Polycyclic aromatic hydrocarbon affects acetic acid production during ...
Polycyclic aromatic hydrocarbon affects acetic acid production during anaerobic fermentation of waste activated sludge by altering activity and viability of acetogen
Jingyang Luo, Yinguang Chen*, Leiyu Feng* State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China
*Corresponding author E-mail: [email protected], [email protected] Tel: 86-21-65981263 Fax: 86-21-65986313
Document prepared: June 2, 2016 Number of pages: 11 Number of figures: 4 Number of tables: 5
S1
MATERIALS AND METHODS Characteristics of the different sludge withdrawn from another WWTP A different sludge withdrawn from another WWTP with the entry of approximate 20% industrial wastewater was used to test the phenanthrene effect on different WAS anaerobic fermentation for acetic acid production. Its main characteristics are as follows: pH 6.7 ± 0.1, TSS 14530 ± 300 mg/L, VSS 10200 ± 125 mg/L, SCOD 420 ± 30 mg/L, TCOD 14850 ± 365 mg/L, total carbohydrate 1105 ± 130 mg COD/L, total protein 7935 ± 205 mg COD/L, and lipid and oil 255 ± 20 mg COD/L. Effects of phenanthrene on the processes involved in anaerobic fermentation Phenanthrene effects on the solubilization and hydrolysis of WAS under the alkaline condition were examined respectively based on the apparent concentrations of soluble proteins and carbohydrates in WAS fermentation liquid and the hydrolysis efficiencies of bovine serum albumin (BSA, average molecular weight (Mw) 67000, model protein) and dextran (Mw 23800, model carbohydrate) in the synthetic wastewater. The wastewater was synthesized by 900 mL tap water with the mixture of BSA (3.0 g) and dextran (0.5 g) (the mass ratio of BSA and dextran was almost the same with that of proteins to carbohydrates in raw WAS) and 100 mL WAS (withdrawn from the control and external phenanthrene addition (100 mg/kg) reactors and served as inoculum) with a final sludge concentration of about 1300 mg/L. The pH was controlled at 10 by the addition of 4 M NaOH or 4 M HCl.
After being flushed
with nitrogen gas for 10 min to remove oxygen, all reactors were capped with rubber stoppers, sealed and stirred at 20 ± 1 oC and 100 rpm. The hydrolysis efficiencies of BSA and dextran were expressed by the following equation.
( intitial − C residue ) /C intitial ×100
Hydrolysis efficiency (%) = C
(1)
where Cinitial and Cresidue represent the concentration of BSA and dextran in the synthetic wastewater before
S2
and after the fermentation tests, respectively. The same operations were conducted for the exploration of phenanthrene effect on the acidification of hydrolyzed products except that the substrates were replaced by mixing 3.0 g L-glutamate and 0.5 g glucose. Based on the analysis of SCFAs concentration and composition in the fermentation reactors, the influence of phenanthrene on the acidification during anaerobic fermentation was revealed. Homoacetogenic bacteria are microorganisms utilizing H2 and CO2 as substrates for acetic acid formation. In order to investigate the contribution of homoacetogenic bacteria to acetic acid production in the presence of phenanthrene, the synthetic gas including 70% nitrogen, 20% hydrogen and 10% carbon dioxide (v/v) was fed into 600 mL serum bottles which contained 250 mL sludge from the control and phenanthrene-added (100 mg/kg) reactors, respectively. nitrogen flush were set as controls.
