performance and microbial community profiles in an anaerobic reactor treating with simulated pta...

Accepted Manuscript

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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http://dx.doi.org/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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504

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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_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_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_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_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_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_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_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


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UAFB_TA_37_A23 KJ476544 Aciduliprofundum boonei T469 Thermoplasmata 81 CP001941 1/15


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_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_A27 KJ476555 Methanoculleus palmolei DSM 4273 Methanomicrobia 93 Y16382 1/40

performance and microbial community profiles in an anaerobic reactor treating with simulated pta...

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