performance and microbial community profiles in an anaerobic reactor treating with simulated pta...
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Performance and microbial community profiles in an anaerobic reactor treating withsimulated PTA wastewater: From mesophilic to thermophilic temperature
Xiang-kun Li , Kai-li Ma , Ling-wei Meng , Jie Zhang , Ke Wang
PII: S0043-1354(14)00318-2
DOI: 10.1016/j.watres.2014.04.033
Reference: WR 10635
To appear in: Water Research
Received Date: 18 November 2013
Revised Date: 15 April 2014
Accepted Date: 18 April 2014
Please cite this article as: Li, X.-k., Ma, K.-l., Meng, L.-w., Zhang, J., Wang, K., Performance andmicrobial community profiles in an anaerobic reactor treating with simulated PTA wastewater: Frommesophilic to thermophilic temperature, Water Research (2014), doi: 10.1016/j.watres.2014.04.033.
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Performance and microbial community profiles in an anaerobic reactor treating 1
with simulated PTA wastewater: From mesophilic to thermophilic temperature 2
Xiang-kun Li*, Kai-li Ma, Ling-wei Meng, Jie Zhang, Ke Wang* 3
School of Municipal and Environmental Engineering, State Key Laboratory of Urban 4
Water Resource and Environment, Harbin Institute of Technology, 73 Huanghe Road, 5
Harbin 150090, China. 6
*Corresponding author: Tel.:+86 13836032688. Fax: +86451 86282762. 7
E-mail address: [email protected] (Xiang-kun Li), [email protected] (Wang Ke). 8
ABSTRACT 9
Performance and microbial community profiles in a hybrid anaerobic reactor treating 10
synthetic PTA wastewater (contained the major pollutants terephthalate and benzoate) 11
were studied over 220 days from 33°C to 52°C. Results indicated that PTA treatment 12
process was highly sensitive to temperature variations in terms of COD removal. 13
Operation at 37°C showed the best performance as well as the most diverse microbial 14
community revealed by 16S rRNA gene clone library and T-RFLP (terminal 15
restriction fragment length polymorphism). Finally, the anaerobic process achieved a 16
total COD removal of 77.4%, 91.9 %, 87.4% and 66.1% at 33, 37, 43 and 52°C. 17
While the corresponding TA removal were 77.6%, 94.0%, 89.1% and 60.8%, 18
respectively. Sequence analyses revealed acetoclastic Methanosaeta was 19
preponderant at 37°C, while hydrogenotrophic genera including Methanobrevibacter 20
and Methanofollis were more abundant at other temperatures. For bacterial 21
community, 16 classes were identified. The largely existent Syntrophorhabdus 22
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members (belonging to δ-Proteobacteria) at 37°C was likely to play an important role 23
in mesophilic anaerobic wastewater treatment system contained terephthalate. 24
Meanwhile, β-Proteobacteria seemed to be favored in an anaerobic system higher 25
than 43°C. 26
Keywords: 27
PTA wastewater 28
Temperature influence 29
Microbial community 30
Phylogenetic analysis 31
T-RFLP 32
1. Introduction 33
Purified terephthalic acid (1,4-benzenedicarboxylic acid), or PTA, is an important raw 34
material widely used in petrochemical industry to make various synthetic products 35
including polyethylene terephthalate bottles, polyester textile fiber, polyester films, 36
pesticide, etc. (Joung et al. 2009). During PTA manufacturing processes, high strength 37
PTA-contained wastewater is generated, with a ratio of 3~10 m3 wastewater per ton 38
PTA manufactured. Aromatic pollutants including terephthalate (TA), 39
4-carboxybenzaldehyde (4-CBA), benzoic acid (BA), and p-toluic acid are found in 40
PTA wastewater (Kleerebezem et al. 1999, Macarie et al. 1992), which can invoke 41
serious environmental problems including various toxicity (Daramola et al. 2011). 42
Generally, TA is identified as the major component in the wastewater (accounts for 43
over 25% of the equivalent COD, Cheng et al. (1997)) . Considering its low 44
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biodegradability, the research of PTA wastewater anaerobic treatment is mainly 45
focused on the degradation of TA. 46
During producing process, PTA wastewater has a wide temperature range, usually 47
higher than 45°C, sometimes can be as high as 90°C (Lei et al. 2003), which is 48
favorable to both mesophilic and thermophilic anaerobic systems. Over recent years, 49
PTA wastewater is generally treated by anaerobic biological processes under 50
mesophilic conditions, achieving a COD removal ranging from 45% to 92% (Joung et 51
al. 2009, Patel et al. 2005, Piterina et al. 2012, Zhu et al. 2010). Chen et al. (2004) 52
testified the feasibility of thermophilic anaerobic treatment of PTA wastewater and 53
got a favorable result. Lykidis et al. (2010) uses a hyper-mesophilic temperature (46–54
50°C) operated reactor to further analyze syntrophic interaction pathway in 55
TA-degradation. For anaerobic treatment, it is of great necessity to choose an 56
appropriate treatment temperature based on the characteristic of processing water, 57
which may not only reduce the energy consumption, but also save operation cost. 58
In previous work, δ-Proteobacteria were identified as the microorganism 59
responsible for TA degradation under mesophilic condition (Qiu et al. 2004, Wu et al. 60
2001), while Desulfotomaculum related group has been assumed to be dominant 61
under thermophilic condition (Chen et al. 2004). Additionally, the syntrophic 62
methanogenic counterparts have also been characterized in previous work (Lykidis et 63
al. 2010). 64