A corresponding set of reactors with pure
By monitoring the hydrogen consumption and acetic acid
concentration, the effect of phenanthrene on homoacetogenic bacteria was examined. As to the methanogenesis, it has been demonstrated previously that the methanogenesis of acidification products during anaerobic fermentation was inhibited when the fermentation pH was controlled at 10 which had been demonstrated to be the optimal one for SCFAs production from WAS.1 Thus, in this study the effect of phenanthrene on the methanogenesis process of WAS was not considered. DNA extraction and real-time quantification PCR of acetate-related genes For each set, DNA was extracted from concentrated Proteiniphilum acetatigenes of three replicate reactors using the FastDNA Kit (BIO 101, Vista, CA) according to the manufacture’s instruction, and then pooled together. In real-time PCR assays, the primers, PCR reactions mixtures and procedures were shown below (Table S1, S2 and S3). All PCR assays were performed using three replicates per sample, and contained the control reactions without template DNA. The copy number of total genes was calculated
S3
using a standard curve generated by using 10-fold serial dilutions of linearized plasmids containing the gene fragment as a template. Procedures of Illumina MiSeq sequencing for the microbial community analysis The fermentation mixture was firstly centrifuged for 5 min at 10000 rpm, and the total genomic DNA was extracted using the CW2091 DNA kit. Next generation sequencing library preparations and Illumina MiSeq sequencing were conducted at GENEWIZ, Inc. (Beijing, China). DNA samples were quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad) and DNA quality was checked on a 0.8% agarose gel. Sequencing library was constructed using a MetaVx™ Library Preparation kit (GENEWIZ, Inc., South Plainfield, NJ, USA). Briefly, 5-50 ng DNA was used to generate amplicons that cover V3, V4, and V5 hypervariable regions of bacteria and archaea 16S rRNA genes. Indexed adapters were added to the ends of the 16S rDNA amplicons by limited cycle PCR. DNA libraries were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit and real time PCR (Applied Biosystems, Carlsbad, CA, USA). DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was performed using a 2×250 paired-end configuration. Image analysis and base calling were conducted by the MiSeq Control Software (MCS) on the MiSeq instrument. Initial taxonomy analysis was carried out on Illumina base-space cloud computing platform. The raw pyrosequencing data that obtained from this study were deposited to the NCBI Sequence Read Archive with accession number SRR1659082 and SRR1648074 (SRA, http://trace.ncbi.nlm.nih.gov/Traces/sra_sub/sub.cgi?login=pda). Determination of membrane potential of Proteiniphilum acetatigenes The analysis of membrane potential of acetogen cells was determined by flow cytometry with DiBAC4(3). DiBAC4(3) emits no fluorescence signal outside cell. When the cell membrane is structurally damaged, the dye enters the cytosol and the fluorescence intensity increases. In the present study, the cells S4
of Proteiniphilum acetatigenes were withdrawn from the fermentation reactors, and then centrifuged and concentrated. The concentrated cells were firstly washed with HEPES buffered solution (HBS) containing (in mg/L) NaCl 8482, KCl 372, MgCl2 95, CaCl2 111, D-glucose 990 and HEPES 2383 for three times and then loaded with DiBAC4(3) at 37 °C for 40 min, and finally placed in a chamber filled with HBS. Fluorescence intensity at an excitation wavelength of 490 nm was obtained by cytometer (BD FACSVerse, America) and analyzed with Flowjo.7.6.2 software (Peking University, China).
S5
Table S1. Primer sequences and annealing temperatures of PCR and qPCR assays
Primer
Target gene
PTA-Fw
PTA
PTA-Rv
Primer/Probe sequence
Annealing
(5’- 3’)
Temperature
AAGGGAAGTGCACAACATGA
60 ºC
(2)
60 ºC
(3)
Reference
ACTACCAGGTGCTTTTAAATTTGC
ACK-Fw
AK
ACK-Rv
CTCAGATGCTGGGCAAACCT ACAAATACTTGCTCCGTTTCCAA
Table S2. PCR reaction mixture Reaction Component
Concentration
Volume (µL)
SybrGreen qPCR Master Mix
2X
12.5
Primer F
10 µM
0.5
Primer R
10 µM
0.5
ddH2O
9.5
Template (cDNA)
2
Total
25
Table S3. PCR reaction procedures Time and Temperature Thermal cycle
Each of 40 cycles Initial steps Melt Hold
Anneal/Extend Cycle
Real-Time Quantitative PCR
10 min
15 sec
1 min
(ABI 7500)
95 ºC
95 ºC
60 ºC
S6
Table S4. Contribution of degraded PAH to the acetic acid production in WAS fermentationa Organic
Molecule weight
Degradation
COD conversion
Theoretical production
compounds
(g/mol)
efficiency (%)
factor
of acetic acid from degraded PAHb
Phenanthrene
178
47.5 ± 5.0
/
1.6 ± 0.2
Acetic acid
60
/
1.07
/
Bioconversion equation
C14H10
7 C2H4O2,
Acetic acid = 100 mg/kg × 13.3 g/L × 60 g/mol × 7 × 1.07 × 47.5% / 178 g/mol=1.6 mg COD/L a b
The concentration of PAH is 100 mg/kg dry sludge. The unit is (mg COD/L).
