Embed Size (px)
Citation preview
Bioresource Technology 192 (2015) 507–513
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier .com/locate /bior tech
Impact of high external circulation ratio on the performance of anaerobicreactor treating coal gasification wastewater under thermophiliccondition
http://dx.doi.org/10.1016/j.biortech.2015.05.1060960-8524/� 2015 Elsevier Ltd. All rights reserved.
⇑ Corresponding author at: School of Municipal and Environmental Engineering,Harbin Institute of Technology, Harbin 150090, China. Tel.: +86 451 87649777; fax:+86 451 86283082.
E-mail address: [email protected] (H. Han).
Shengyong Jia, Hongjun Han ⇑, Haifeng Zhuang, Baolin Hou, Kun LiState Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
h i g h l i g h t s
� External circulation anaerobic reactor was investigated to treat CGW.� External circulation ratio (R) played significant roles on pollutants removals.� Increasing R significantly improved the biodegradability of CGW.� R influenced anaerobic granular sludge and particle size distribution.� Pollutants removals were related to the main bacterial community shift at each R.
a r t i c l e i n f o
Article history:Received 21 April 2015Received in revised form 26 May 2015Accepted 27 May 2015Available online 10 June 2015
Keywords:Coal gasification wastewaterExternal circulationAnaerobic reactorHigh-throughput sequencingBiodegradability improvement
a b s t r a c t
A laboratory-scale external circulation anaerobic reactor (ECAR) was developed to treat actual coal gasi-fication wastewater. The external circulation ratio (R) was selected as the main operating variable foranalysis. From the results, with the hydraulic retention time of 50 h, pH > 8.0 and R of 3, the COD, totalphenols, volatile phenol and NH4
+-N removal efficiencies were remarkably increased to 10 ± 2%,22 ± 5%, 18 ± 1%, and �1 ± 2%, respectively. Besides, increasing R resulted in more transformation frombound extracellular polymeric substances (EPS) to free EPS in the liquid and the particle size distributionof anaerobic granular sludge accumulated in the middle size range of 1.0–2.5 mm. Results showed thegenus Saccharofermentans dominanted in the ECAR and the bacterial community shift was observed atdifferent external circulation ratio, influencing the pollutants removal profoundly.
� 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Due to increasing awareness on environmental issues and tightenvironmental regulations, treatment of industrial wastewater hasbeen the key aspect of research on wastewater treatment (Asadiet al., 2012). Coal gasification wastewater (CGW) represents unpre-dictable toxicological and eco-toxicological effects for the diversi-fied high concentrated constituents, such as phenolic compounds,ammonia, heterocyclic and polycyclic aromatic hydrocarbons, longchain hydrocarbons, thiocyanate (SCN�) and cyanide (Wang et al.,2011), and if disposed of without adequate treatment can causeserious environmental pollution. In addition to the toxicity, this
wastewater is characterized by the extremely low biodegradabil-ity, resulting in great difficulty to the biological treatment.
Anaerobic process is an attractive option for the treatment ofhigh-strength wastewater (Chan et al., 2009) and variations ofinnovative processes have been investigated to treat CGW.Two-continuous up-flow anaerobic sludge bed (UASB) reactorshave been investigated to treat CGW, which represented signifi-cantly improved biodegradability (Wang et al., 2011). Powderedactivated carbon was added to weaken the inhibition of phenoliccompounds to the CGW treatment in a UASB reactor (Wang andHan, 2012). Oxygen-limited aeration was conducted in an anaero-bic expanded-bed granular activated carbon reactor and this recov-ery strategy could be helpful to relieve the inhibitory effect ofphenolic compounds and restore metabolic activity of anaerobicbacteria (Wang et al., 2014). In addition, bio-augmented anaerobicprocess has also been investigated by methanol addition toimprove the biodegradability for CGW (Wang et al., 2010).
Table 1Operational parameters of continuous experiments.