Nevertheless, the influence of temperature variation from mesophilic to 65
thermophilic conditions on performance of PTA wastewater anaerobic treatment 66
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remains unclear. Furthermore, characteristics of microbial community structure with 67
stepwise increased temperature are rarely investigated, especially changes in 68
diversity and abundance. For these reasons, a lab-scale UAFB (up-flow anaerobic 69
fixed bed) reactor was operated to treat TA containing wastewater. The goal of this 70
study is to investigate the long term performance of anaerobic reactor on treating 71
synthetic PTA wastewater under the conditions of increasing temperature step by step 72
from 33°C to 55°C. Furthermore, 16S rRNA gene based methods including clone 73
library and T-RFLP were used to evaluate microbial succession and to figure out the 74
dominant microbes associated to TA degradation at different operating temperatures. 75
2. Materials and methods 76
2.1. Anaerobic TA-degrading reactor and synthetic PTA wastewater 77
A laboratory-scale UAFB reactor was used to enrich anaerobic microbial consortia 78
degrading TA, with a working volume of 700mL and 200mL padding. To solve the 79
blockage problem of pumping tubes at low liquid velocity, a recycle system was also 80
set up. Both the influent and recycle process were controlled by peristaltic pumps. 81
Temperature inside the reactor was maintained with a circulated acrylic plastic tube, 82
cycling by continuous heated water from a thermostat water bath. The reactor used in 83
this study is shown in Fig 1. 84
Fig 1 85
Seeding sludge was obtained from a full scale Cyclic Activated Sludge System 86
(CASS) process treating municipal wastewater. The reactor was initially fed with a 87
synthetic glucose wastewater at room temperature. Operation temperature was raised 88
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at a rate of 2°C/d until it elevated to 33°C. After the domestication of glucose 89
wastewater, a synthetic PTA wastewater containing TA 600 mg/L, benzoate 200 mg/L, 90
acetic acid (HAC) 450 mg/L and ethyl acetate 400 mg/L were fed with a hydraulic 91
retention time (HRT) of 32 h, NH4Cl and KH2PO4 were added following the ratio of C: 92
N: P = 350 : 5: 1. 1µL trace metal solution contained FeSO4·7H2O 5000 mg/L, 93
EDTA·Na2 5000 mg/L, CuSO4·5H2O 100 mg/L, MnCl2·4H2O 500 mg/L, 94
CoCl2·6H2O 202 mg/L, H3BO4 1000 mg/L, ZnCl2 1050 mg/L, KI 80 mg/L was added 95
for 1L synthetic PTA wastewater. After 30 days of continuous operation, the synthetic 96
feed was replaced with TA (1000 mg/L) and BA (200 mg/L) as the carbon source, in 97
order to further increase the TA removal capacity and enrich the microbial consortia 98
degrading TA. Throughout the study, the hydraulic retention time (HRT) and 99
volumetric organic loading rate (VLR) were changed several times (see Table. 1) to 100
achieve an optimum operational state. 101
Duplicate sludge samples collected at days 72, 105, 172 and 216 were stored at 102
-80°C, until further microbial community analysis. 103
Table 1 104
2.2. Chemical and solid content analysis 105
Reactor performance was evaluated by monitoring the removal efficiency of TA and 106
COD. The concentration of TA was determined by ultraviolet spectrophotometry 107
(Bing and Liqiang 2007, Yang 2002), while COD was measured according to 108
standard methods(SEPA 2002). Parameters such as pH, dissolved oxygen (DO) and 109
alkalinity were monitored as well. 110
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2.3. DNA extraction and PCR amplification 111
Genomic DNA were extracted according to a protocol reported previously (Zeng et al. 112
2008). The concentration of DNA was determined by Nano Drop® 113
Spectrophotometer ND-1000 (Thermo Fisher Scientific, USA). 114
PCR amplification was performed for further clone sequencing. The 50 µL reaction 115
volume contained 1×EasyTaq Buffer, 200 µM dNTPs, 2 mM MgCl2, 0.2 µM of each 116
primer and 2.5~5 U EasyTaq DNA polymerase. Primer sets used in this study are27F 117
(5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’- 118
GGTTACCTTGTTACGACTT-3’) for bacteria (Weisburg et al. 1991), 25F 119
(5’-CYGGTTGATCCTGCCRG-3’) and 958R 120
(5’-YCCGGCGTTGAMTCCAATT-3’) for archaea (El-Mashad et al. 2004). The 121
DNA amplification of both bacteria and archaea were started with an initial 122
denaturation for 10 min at 95°C, followed by 31 cycles of denaturation (94°C, 30 s), 123
annealing (56°C, 30 s) and extension (72°C, 60 s), ended with a final extension step of 124
10 min at 72°C. The PCR products were further examined by electrophoresis on 0.8% 125
agarose gels, and purified by Axygen PCR cleanup purification kit (Axygen 126
Biotechnology Ltd. Hangzhou, China). 127
2.4. 16S rRNA gene clone libraries construction and phylogenetic analysis 128
Detailed descriptions for cloning are shown elsewhere (Zhao et al. 2012). Selected 129
clones containing the PCR product inserts were sequenced at Sangon sequencing 130
company with primer M13F (5’-CGCCAGGGTTTTCCCAGTCACGAC-3’). 131
The returned 16S rRNA gene sequences were initially compared with sequences in 132
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public databases at http://blast.ncbi.nlm.nih.gov/Blast.cgi. Phylogenetic analysis of 133
retrieved 16S rRNA sequences was then conducted by a neighbor-joining tree with the 134
Kimura 2-parameter model method using MEGA 5.0. Bootstrap value was calculated 135