The data are the averages and their standard deviations in triplicate tests.
Table S5. Abundance of Bacteria in anaerobic reactors related with acetate formation at genus level Genus
Affiliated phylum
Abundance (%) Control
Phenanthrene reactor
Reference a
Lactobacillus
Firmicutes
1.44
2.74
(4)
Proteiniclasticum
Firmicutes
2.57
3.09
(5)
Ruminococcus
Firmicutes
0.011
0.20
(6)
Anaerovorax
Firmicutes
0.42
0.87
(7)
Proteiniborus
Firmicutes
0.009
0.015
(8)
Bacteroides
Bacteroidetes
0.008
0.02
(9)
Paludibacter
Bacteroidetes
0.03
0.06
(10)
Parabacteroides
Bacteroidetes
0.008
0.04
(10)
Caldilinea
Chloroflexi
1.95
2.88
(11)
Collinsella
Actinobacteria
0.005
0.13
(12)
a
: The dosage of phenanthrene in WAS fermentation reactor was 100 mg/kg.
S7
Total proteins Other SCFAs
Total carbohydrates Others CO2
Acetic acid
20
Carbon (g)
15
10
5
0 Initial
0
50
100
200
500
Phenanthrene dosage (mg/kg)
Figure S1. Carbon balance analysis of the current fermentation systems (The other SCFAs include propionic, butyric and valeric acids, and the others mainly refer to lipid, lactic acid, ethanol and some unknown organic carbons. Methane is not detected in the present study).
H2/CO2
Homoacetogenesis Solubilization & Hydrolysis
WAS
Methanogenesis
Acidification
Hydrolyzed substrates
Acetic acid
Methane
Figure S2. Pathways for acetic acid production from WAS during anaerobic fermentation
S8
400 Soluble protein
(B) 100
Soluble carbohydrate
(C) 600 BSA Dextran
Control C o n c e n tra tio n o f a c e tic a c id (m g C O D / L )
(A) 1600
800
200
100
400
D egrad atio n efficien cy (% )
300
S o lu b le c a rb o h y d ra te ( m g / L )
S o lu b le p ro te in ( m g /L )
90
1200
80
70
60
0
0 0
50 100 200 Dosage of phenanthrene (mg/kg)
500
50 Control
100
Dosage of phenanthrene (mg/kg)
Phenanthrene（ 100mg/kg）
500
400
300
200 4
6
8
Operation time (d)
Figure S3. Effects of phenanthrene on (A) solubilization (B) hydrolysis and (C) acidification during anaerobic fermentation. Error bars represent standard deviations of triplicate tests.
Figure S4. Venn analysis of the microbial community in the fermentation reactors based on OUT (3%
S9
REFERENCE 1.
Chen, Y. G.; Jiang, S.; Yuan, H. Y.; Zhou, Q.; Gu, G. W., Hydrolysis and acidification of waste activated
sludge at different pHs. Water Res 2007, 41, (3), 683-689. 2.
Bassi, D.; Fontana, C.; Zucchelli, S.; Gazzola, S.; Cocconcelli, P. S., TaqMan real time-quantitative PCR
targeting the phosphotransacetylase gene for Clostridium tyrobutyricum quantification in animal feed, faeces, milk and cheese. Int Dairy J 2013, 33, (1), 75-82. 3.
Stevenson, D. M.; Weimer, P. J., Expression of 17 genes in Clostridium thermocellum ATCC 27405 during
fermentation of cellulose or cellobiose in continuous culture. Appl Environ Microb 2005, 71, (8), 4672-4678. 4.
Elferink, S. J. O.; Krooneman, J.; Gottschal, J. C.; Spoelstra, S. F.; Faber, F.; Driehuis, F., Anaerobic
conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri. Appl Environ Microb 2001, 67, (1), 125-132. 5.
Chen, Y.; Yu, B.; Yin, C.; Zhang, C.; Dai, X.; Yuan, H.; Zhu, N., Biostimulation by direct voltage to
enhance anaerobic digestion of waste activated sludge. RSC Advances 2016, 6, (2), 1581-1588. 6.