Stage Period (d) HRT (h) pH R
I 1–30 30 >8.0 131–60 40 >8.0 161–90 50 >8.0 191–120 60 >8.0 1
II 121–150 50 >8.0 1151–180 50 6.5–7.5 1181–210 50 6.5–7.5 1
III 211–270 50 >8.0 1271–330 50 >8.0 2331–390 50 >8.0 3391–450 50 >8.0 4
HRT, hydraulic retention time; R was defined as the ratio of the recirculation flowrate to that of the influent flow rate.
508 S. Jia et al. / Bioresource Technology 192 (2015) 507–513
However, the major obstacles of these processes were big foot-print (two stages UASB) and high agent consumption (powderedactivated carbon and methanol). In order to resolve these prob-lems, external circulation anaerobic reactor (ECAR) was developedon the basis of UASB reactor to further relieve the inhibition oftoxic and refractory compounds to the bacterial activity andimprove the biodegradability. Particularly, the external circulationwas defined as the approach that effluent of sludge water from theUASB reactor was used to recycle outside. It was suggested thatexternal circulation could speed the upflow velocity (Vup) in thereactor at a specific HRT, decrease the burden of solid–liquid sep-aration of three-phase separator, enhance the contact betweenCGW and anaerobic granular sludge (AGS), and improve the effec-tiveness of treatment (Yu and Lu, 2014). Moreover, to some extent,the influent loading has been gradually decreased as the externalcirculation ratio increased. Additionally, thermophilic conditionshould be paid more attention using ECAR, due to the residual heatfrom the steaming ammonia process, resulting in high temperaturein the range of 40–50 �C in CGW.
Up to date, very few works have evaluated the effect of highexternal circulation ratio (P1, external circulation flow rate:influ-ent flow rate) on the anaerobic CGW treatment. Hence the presentstudy was undertaken to explore the effect of external circulationratio on COD, total phenols (TPh), volatile phenol (VP) and NH4
+-Nremoval and biodegradation improvement for CGW. Before these,the optimum hydraulic retention time (HRT) and pH range wereinvestigated. Besides, the influence of external circulation ratioon the extracellular polymeric substances (EPS), particle size distri-bution (PSD) of AGS, specific TPh utilization rate (STPh-UR), speci-fic VP utilization rate (SVP-UR) and specific methanogenic activity(SMA) were discussed. Additionally, according to thehigh-throughput sequencing technology, relationships betweenmain bacterial community and pollutants removal at each externalcirculation ratio were explored.
2. Methods
2.1. Experimental setup, inoculums and CGW characteristics
The ECAR was made of plexiglas with the working volume of34.5 L and operated around 45–50 �C. The inoculated anaerobicactivated sludge was taken from the UASB tank treating CGW fromthe wastewater treatment plant (UASB – oxygen limited aerationprocess–three stages anoxic/oxic process–ozone oxidation pro-cess–biological aerated filter) and the suspended solids in theECAR were about 10 g/L.
The CGW used in this study mainly included (in mg/L): COD of3200–3500, BOD of 700–850, TPh of 750–850, VP of 350–550,NH4
+-N of 250–300, volatile acids of 10–18, sulfide of 30–60,SCN� of 55–110, CN� of 1–7 and total alkalinity of 13–16 mM/L,pH of 8.0–9.5.
2.2. Experimental procedures
The influent and recirculation were obtained by peristalticpumps (BT100 2 J, Longer pump, China). The continuous experi-ment has been divided into 3 stages and the parameters of HRT,pH and external circulation ratio were investigated in stage I (days1–120), II (days 121–210) and III (days 211–450), respectively.Dilute sulfuric acid was used to adjust the pH of influent CGW.The continuous operational strategy was outlined in Table 1.
In order to evaluate the influence of external circulation ratio onthe AGS, the concentrations of EPS in terms of protein and polysac-charide were determined every 5 days, which thought to bedetached from the surface of AGS. Besides, the PSD of AGS was also
determined on the last day of each period in stage III. The concen-trations of COD, BOD, TPh, VP and NH4
+-N were analyzed every2 days. STPh-UR, SVP-UR and SMA were analyzed every 5 days.Additionally, in order to represent the data more objectively, allthe parameters described in above were determined in triplicate;furthermore, the data in stages I and II (Fig. 1) and STPh-UR,SVP-UR, SMA and EPS in stages III (Table 2 and Fig. 3a) were repre-sented as mean values ± the standard error; while the COD, BOD,TPh, VP, NH4
+-N and PSD in stages III were represented as mean val-ues (Figs. 2 and 3b).