based on 1000 replications. Subsequent editing work was completed in Micro office 136
Word 2007. The sequences reported in this study were deposited in the GenBank 137
under accession number KJ476541-KJ476628. 138
2.5. T-RFLP analysis 139
PCR amplification for T-RFLP was the same as that described at part 2.3, except that 140
the forward primer was labeled with 6-FAM. The 30 µL reaction system contained 17 141
µL purified PCR products, 2 µL 10×buffer and 1 µL restriction enzyme MspI (10 142
U/µL). The digestion mixture was incubated at 37°C overnight and heat terminated at 143
65°C for 20 min before further ABI gene scan. 144
Initial data analysis with Gene Marker V1.6 was completed by Sangon company 145
(Shanghai, China). T-RFLP profiles alignment were then implemented by hierarchical 146
clustering method described by Abdo et al. (2006). Fragments smaller than 50 bp or 147
larger than 600 bp were excluded from analysis. T-RFs with abundance less than 1% 148
of total intensity were also neglected in this study (Horz et al. 2000, Jin et al. 2011, 149
Zhao et al. 2012). The program package used for T-RFLP data analysis is available at 150
http://www.ufz.de/index.php?en=22174. 151
To further contact the T-RFLP analysis with phylogenetic assignments, dominant 152
clone sequences obtained in clone libraries were analyzed by finding the first MspI 153
enzymatic digestion site downstream from 27F with DNAman (Li et al. 2011, Zhao et 154
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al. 2012). All electropherograms of Msp I-digested T-RFLP fingerprints were drawn 155
by software origin 8.1 (http://www.3987.com/xiazai/3/98/17622.html). 156
3. Results 157
3.1. General performance under different temperatures 158
Long term results (210 days) are shown in Figure 2. In the earlier period (from 159
start-up to days 30), four carbon sources: TA, BA, HAC and ethyl acetate were fed 160
into the reactor to enrich the microbial diversity. In this period, the reactor showed a 161
general removal of 42.8% for TA and 64.0% for COD. The low TA removal efficiency 162
was believed to be mainly related with biological inhibition, which will be discussed 163
later. 164
Figure 2 165
Since days 31, HAC and ethyl acetate were removed from the feed, leaving TA 166
(1000 mg/L) and BA (200 mg/L) as carbon source. 40 days later, total removal of TA 167
and COD reached to 78.4% and 78.8%, respectively. At days 72, the temperature was 168
raised to 37°C, and the effluent COD concentration showed an obvious drop three 169
days later. After a month, the concentration of COD in effluent was stabilized around 170
100 mg/L, and the total COD removal efficiency was maintained at 91.9%. 171
Meanwhile, a slightly higher TA removal (94.0%) was observed. 172
According to Razo-Flores et al. (2006), a VLR greater than 3 kg COD/(m3·d) is 173
usually pursued in most real cases. Therefore, we doubled the concentration of TA and 174
BA to elevate VLR from 2.1 kg COD/(m3·d) to 4.5 kg COD/(m3·d) from days 111 to 175
140. The effluent COD concentration increased sharply when subjected to shock 176
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variation in VLR, and then slowly dropped to 108.6 mg/L. 177
Subsequently, operational temperature was increased stepwise to investigate the 178
thermophilic performance of PTA wastewater treatment. The reactor was firstly 179
operated under 43°C during days 141 and 172, an apparent decrease in COD removal 180
(from 93.7% to 87.4%) was observed in this phase. Since days 174, it was 181
maintained at 47°C as a buffer stage for one week before following operation at 52°C. 182
Obviously, during this period, COD removal met a sudden drop from 88.8% to 58.3%. 183
From days 182 to days 220, the reactor was conducted at 52°C, and a better removal 184
capacity was achieved in this stage, with COD removal fluctuating at 70(±5)%. 185
However, it was still lower than that at 37°C and 43°C. 186
3.3. Phylogenetic analysis based on 16S rRNA gene clone libraries 187
For further understanding of particular TA-degrading consortia at different operating 188
temperatures, phylogenetic composition of both archaea and bacteria were examined 189
through clone library construction. In total, 34, 38, 28, 31 bacterial clones and 43, 45, 190
39, 40 archaeal clones were selected for phylogenetic analysis. 191
Figure 3 192
Figure 4 193
Figure 3 shows that a significant proportion of archaea clones are closely related to 194
the acetoclastic Methanosaeta group, while another large part of clones are related to 195
hydrogen utilizing Methanobrevibacter genus, and the remaining ones are assigned to 196
Thermogymnomonas acidicola and Aciduliprofundum boonei. Both of them are 197
thermoacidophilic strains. The former is isolated from a solfataric soil in Japan and 198
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available at a temperature range of 38~68°C (Itoh et al. 2007), and the latter is isolated 199
from hydrothermally heated black smoker wall at the Mid Atlantic Ridge (Schouten et 200
al. 2008). 201
Phylogenetic tree in Figure 4 indicated that the 131 clones obtained from four 202
samples were affiliated with 16 different bacterial divisions after sequencing and 203
alignment. Most of them are gram-negative, and only few clones assigning to 204
Spirochaetes are gram-negative. The distribution profiles of microbial community in 205
UAFB reactor treating PTA wastewater in response to temperature increase are 206
demonstrated in Figure 5. 207
Comparison of the 16S rRNA gene sequences with the NCBI database revealed a 208
