in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity, International Journal of Hydrogen Energy, https://doi.org/10.1016/ j.ijhydene.2020.01.138