Four anaerobic activated sludge samples were collected fromthe bioreactor on the last day at each external circulation ratio(day 270, 330, 390 and day 450) for the bacterial communityanalysis.
2.3. Analytical methods
COD, BOD, TPh, VP and NH4+-N were measured according to
Standard Methods (section 5220 D, 5210 B, 5530 D, 6420 B and4500 G, respectively) (AHPA, 1998). DO and pH values were deter-mined daily with a hybrid meter (30 d, HACH, USA). EPS were usedin the protein and polysaccharide assays (Taimur Khan et al.,2013). The measurements of PSD of AGS were conducted using alaser particle size analyzer (Mastersizer 2000, Malvern, UK).Biogas production was determined by a gasflow meter andmethane content was analyzed using a 3 M NaOH solution.
Triplicate activated sludge samples at each external circulationratio were collected and stored at �80 �C before use. Genomic DNAof the 4 sludge samples was extracted using the method of Maet al. (2015), the DNA concentration was determined using aNanoDrop (NanoDrop Technologies Inc., Wilmington, DE, USA).The extracted DNA was amplified by using a set of primers with341F primer (CCTACACGACGCTCTTCCGATCTNCCTACGGGNGGCWGCAG) and 805R primer (GACTGGAGTTCCTTGGCACCCGAGAATTCCAGACTACHVGGGTAT CTAATCC) targeting the V3–V4 region ofbacterial 16S rRNA gene. The PCR conditions were set as follows:initial denaturation at 94 �C for 3 min, followed by 5 cycles ofdenaturation at 94 �C for 30 s, annealing at 45 �C for 20 s, andextension at 65 �C for 30 s; and then 20 cycles of denaturation at94 �C for 20 s, annealing at 55 �C for 20 s, and extension at 72 �Cfor 30 s and then final extension at 72 �C for 5 min.
The PCR products were determined by pyrosequencing usingMiseq Illumina by Sangon Biotech (Shanghai, China) Co., Ltd. Toobtain the effective sequencing data, raw pyrosequencing resultswere processed by the method of Zhang et al. (2015).
The resulting high quality sequences were processed to gener-ate operational taxonomic units (OTUs) by CD-HIT at the 97%sequence similarity threshold. The taxonomic assignment was
-20
-15
-10
-5
0
5
10
15
20
pH>8.0 7.5>pH>6.5
Pollutants
Rem
ov
al e
ffic
ien
cy (
%)
COD TPh VP
NH4
+-N
BOD/COD
-1.0-0.9-0.8-0.7-0.6-0.5-0.4-0.3-0.2-0.10.00.10.20.30.40.50.60.70.80.91.0
BO
D/C
OD
-35
-30
-25
-20
-15
-10
-5
0
5
10
15
20
25
30
30 h 40 h 50 h 60 h
BO
D/C
OD
Rem
ov
al e
ffic
ien
cy (
%)
BOD/COD
NH4
+-N
VPTPhCOD
Pollutants
-1.0-0.9-0.8-0.7-0.6-0.5-0.4-0.3-0.2-0.10.00.10.20.30.40.50.60.70.80.91.0
(a)
(b)
Fig. 1. Removal of COD, TPh, VP and NH4+-N and BOD/COD ratio variations with the increasing HRT (a) and pH adjustment (b). (Values represent the mean values in each
period in stages I and II, error bars represent standard deviation of triplicate tests.)
Table 2Specific TPh utilization rate (STPh-UR), specific VP utilization rate (SVP-UR) andspecific methanogenic activity (SMA) tests at different external circulation ratio.
R STPh-UR SVP-UR SMAmgTPh/(g VSS d) mgVP/(g VSS d) mgCOD-CH4/(g VSS d)
1 103 ± 16a 72 ± 14 85 ± 112 67 ± 12 34 ± 14 100 ± 163 135 ± 29 53 ± 12 118 ± 154 105 ± 8 41 ± 4 109 ± 12
a Values represent the mean values ± the standard error in each period in stageIII.