remarkable shift in community composition with temperature. Among these classes, 209
Proteobacteria was considered as the dominant category in anaerobic system, and five 210
Proteobacteria classes including α, β, γ, δ and ε were present in the phylogenetic tree. 211
For sample taken at days 105 (37°C), δ-Proteobacteria was undoubtedly the most 212
dominant class with a 50.0% share, while its abundance at 33°C, 43°C and 52°C were 213
12.5%, 42.9%, 16.1%, respectively. Of the δ-Proteobacteria at 37°C, 214
Syntrophorhabdaceae was the most abundant family and intensively assigned to 215
genera Syntrophorhabdus with 23.7%. In addition, Syntrophaceae also shared a 216
proportion of 13.2%, and was closely assigned to Syntrophus and Smithella genus. 217
Apart from δ-Proteobacteria, β-Proteobacteria was another dominant class present 218
in the system. From the sequencing results, levels of β-Proteobacteria fluctuated as 219
significantly as that of δ-Proteobacteria. The most dominant clone 220
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(UAFB_TA_33_clone37, 6/34) was closely affiliated with species Macromonas 221
bipunctata , isolated from activated sludge(Dubinina and Grabovich 1984), which is 222
a hydrogen utilization genus Hydrogenophaga and presumed to be also beneficial for 223
anaerobic biodegradation process. The remaining clones at low abundance were 224
dispersedly distributed, as demonstrated in Figure 4. 225
In addition to the quantitative shift in dominant clones, qualitative variations in 226
community diversity were also observed. Clones affiliated with species Candidatus 227
Methylomirabilis oxyfera were detected only existed at 33°C. When gradually rise the 228
temperature from 33°C to 37°C, four new classes appeared, namely Clostridia, 229
Anaerobilineae, Syergistia and α-Proteobacteria. With temperature increase, some 230
thermophilic bacteria came up at 43°C, including Thermus and Melioribacter roseus 231
(developed from hot water produced by an oil exploring well. Podosokorskaya et al. 232
(2013)). When operated at 52°C after a period, the microbial structure was once 233
again different from that at 43°C. Details are shown in Figure 5. 234
Figure 5 235
3.4. Temperature effects on microbial diversity revealed by T-RFLP technology 236
T-RFLP has become a powerful tool for rapid comparative community analysis. 237
Especially when it is complemented with clone library construction and analysis, 238
unique polymorphic ‘‘peaks’’ in the electropherograms can be attributed more 239
precisely to target sequence information and species prediction. Nevertheless, 240
T-RFLP is often considered a semi-quantitative technique due to biases associated 241
with DNA extraction and PCR amplification. In most cases, the output can be used 242
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in combination with sequence information, to monitor changes in the abundance of 243
specific populations (Padmasiri et al. 2007, Timmis et al. 2010). 244
Distribution of community structures for bacteria were analyzed by T-RFLP using 245
samples taken from steady operation stage at 33°C, 37°C, 43°C and 52°C (see Figure 246
6). In total, 24 distinct terminal restriction fragments (T-RFs) were detected. 247
Fragments that occupy a relatively high abundance among the four different samples 248
were mainly considered, including 78 bp,88 bp,124 bp,162 bp,274 bp,466 bp, 249
484 bp, 508 bp and 524 bp. 250
All clones were simultaneously analyzed using in vitro T-RF. Phylogenetic 251
affiliation of the distinct T-RFs was investigated by finding the first MspI enzymatic 252
digestion site downstream from 27F. In general, most of the detected T-RFs could be 253
assigned to defined phylogenetic groups. Detailed results are displayed in Figure 4. 254
Obviously, variation of temperature from 33°C to 52°C caused an apparent shift in 255
the microbial community distribution in terms of T-RFs. Among the T-RFs, 162 bp, 256
483 bp and 508 bp were observed to be the major peaks in favor of TA-degradation. 257
The most dominant 162 bp T-RF observed at mesophilic ranges are related to the 258
Syntrophorhabdus aromaticivorans group, it accounted for 8.69% and 23.68% of 259
total bacterial abundance at 33°C and 37°C respectively. Though T-RF of 508 bp 260
indicated a same class as 162 bp, its closest assignment was proved to be 261
Syntrophaceae, including species Syntrophus aciditrophicus SB, Smithella propionica, 262
et al. Another dominant T-RF of 484 bp represented β-Proteobacteria, which 263
accounted for a large proportion at thermophlic conditions of both 43°C and 52°C. 264
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The specific abundance for T-RF of 484 bp at 33°C, 37°C, 43°C and 52°C were 265
26.57%, 14.56%, 65.46% and 51.50%, respectively. 266
Besides, it was further observed that some T-RFs such as 139 bp, 91 bp could 267
represent more than one phylogenic group, while some other T-RFs such as 62 bp 268
could be assigned to none of the clone sequences. 269
Figure 6 270
4. Discussion 271
Effect of temperature changes on TA-degradation from mesophilic to thermophilic 272
was determined in this study. Results of the present study demonstrated that 273
significant removal was achieved at 37°C with a total pollutant loading rate of 2.1 kg 274
COD/(m3•d) and HRT of 18.9 h. All the four tested temperatures (33, 37, 43, 52°C) 275
could cope with temperature changes. However, temperature increase showed a 276