Ambuchi, J. J.; Liu, J.; Wang, H.; Shan, L.; Zhou, X.; Mohammed, M. O.; Feng, Y., Microbial community
structural analysis of an expanded granular sludge bed (EGSB) reactor for beet sugar industrial wastewater (BSIW) treatment. Appl Microbiol Biot 2016, 1-11. 7.
Duarte, I.; Oliveira, L.; Saavedra, N.; Fantinatti-Garboggini, F.; Oliveira, V.; Varesche, M., Evaluation of
the microbial diversity in a horizontal-flow anaerobic immobilized biomass reactor treating linear alkylbenzene sulfonate. Biodegradation 2008, 19, (3), 375-385. 8.
Niu, L.; Song, L.; Dong, X., Proteiniborus ethanoligenes gen. nov., sp. nov., an anaerobic protein-utilizing
bacterium. Int J Syst Evol Micr 2008, 58, (1), 12-16. 9.
Bryant, M.; Small, N.; Bouma, C.; Chu, H., Bacteroides ruminicola n. sp. and Succinimonas amylolytica
the new genus and species: species of succinic acid-producing anaerobic bacteria of the bovine rumen. Journal of Bacteriology 1958, 76, (1), 15. 10. Nelson, M. C.; Morrison, M.; Yu, Z., A meta-analysis of the microbial diversity observed in anaerobic digesters. Bioresource Technol 2011, 102, (4), 3730-3739. 11. Grégoire, P.; Bohli, M.; Cayol, J.-L.; Joseph, M.; Guasco, S.; Dubourg, K.; Cambar, J.; Michotey, V.; Bonin, P.; Fardeau, M.-L., Caldilinea tarbellica sp. nov., a filamentous, thermophilic, anaerobic bacterium isolated from a deep hot aquifer in the Aquitaine Basin. Int J Syst Evol Micr 2011, 61, (6), 1436-1441. 12. Kageyama, A.; Benno, Y.; Nakase, T., Phylogenetic and phenotypic evidence for the transfer of S10
Eubacterium aerofaciens to the genus Collinsella as Collinsella aerofaciens gen. nov., comb. nov. Int J Syst Evol Micr 1999, 49, (2), 557-565.
S11
Jingyang Luo, Yinguang Chen*, Leiyu Feng* State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China
*Corresponding author E-mail: [email protected], [email protected] Tel: 86-21-65981263 Fax: 86-21-65986313
Document prepared: June 2, 2016 Number of pages: 11 Number of figures: 4 Number of tables: 5
S1
MATERIALS AND METHODS Characteristics of the different sludge withdrawn from another WWTP A different sludge withdrawn from another WWTP with the entry of approximate 20% industrial wastewater was used to test the phenanthrene effect on different WAS anaerobic fermentation for acetic acid production. Its main characteristics are as follows: pH 6.7 ± 0.1, TSS 14530 ± 300 mg/L, VSS 10200 ± 125 mg/L, SCOD 420 ± 30 mg/L, TCOD 14850 ± 365 mg/L, total carbohydrate 1105 ± 130 mg COD/L, total protein 7935 ± 205 mg COD/L, and lipid and oil 255 ± 20 mg COD/L. Effects of phenanthrene on the processes involved in anaerobic fermentation Phenanthrene effects on the solubilization and hydrolysis of WAS under the alkaline condition were examined respectively based on the apparent concentrations of soluble proteins and carbohydrates in WAS fermentation liquid and the hydrolysis efficiencies of bovine serum albumin (BSA, average molecular weight (Mw) 67000, model protein) and dextran (Mw 23800, model carbohydrate) in the synthetic wastewater. The wastewater was synthesized by 900 mL tap water with the mixture of BSA (3.0 g) and dextran (0.5 g) (the mass ratio of BSA and dextran was almost the same with that of proteins to carbohydrates in raw WAS) and 100 mL WAS (withdrawn from the control and external phenanthrene addition (100 mg/kg) reactors and served as inoculum) with a final sludge concentration of about 1300 mg/L. The pH was controlled at 10 by the addition of 4 M NaOH or 4 M HCl.