S. Jia et al. / Bioresource Technology 192 (2015) 507–513 509
performed with the RDP classifier with a confidence cutoff of 0.5.The average data were calculated for each sample before analyzingthe unique and shared OTUs/genera.
3. Results and discussion
3.1. HRT and pH
It has been suggested that the parameter of HRT representedsignificant influence on the hydraulic conditions and reaction timeamong different reactants within the reactor (Rosman et al., 2014).Effects of HRT on COD, TPh, VP and NH4
+-N removal and BOD/CODimprovement are shown in Fig. 1a. With the increasing HRT from30 to 60 h, result showed COD removal up to 15% and BOD/CODhigher than 0.55, which relied on the prolonged reaction time
and abundant increase in bacterial density and accumulation ofrod-shaped bacteria dispersed on the AGS surface (Figure notshown), these were consistent with previous studies (Prakashand Gupta, 2000; Ramakrishnan and Gupta, 2008). It was foundthat VP removal stayed in a high and stable level at HRT of 50and 60 h, giving the average efficiencies of 20%. However, suddendecreases of TPh removal occurred at the HRT of 60 h, resultingin values as low as 9%, which clarified too high HRT played theadverse effect on TPh removal.
Result showed negative NH4+-N removal in ECAR, probably due
to lower NH4+-N degradation rate than generation rate for the pres-
ence of organic nitrogen and SCN� in the influent, since the formerwas transformed into NH4
+-N and the latter into NH4+-N, CO2 and
SO42� during the hydrolysis process (Vázquez et al., 2006). While
a sudden improved NH4+-N removal occurred when the HRT was
increased from 30 to 50 h, probably owing to the prolong timefor the degradation.
For the high alkalinity of the actual CGW, influence of pH shouldbe noticed. As shown in Fig. 1b, it is clearly shown that the overallperformance with higher pH (>8.0) was better than in range of 6.5–7.5, the reasons might be that the anaerobic bacteria have accli-mated the high pH condition representing high activity; however,with neutral pH, some bacterial activity was inhibited. Liang et al.(2013) has demonstrated that all the pH (2.5–10.5) in the anaero-bic systems moved towards the neutral direction, but in this study,the effluent pH increased to be higher than influent due to thecomplex oxidation-reduction processes.
0
500
1000
1500
2000
2500
BOD
R=4R=3R=2BO
D (
mg
/L)
R=1
0
500
1000
1500
2000
2500
3000
3500
CO
D (
mg
/L)
0
5
10
15
Rem
ov
al e
ffic
ien
cy (
%)
0.0
0.2
0.4
0.6
0.8
1.0
BOD/COD
BO
D/C
OD
0
100
200
300
400
500
600
700
800
900
1000
Influent Effluent Time(d)
R=4R=3R=2
TP
h (
mg
/L)
R=10
5
10
15
20
25
30
35
450360330270
Removal efficiency
Rem
ov
al e
ffic
ien
cy (
%)
2100
50
100
150
200
250
300
350
Influent Effluent
R=4R=3R=2
NH
4+-N
(mg
/L)
Time (d)
R=1
270210 330 360 450-15
-10
-5
0
5
10
Removal efficiency
Rem
ov
al e
ffic
ien
cy (
%)
0
100
200
300
400
500
600
R=4R=3R=2
VP
(m
g/L
)
R=10
5
10
15
20
25
30
Rem
ov
al e
ffic
ien
cy (
%)
(a) (c)
(b) (d)
Fig. 2. Removal of (a) COD, (b) TPh, (c) VP and (d) NH4+-N and BOD/COD ratio variations with the increasing external circulation ratio (R).
510 S. Jia et al. / Bioresource Technology 192 (2015) 507–513
Relying on the above results, the optimal parameters were HRTof 50 h and pH of higher than 8.0. Notwithstanding the ECAR per-formance was improved, the overall pollutants removal andBOD/COD ratio remained low. Therefore, further investigationrelated to external circulation was carried out.