drastic effect on the operational performance and microbial community. 277
Generally, when various pollutants coexist in an anaerobic system, due to the 278
excellent biodegradability, small molecules like fatty acids and esters are usually 279
more competitive in substrate utilization for biological metabolism and microbial 280
growth. Anaerobic degradation of TA has been proved to be adversely affected by the 281
presence of BA (Fajardo et al. 1997, Kleerebezem et al. 1999). Hence the low 282
treatment efficiency at the beginning 30 days can be explained. With temperature 283
elevated to 37°C, operational efficiency of the system gradually kept at a high level. 284
This is deemed to be the contribution of the booming growing mesophilc microbes 285
correlating with TA degradation. Compared with 37°C, the operation at 43°C 286
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seemed to make little difference in terms of COD and TA removal. Following 287
operation at 43°C, 47°C was stressed as a buffer stage to avoid the possible 288
inadaptation caused by sudden changes of temperature directly from 43°C to 52°C. 289
However, a drastic increase of effluent COD at 47°C was observed, which is similar 290
to previous reports (Boušková et al. 2005, Choorit and Wisarnwan 2007). Anaerobic 291
treatment system is generally believed sensitive to temperature shocks. Considering 292
45°C is at the range between mesophilic and thermophilic temperature, once operated 293
at higher than 45°C, the dominant mesophilic microorganisms can't immediately 294
adapt to a new higher temperature range. Meanwhile, thermophilic microorganism 295
may hardly recover from a long time’s inhibition, which led to the poor treatment 296
efficiency during this temperature range (45-50°C). Though the efficiency partly 297
recovered in the subsequent operation at 52°C, it is still far beyond that at 37°C. 298
Despite a high efficiency under thermophilic conditions has been achieved 299
previously(Chen et al. 2004), in this research, the removals of COD and TA under 300
thermophilic conditions seemed a little inefficient. Several reasons can be taken into 301
account for the results. Despite the reaction rates at thermophilic conditions are higher 302
than those at mesophilic conditions theoretically, the achievement of maximum 303
growth rate is simultaneously affected by the substrate available in the reactor. 304
According to Abeynayaka and Visvanathan (2011), limited substrate may enhance 305
decay rate and facilitate the release of microbial products like cell lysate, which are 306
supposed to be responsible for the high COD values in the supernatant. The relative 307
inefficiency at 52°C might also be largely attributed to the inferior adaptability of 308
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microbial community at utilizing the same range of substrates under thermophilic 309
temperature, which is probably due to a too quick change of temperature. Moreover, 310
the relatively short term of experimental phases cannot definitely ensure a complete 311
steady state for the current phase. This might also induce the instability and poor 312
efficiency in operation. These results seemed to indicate that in an anaerobic system, 313
within an identical period, a high efficiency is more accessible under mesophilic 314
rather than thermophilic conditions. 315
Sequencing analysis indicated that temperature had significant effect on microbial 316
community structure in TA degradation system. Archaea genera found in the reactor 317
were closely related to acetotrophic methanogens (Methanosaetaceae) and 318
hydrogenotrophic methanogens. Acetoclastic Methanosaeta preponderant at 319
mesophilic condition (37°C) was replaced by hydrogenotrophic genera including 320
Methanobrevibacter and Methanofollis at thermophilic temperatures (43°C and 52°C). 321
The detection of thermoacidophilic related species Thermogymnomonas acidicola 322
and Aciduliprofundum boonei supports that these methanogens might be crucial for 323
aromatics degradation at an extreme thermoacidophilic condition(Itoh et al. 2007, 324
Schouten et al. 2008). The observation of Methanofollis and Methanregula group 325
suggests that these methanogens are also important in suspended growth system, by 326
utilizing H2/CO2, as well as formate to produce methane (Demirel and Scherer 2008). 327
Besides, changes in temperature from mesophilic to thermophilic seemed to affect 328
microbial diversity more significantly on bacterial than archaea. Molecular analysis 329
showed diversified bacterial class were present in this anaerobic system, including 330
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Proteobacteria, Clostridia, Anaerobilineae, Syergistia, Ignavibacteria and some 331
thermophilic classes (Figure 4). 332
In view of these results, various microbes are capable of degrading TA to acetate, 333
CO2, H2O, and further mineralize these compounds by methanogenic counterpart. 334
The syntrophic phenol degrading species belonging to δ-Proteobacteria were assumed 335
to be the dominant mesophilic microbes promoting TA degradation. Phylogenetic 336
analysis revealed that 23.7% clones of 37°C were highly affiliated with strain 337
Syntrophorhabdus aromaticivorans. As it was reported to be is a phenol, p-cresol, 338
isophthalate and benzoate degrading microbe(Qiu et al. 2008), the predominant 339
abundance of Syntrophorhabdus aromaticivorans at 37°C may well suggest that 340