After being flushed
with nitrogen gas for 10 min to remove oxygen, all reactors were capped with rubber stoppers, sealed and stirred at 20 ± 1 oC and 100 rpm. The hydrolysis efficiencies of BSA and dextran were expressed by the following equation.
( intitial − C residue ) /C intitial ×100
Hydrolysis efficiency (%) = C
(1)
where Cinitial and Cresidue represent the concentration of BSA and dextran in the synthetic wastewater before
S2
and after the fermentation tests, respectively. The same operations were conducted for the exploration of phenanthrene effect on the acidification of hydrolyzed products except that the substrates were replaced by mixing 3.0 g L-glutamate and 0.5 g glucose. Based on the analysis of SCFAs concentration and composition in the fermentation reactors, the influence of phenanthrene on the acidification during anaerobic fermentation was revealed. Homoacetogenic bacteria are microorganisms utilizing H2 and CO2 as substrates for acetic acid formation. In order to investigate the contribution of homoacetogenic bacteria to acetic acid production in the presence of phenanthrene, the synthetic gas including 70% nitrogen, 20% hydrogen and 10% carbon dioxide (v/v) was fed into 600 mL serum bottles which contained 250 mL sludge from the control and phenanthrene-added (100 mg/kg) reactors, respectively. nitrogen flush were set as controls.
A corresponding set of reactors with pure
By monitoring the hydrogen consumption and acetic acid
concentration, the effect of phenanthrene on homoacetogenic bacteria was examined. As to the methanogenesis, it has been demonstrated previously that the methanogenesis of acidification products during anaerobic fermentation was inhibited when the fermentation pH was controlled at 10 which had been demonstrated to be the optimal one for SCFAs production from WAS.1 Thus, in this study the effect of phenanthrene on the methanogenesis process of WAS was not considered. DNA extraction and real-time quantification PCR of acetate-related genes For each set, DNA was extracted from concentrated Proteiniphilum acetatigenes of three replicate reactors using the FastDNA Kit (BIO 101, Vista, CA) according to the manufacture’s instruction, and then pooled together. In real-time PCR assays, the primers, PCR reactions mixtures and procedures were shown below (Table S1, S2 and S3). All PCR assays were performed using three replicates per sample, and contained the control reactions without template DNA. The copy number of total genes was calculated
S3
using a standard curve generated by using 10-fold serial dilutions of linearized plasmids containing the gene fragment as a template. Procedures of Illumina MiSeq sequencing for the microbial community analysis The fermentation mixture was firstly centrifuged for 5 min at 10000 rpm, and the total genomic DNA was extracted using the CW2091 DNA kit. Next generation sequencing library preparations and Illumina MiSeq sequencing were conducted at GENEWIZ, Inc. (Beijing, China). DNA samples were quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad) and DNA quality was checked on a 0.8% agarose gel. Sequencing library was constructed using a MetaVx™ Library Preparation kit (GENEWIZ, Inc., South Plainfield, NJ, USA). Briefly, 5-50 ng DNA was used to generate amplicons that cover V3, V4, and V5 hypervariable regions of bacteria and archaea 16S rRNA genes. Indexed adapters were added to the ends of the 16S rDNA amplicons by limited cycle PCR. DNA libraries were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit and real time PCR (Applied Biosystems, Carlsbad, CA, USA). DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was performed using a 2×250 paired-end configuration. Image analysis and base calling were conducted by the MiSeq Control Software (MCS) on the MiSeq instrument. Initial taxonomy analysis was carried out on Illumina base-space cloud computing platform. The raw pyrosequencing data that obtained from this study were deposited to the NCBI Sequence Read Archive with accession number SRR1659082 and SRR1648074 (SRA, http://trace.ncbi.nlm.nih.gov/Traces/sra_sub/sub.cgi?login=pda). Determination of membrane potential of Proteiniphilum acetatigenes The analysis of membrane potential of acetogen cells was determined by flow cytometry with DiBAC4(3). DiBAC4(3) emits no fluorescence signal outside cell. When the cell membrane is structurally damaged, the dye enters the cytosol and the fluorescence intensity increases. In the present study, the cells S4
of Proteiniphilum acetatigenes were withdrawn from the fermentation reactors, and then centrifuged and concentrated. The concentrated cells were firstly washed with HEPES buffered solution (HBS) containing (in mg/L) NaCl 8482, KCl 372, MgCl2 95, CaCl2 111, D-glucose 990 and HEPES 2383 for three times and then loaded with DiBAC4(3) at 37 °C for 40 min, and finally placed in a chamber filled with HBS. Fluorescence intensity at an excitation wavelength of 490 nm was obtained by cytometer (BD FACSVerse, America) and analyzed with Flowjo.7.6.2 software (Peking University, China).