Syntrophorhabdaceae members likely play an important role in anaerobic treatment of 341
industrial wastewater containing aromatic compounds. Meanwhile, it may also trigger 342
the efficient degradation of TA in obligate syntrophic association with a 343
hydrogenotrophic methanogen. The presence of a hydrogen generation population 344
Pelobacter (2.6%) at 37°C is also of great advantage for further methanogens step. As 345
Pelobacter plays an important role in degrading VFAs to acetic acid and hydrogen, 346
under a low acetic acid concentration and hydrogen partial pressure(Schink and 347
Pfennig 1982). In addition, β-Proteobacteria was also largely detected at both 33°C 348
and 52°C. 349
Further T-RFLP analysis results revealed changes in microbial abundance. In 350
previous phylogenetic analysis, β-Proteobacteria was major detected at 33°C and 351
52°C, whereas little present at 43°C. However, the abundance of β-Proteobacteria at 352
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43°C revealed by T-RFLP technology shows a totally different tendency. As is 353
illustrated in Figure 6, the β-Proteobacteria representative peak 484bp is largely 354
abundant at 43°C (66.46%) and 52°C (51.50%), and less abundant at 33°C with a 355
percentage of 26.57%. These data seemed to indicate that β-Proteobacteria was 356
especially favored at temperature higher than 43°C. This discrepancy is attributed to 357
inaccuracy of clone library in characterizing the abundance of individual clone, 358
especially when insufficient white clones are selected for sequencing. Conversely, the 359
relative abundance of individual sequence can be determined by calculating the ratio 360
between the area of each peak and the total area of all peaks in one sample through 361
T-RFLP fingerprint. The combination of these two methods may well reveal the 362
microbial diversity and their changes in abundance. 363
5. Conclusions 364
This study investigated the changes of microbial community structure in an UAFB 365
reactor treating TA-rich wastewater in response to temperature variation from 33°C 366
to 52°C. In total, four temperatures 33°C, 37°C, 43°C and 52°C are discussed. The 367
reactor performance showed a most efficient performance at 37°C, followed by 368
43°C, 33°C and 52°C. Furthermore, the combination biological analysis of 16S 369
r-DNA gene clone library and T-RFLP technology also well characterized the 370
microbial diversity and their changes in abundance toward a stable condition within 371
temperatures variation. T-RF of 162 bp, 483 bp and 508 bp were regarded as the 372
major peaks that might be in favor of TA degradation. Meanwhile, δ-Proteobacteria 373
was the most dominant bacterial group in mesophilic anaerobic treatment process, 374
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whereas β-Proteobacteria appeared to be favored at temperature higher than 43°C. 375
Acknowledgments 376
This work was supported by “National Science Foundation of China” (Grant No. 377
51178137). The assistance from laboratory members is greatly appreciated. 378
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Figure Captions 506
Figure. 1. Scheme of the hybrid anaerobic reactor. 507
Figure. 2. Time profiles of the pollutant concentrations and the removal efficiencies 508
in terms of TA and COD along with temperature. 509
Figure. 3. Evolutionary relationship of the archaea 16S rRNA gene sequences 510
received from sludge of four operational temperatures. The phylogenetic tree was 511
constructed with the neighbor-joining method. Bootstrap values smaller than 50% 512
(based on 1000 bootstrap re-samplings) were hided at nodes. 513
Figure. 4. Evolutionary relationship of the bacterial 16S rRNA gene sequences 514
received from sludge of four operational temperatures. The phylogenetic tree was 515
constructed with the neighbour-joining method. Bootstrap values smaller than 50% 516
(based on 1000 bootstrap re-samplings) were hided at nodes. Scale bar represents 2% 517
sequence divergence. 518
Figure. 5. Bacterial composition at the phylum level retrieved from TA degrading 519
anaerobic reactor of different temperatures. The statistic is done by way of 520
phylogenetic analysis based on 16S rRNA gene clone libraries. 521
Figure. 6. Relative abundance of the MspI-digested T-RFs obtained from bacterial 522
community operated under temperature 33°C, 37°C, 43°C and 52°C. Numbers in the 523
legend indicate the length of T-RFs in base pairs. Those T-RFs of less than 1% of the 524
total were grouped together as a single group donated by others<1%. The deviation 525
was set as 2 bp. 526
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Table 1 Parameters and performances of the UAFB reactor during continuous experiment
Parameters and Performances
Time Period 0~40 41~75 76~110 111~140 141~172 188~220
Temperature/°C 33 33 37 37 43 52 HRT/h 24 18.9 18.9 18.9 18.9 18.9
Nv/kg COD·m-3·d-1 1.6 1.9 2.1 4.5 2.1 2.1
COD Removal efficiency/% 65.9 77.4 91.9 93.7 87.4 66.1 TA Removal efficiency/% 51.1 77.6 94.0 93.3 89.1 60.8
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• The effect of process temperature (33, 37, 43 and 52°C) on anaerobic degradation of terephthalate and microbial community
• Acetoclastic Methanosaeta preponderant at 37°C was partly replaced by hydrogenotrophic methanogens at other temperatures.
• Higher richness of δ-Proteobacteria at 37°C associated to better reactor performance at mesophilic conditions.
• β-Proteobacteria appeared to be favored at temperature higher than 43°C.