S5
Table S1. Primer sequences and annealing temperatures of PCR and qPCR assays
Primer
Target gene
PTA-Fw
PTA
PTA-Rv
Primer/Probe sequence
Annealing
(5’- 3’)
Temperature
AAGGGAAGTGCACAACATGA
60 ºC
(2)
60 ºC
(3)
Reference
ACTACCAGGTGCTTTTAAATTTGC
ACK-Fw
AK
ACK-Rv
CTCAGATGCTGGGCAAACCT ACAAATACTTGCTCCGTTTCCAA
Table S2. PCR reaction mixture Reaction Component
Concentration
Volume (µL)
SybrGreen qPCR Master Mix
2X
12.5
Primer F
10 µM
0.5
Primer R
10 µM
0.5
ddH2O
9.5
Template (cDNA)
2
Total
25
Table S3. PCR reaction procedures Time and Temperature Thermal cycle
Each of 40 cycles Initial steps Melt Hold
Anneal/Extend Cycle
Real-Time Quantitative PCR
10 min
15 sec
1 min
(ABI 7500)
95 ºC
95 ºC
60 ºC
S6
Table S4. Contribution of degraded PAH to the acetic acid production in WAS fermentationa Organic
Molecule weight
Degradation
COD conversion
Theoretical production
compounds
(g/mol)
efficiency (%)
factor
of acetic acid from degraded PAHb
Phenanthrene
178
47.5 ± 5.0
/
1.6 ± 0.2
Acetic acid
60
/
1.07
/
Bioconversion equation
C14H10
7 C2H4O2,
Acetic acid = 100 mg/kg × 13.3 g/L × 60 g/mol × 7 × 1.07 × 47.5% / 178 g/mol=1.6 mg COD/L a b
The concentration of PAH is 100 mg/kg dry sludge. The unit is (mg COD/L).
The data are the averages and their standard deviations in triplicate tests.
Table S5. Abundance of Bacteria in anaerobic reactors related with acetate formation at genus level Genus
Affiliated phylum
Abundance (%) Control
Phenanthrene reactor
Reference a
Lactobacillus
Firmicutes
1.44
2.74
(4)
Proteiniclasticum
Firmicutes
2.57
3.09
(5)
Ruminococcus
Firmicutes
0.011
0.20
(6)
Anaerovorax
Firmicutes
0.42
0.87
(7)
Proteiniborus
Firmicutes
0.009
0.015
(8)
Bacteroides
Bacteroidetes
0.008
0.02
(9)
Paludibacter
Bacteroidetes
0.03
0.06
(10)
Parabacteroides
Bacteroidetes
0.008
0.04
(10)
Caldilinea
Chloroflexi
1.95
2.88
(11)
Collinsella
Actinobacteria
0.005
0.13
(12)
a
: The dosage of phenanthrene in WAS fermentation reactor was 100 mg/kg.
S7
Total proteins Other SCFAs
Total carbohydrates Others CO2
Acetic acid
20
Carbon (g)
15
10
5
0 Initial
0
50
100
200
500
Phenanthrene dosage (mg/kg)
Figure S1. Carbon balance analysis of the current fermentation systems (The other SCFAs include propionic, butyric and valeric acids, and the others mainly refer to lipid, lactic acid, ethanol and some unknown organic carbons. Methane is not detected in the present study).
H2/CO2
Homoacetogenesis Solubilization & Hydrolysis
WAS
Methanogenesis
Acidification
Hydrolyzed substrates
Acetic acid
Methane
Figure S2. Pathways for acetic acid production from WAS during anaerobic fermentation
S8
400 Soluble protein
(B) 100
Soluble carbohydrate
(C) 600 BSA Dextran
Control C o n c e n tra tio n o f a c e tic a c id (m g C O D / L )
(A) 1600
800
200
100
400
D egrad atio n efficien cy (% )
300
S o lu b le c a rb o h y d ra te ( m g / L )
S o lu b le p ro te in ( m g /L )
90
1200
80
70
60
0
0 0
50 100 200 Dosage of phenanthrene (mg/kg)
500
50 Control
100
Dosage of phenanthrene (mg/kg)
Phenanthrene（ 100mg/kg）
500
400
300
200 4
6
8
Operation time (d)
Figure S3. Effects of phenanthrene on (A) solubilization (B) hydrolysis and (C) acidification during anaerobic fermentation. Error bars represent standard deviations of triplicate tests.