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Seq ID Access Number Closest affiliation to cultivated strains Class Identify % Access Number Abundance
UAFB_TA_33_clone3 KJ476556 Parabacteroides distasonis Bacteroidia 82 AB238922 1/34
UAFB_TA_33_clone6 KJ476557 Sulfurospirillum barnesii SES-3 Epsilon-Proteobacteria 87 CP003333 1/34
UAFB_TA_33_clone9 KJ476558 Candidatus Methylomirabilis oxyfera Actinobacteridae 82 FP565575 3/34
UAFB_TA_33_clone10 KJ476559 Halothiobacillus kellyi Gamma-Proteobacteria 91 AF170419 5/34
UAFB_TA_33_clone13 KJ476560 Solitalea canadensis DSM 3403 Sphingobacteriia 87 CP003349 1/34
UAFB_TA_33_clone14 KJ476561 Thauera humireducens Beta-Proteobacteria 99 JQ038037 1/34
UAFB_TA_33_clone15 KJ476562 Dechlorosoma suillum PS Beta-Proteobacteria 92 CP003153 1/34
UAFB_TA_33_clone17 KJ476563 Syntrophorhabdus aromaticivorans UI Delta-Proteobacteria 94 AB212873 1/34
UAFB_TA_33_clone22 KJ476564 Schlegelella thermodepolymerans Beta-Proteobacteria 97 AY152824 5/34
UAFB_TA_33_clone25 KJ476565 Geobacter metallireducens GS-15 Delta-Proteobacteria 83 CP000148 1/17
UAFB_TA_33_clone26 KJ476566 Hydrogenophaga pseudoflava Beta-Proteobacteria 99 AF078770 1/34
UAFB_TA_33_clone29 KJ476567 Ignavibacterium album JCM 16511 Ignavibacteria 84 CP003418 1/34
UAFB_TA_33_clone33 KJ476568 Exilispira thermophila Spirochaetale 84 AB364473 3/34
UAFB_TA_33_clone34 KJ476569 Candidatus Solibacter usitatus Ellin6076 Erysipelothrix 91 CP000473 1/34
UAFB_TA_33_clone36 KJ476570 Denitratisoma oestradiolicum Beta-Proteobacteria 95 AY879297 1/34
UAFB_TA_33_clone37 KJ476571 Macromonas bipunctata Beta-Proteobacteria 99 AB077037 3/17
UAFB_TA_37_clone1 KJ476572 Rikenella microfusus Bacteroidia 83 L16498 1/19
UAFB_TA_37_clone4 KJ476573 Owenweeksia hongkongensis DSM 17368 Sphingobacteriia 87 CP003156 1/38
UAFB_TA_37_clone6 KJ476574 Candidatus Cloacamonas acidaminovorans Spirochaetale 89 CU466930 1/38
UAFB_TA_37_clone7 KJ476575 Hydrogenophaga pseudoflava Beta-Proteobacteria 99 AF078770 1/38
UAFB_TA_37_clone8 KJ476576 Moorella thermoacetica ATCC 39073 Spirochaetale 83 CP000232 1/38
UAFB_TA_37_clone9 KJ476577 Syntrophus aciditrophicus SB Delta-Proteobacteria 92 CP000252 1/19
UAFB_TA_37_clone10 KJ476578 Syntrophorhabdus aromaticivorans UI Delta-Proteobacteria 96 AB212873 1/38
UAFB_TA_37_clone12 KJ476579 Leptolinea tardivitalis Anaerolineae 92 AB109438 1/38
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UAFB_TA_37_clone13 KJ476580 Defluviicoccus vanus Alpha-Proteobacteria 91 AF179678 1/19
UAFB_TA_37_clone14 KJ476581 Smithella propionica Delta-Proteobacteria 97 AF126282 1/19
UAFB_TA_37_clone15 KJ476582 Desulfatibacillum alkenivorans AK-01 Delta-Proteobacteria 84 CP001322 1/38
UAFB_TA_37_clone16 KJ476583 Halothiobacillus neapolitanus c2 Gamma-Proteobacteria 92 CP001801 1/38
UAFB_TA_37_clone17 KJ476584 Burkholderia phytofirmans PsJN Beta-Proteobacteria 94 CP001052 1/38
UAFB_TA_37_clone18 KJ476585 Erysipelothrix inopinata Erysipelothrix 91 AJ550617 1/38
UAFB_TA_37_clone19 KJ476586 Thermovirga lienii DSM 17291 Synergistia 89 CP003096 1/38
UAFB_TA_37_clone21 KJ476587 Acetomicrobium faecale Clostridia 88 FR749980 1/38
UAFB_TA_37_clone22 KJ476588 Syntrophorhabdus aromaticivorans UI Delta-Proteobacteria 96 AB212873 1/19
UAFB_TA_37_clone24 KJ476589 Desulfovibrio aminophilus Delta-Proteobacteria 91 AF067964 1/38
UAFB_TA_37_clone27 KJ476590 Desulfotomaculum acetoxidans DSM 771 Clostridia 81 CP001720 1/19
UAFB_TA_37_clone28 KJ476591 Chondromyces apiculatus Delta-Proteobacteria 92 AJ233938 1/19
UAFB_TA_37_clone29 KJ476592 Comamonas odontotermitis Beta-Proteobacteria 99 DQ453128 1/38
UAFB_TA_37_clone31 KJ476593 Thermoanaerobaculum aquaticum Delta-Proteobacteria 94 JX420244 1/38
UAFB_TA_37_clone33 KJ476594 Syntrophus gentianae Delta-Proteobacteria 99 X85132 1/38
UAFB_TA_37_clone34 KJ476595 Sulfurovum sp. NBC37-1 Epsilon-Proteobacteria 94 AP009179 1/38
UAFB_TA_37_clone35 KJ476596 Clostridium thermocellum ATCC 27405 Clostridia 90 CP000568 1/38
UAFB_TA_37_clone36 KJ476597 Syntrophorhabdus aromaticivorans UI Delta-Proteobacteria 94 AB212873 3/19
UAFB_TA_37_clone38 KJ476598 Pelobacter carbinolicus DSM 2380 Delta-Proteobacteria 88 CP000142 1/38
UAFB_TA_43_clone6 KJ476599 Smithella propionica strain LYP Delta-Proteobacteria 91 AF126282 3/28
UAFB_TA_43_clone5 KJ476600 Rikenella microfusus strain Q-1 Bacteroidia 83 L16498 1/7
UAFB_TA_43_clone3 KJ476601 Vampirovibrio chlorellavorus Erysipelothrix 85 HM038000 1/28
UAFB_TA_43_clone2 KJ476602 Syntrophorhabdus aromaticivorans UI Delta-Proteobacteria 94 AB212873 3/28
UAFB_TA_43_clone1 KJ476603 Leptolinea tardivitalis Anaerolineae 93 AB109438 1/28
UAFB_TA_43_clone4 KJ476604 Alkaliflexus imshenetskii Sphingobacteriia 87 AJ784993 1/28
UAFB_TA_43_clone15 KJ476605 Syntrophus gentianae Delta-Proteobacteria 99 X85132 1/28
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UAFB_TA_43_clone14 KJ476606 Alkaliflexus imshenetskii Spirochaetale 87 AJ784993 1/28
UAFB_TA_43_clone13 KJ476607 Alkaliflexus imshenetskii Spirochaetale 88 AJ784993 1/14
UAFB_TA_43_clone12 KJ476608 Syntrophorhabdus aromaticivorans UI Delta-Proteobacteria 87 AB212873 1/14
UAFB_TA_43_clone11 KJ476609 Ignavibacterium album JCM 16511 Ignavibacteria 81 CP003418 1/28
UAFB_TA_43_clone10 KJ476610 Acidovorax citrulli AAC00-1 Beta-Proteobacteria 99 CP000512 1/28
UAFB_TA_43_clone25 KJ476611 Phycisphaera mikurensis NBRC 102666 Nitrospirales 81 AP012338 1/28
UAFB_TA_43_clone24 KJ476612 Sulfurovum sp. NBC37-1 Epsilon-Proteobacteria 93 AP009179 3/28
UAFB_TA_43_clone23 KJ476613 Syntrophorhabdus aromaticivorans UI Delta-Proteobacteria 95 AB212873 1/28
UAFB_TA_43_clone28 KJ476614 Syntrophorhabdus aromaticivorans UI Delta-Proteobacteria 96 AB212874 1/14
UAFB_TA_52_clone1 KJ476615 Thermus scotoductus SA-01 Deinococci 72 CP001962 5/31
UAFB_TA_52_clone3 KJ476616 Macromonas bipunctata Beta-Proteobacteria 99 AB077037 3/31
UAFB_TA_52_clone4 KJ476617 Caloramator coolhaasii Clostridia 99 AF104215 1/31
UAFB_TA_52_clone5 KJ476618 Rhizobium selenitireducens Alpha-Proteobacteria 99 EF440185 1/31
UAFB_TA_52_clone6 KJ476619 Syntrophorhabdus aromaticivorans UI Delta-Proteobacteria 94 AB212873 2/31
UAFB_TA_52_clone9 KJ476620 Sulfurospirillum barnesii SES-3 Epsilon-Proteobacteria 87 CP003333 2/31
UAFB_TA_52_clone14 KJ476621 Acinetobacter lwoffii DSM 2403 Gamma-Proteobacteria 98 X81665 1/31
UAFB_TA_52_clone16 KJ476622 Pelobacter carbinolicus DSM 2380 Delta-Proteobacteria 83 CP000142 3/31
UAFB_TA_52_clone21 KJ476623 Acidovorax delafieldii Beta-Proteobacteria 99 AF078764 1/31
UAFB_TA_52_clone25 KJ476624 Thiofaba tepidiphila Gamma-Proteobacteria 99 AB304258 2/31
UAFB_TA_52_clone29 KJ476625 Thermodesulfovibrio yellowstonii DSM Nitrospirales 96 CP001147 2/31
UAFB_TA_52_clone33 KJ476626 Acidovorax temperans Beta-Proteobacteria 99 AF078766 5/31
UAFB_TA_52_clone34 KJ476627 Cytophaga fermentans Cytophagia 87 M58766 2/31
UAFB_TA_52_clone35 KJ476628 Owenweeksia hongkongensis DSM 17368 Sphingobacteriia 86 CP003156 1/31
UAFB_TA_37_A45 KJ476541 Methanosaeta concilii GP6 Methanomicrobia 96 CP002565 4/9
UAFB_TA_37_A9 KJ476542 Thermococcus sibiricus Thermococci 82 CP001463 8/45
UAFB_TA_37_A15 KJ476543 Methanosaeta concilii GP6 Methanomicrobia 98 CP002565 14/45
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UAFB_TA_37_A23 KJ476544 Aciduliprofundum boonei T469 Thermoplasmata 81 CP001941 1/15
UAFB_TA_33_A1 KJ476545 Methanosaeta concilii GP6 Methanomicrobia 98 CP002565 2/21
UAFB_TA_33_A12 KJ476546 Methanobrevibacter smithii ATCC 35061 Methanobacteria 83 CP000678 9/14
UAFB_TA_33_A18 KJ476547 Thermogymnomonas acidicola Thermoplasmata 81 AB269873 1/21
UAFB_TA_33_A29 KJ476548 Thermococcus sibiricus Thermococci 82 CP001463 1/7
UAFB_TA_33_A44 KJ476549 Methanosaeta concilii GP6 Methanomicrobia 98 CP002565 2/21
UAFB_TA_43_A1 KJ476550 Methanosaeta concilii GP6 Methanomicrobia 97 CP002565 7/37
UAFB_TA_43_A11 KJ476551 Methanofollis liminatans GKZPZ Methanomicrobia 99 Y16428 1/37
UAFB_TA_43_A27 KJ476552 Aciduliprofundum boonei T469 Thermoplasmata 82 CP001941 4/37
UAFB_TA_43_A32 KJ476553 Methanobrevibacter smithii ATCC 35061 Methanobacteria 83 CP000678 25/37
UAFB_TA_52_A9 KJ476554 Methanosaeta concilii GP6 Methanomicrobia 99 CP002565 39/40
UAFB_TA_52_A27 KJ476555 Methanoculleus palmolei DSM 4273 Methanomicrobia 93 Y16382 1/40