Figure S4. Venn analysis of the microbial community in the fermentation reactors based on OUT (3%
S9
REFERENCE 1.
Chen, Y. G.; Jiang, S.; Yuan, H. Y.; Zhou, Q.; Gu, G. W., Hydrolysis and acidification of waste activated
sludge at different pHs. Water Res 2007, 41, (3), 683-689. 2.
Bassi, D.; Fontana, C.; Zucchelli, S.; Gazzola, S.; Cocconcelli, P. S., TaqMan real time-quantitative PCR
targeting the phosphotransacetylase gene for Clostridium tyrobutyricum quantification in animal feed, faeces, milk and cheese. Int Dairy J 2013, 33, (1), 75-82. 3.
Stevenson, D. M.; Weimer, P. J., Expression of 17 genes in Clostridium thermocellum ATCC 27405 during
fermentation of cellulose or cellobiose in continuous culture. Appl Environ Microb 2005, 71, (8), 4672-4678. 4.
Elferink, S. J. O.; Krooneman, J.; Gottschal, J. C.; Spoelstra, S. F.; Faber, F.; Driehuis, F., Anaerobic
conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri. Appl Environ Microb 2001, 67, (1), 125-132. 5.
Chen, Y.; Yu, B.; Yin, C.; Zhang, C.; Dai, X.; Yuan, H.; Zhu, N., Biostimulation by direct voltage to
enhance anaerobic digestion of waste activated sludge. RSC Advances 2016, 6, (2), 1581-1588. 6.
Ambuchi, J. J.; Liu, J.; Wang, H.; Shan, L.; Zhou, X.; Mohammed, M. O.; Feng, Y., Microbial community
structural analysis of an expanded granular sludge bed (EGSB) reactor for beet sugar industrial wastewater (BSIW) treatment. Appl Microbiol Biot 2016, 1-11. 7.
Duarte, I.; Oliveira, L.; Saavedra, N.; Fantinatti-Garboggini, F.; Oliveira, V.; Varesche, M., Evaluation of
the microbial diversity in a horizontal-flow anaerobic immobilized biomass reactor treating linear alkylbenzene sulfonate. Biodegradation 2008, 19, (3), 375-385. 8.
Niu, L.; Song, L.; Dong, X., Proteiniborus ethanoligenes gen. nov., sp. nov., an anaerobic protein-utilizing
bacterium. Int J Syst Evol Micr 2008, 58, (1), 12-16. 9.
Bryant, M.; Small, N.; Bouma, C.; Chu, H., Bacteroides ruminicola n. sp. and Succinimonas amylolytica
the new genus and species: species of succinic acid-producing anaerobic bacteria of the bovine rumen. Journal of Bacteriology 1958, 76, (1), 15. 10. Nelson, M. C.; Morrison, M.; Yu, Z., A meta-analysis of the microbial diversity observed in anaerobic digesters. Bioresource Technol 2011, 102, (4), 3730-3739. 11. Grégoire, P.; Bohli, M.; Cayol, J.-L.; Joseph, M.; Guasco, S.; Dubourg, K.; Cambar, J.; Michotey, V.; Bonin, P.; Fardeau, M.-L., Caldilinea tarbellica sp. nov., a filamentous, thermophilic, anaerobic bacterium isolated from a deep hot aquifer in the Aquitaine Basin. Int J Syst Evol Micr 2011, 61, (6), 1436-1441. 12. Kageyama, A.; Benno, Y.; Nakase, T., Phylogenetic and phenotypic evidence for the transfer of S10
Eubacterium aerofaciens to the genus Collinsella as Collinsella aerofaciens gen. nov., comb. nov. Int J Syst Evol Micr 1999, 49, (2), 557-565.
S11