3.2. External circulation ratio
3.2.1. Pollutants removalAs shown in Fig. 2a, COD removal dramatically fluctuated at dif-
ferent external circulation ratio, while the gradually increasingBOD/COD ratios were observed, ranging from 0.50 to 0.77, how-ever, the steady level of BOD/COD ratios was found at R of 4, prob-ably due to the increase of recalcitrance in the reactor. Sharpdecrease of COD removal occurred when external circulation ratioincreased to 2, probably due to the sudden speeding Vup whichchanged the living environment and the AGS structure, but theremoval recovered soon along with operation time. Similar TPhand VP removal were found; efficiencies decreased from 15 ± 3%and 22 ± 4% to 12 ± 2% and 8 ± 2%, respectively. It was speculatedthat increasing external circulation ratio played a more efficientrole in biodegradability improvement than COD removal.
Further increased external circulation ratio improved the TPhremoval owing to the diluted influent, which decreased the loadingrate and reduced the adverse effect of toxic substances on thebiodegradation. However, contrast to R of 1, VP removal was inhib-ited at R of 3 and 4, for the reasons that more polyhydric phenolswere degraded to phenol as part of VP. R of 4 played a negativeeffect on pollutants removal, causing the efficiencies of COD, TPhand VP in the low and stable ranges of 4 ± 1%, 17 ± 2% and13 ± 2%, respectively, due to the inhibition of too much metabolicproduct to the anaerobic digestion. Particularly, compared withinfluent, the phenolic compounds in effluent increased 11 kinds,
in which the 2,4,6-trimethyl phenol, 2,3,6-trimethyl phenol and2-ethyl-5-methyl phenol and other alkylphenols were more refrac-tory to biodegrade.
Obviously, the organic matter should be the main sources ofNH4
+-N increment and external recirculation could add additionalNH4
+-N to the reactor. However, in this study, NH4+-N removal effi-
ciencies represented slight increment, values ranging from �9 ± 1%to 3 ± 1% in the plausible steady period and this could be explainedby the recovery of NH4
+-N degradation bacteria. In addition, duringthe whole stage III, the NO2
�-N and NO3�-N were lower than 1 and
3 mg/L, respectively, thus they were not shown in Fig. 2.
3.2.2. Anaerobic granular sludgeIn addition to the dilution effect, enhanced pollutants removal
was also achieved through the influence of external circulationratio on the AGS formation, changing the structure (Costa et al.,2007). AGS represented specific three-layered structures, and outerlayer constituent such as EPS was usually considered to protect theinner microorganisms against the harsh external environmentalconditions (Mu et al., 2012). Additionally, the process of AGSbuildup comprised two steps: the attachment of biomass and thegrowth of biomass, therefore, lower external circulation ratio inthe start-up stage (R of 1) would facilitate the biomass attachment.
Among the hydrodynamic parameters, Vup is the significantaspect that should be studied seriously. Increasing external circu-lation ratio could speed up the Vup in a specific HRT and it wasreported that high Vup could favor mass transfer among phasesand increase interaction between substrate and activated sludge(Dos Reis and Silva, 2011). In addition, increasing external circula-tion ratio enhanced the collision probability and friction strengthamong AGS, promoting the compactness process. However, toohigh external circulation ratio showed adverse effect, probablydue to disruption to the AGS structure by excessive hydraulic shear
1 2 3 40
20
40
60
80
100 2.5-3.0mm 2.0-2.5mm 1.5-2.0mm 1.0-1.5mm 0.5-1.0mm 0-0.5mm
Perc
ent o
f A
GS
per
size
cla
ss (
%)
R
0
5
10
15
20
Protein Polysaccharide
Prot
ein/
Poly
sacc
hari
de (
mg/
L)
(a)
(b)
Fig. 3. Protein and polysaccharide variations (a) and particle size distribution of the anaerobic granular sludge (AGS) (b) with the increasing external circulation ratio (R).(Values represent averages ± standard deviation at each period in stage III.)
S. Jia et al. / Bioresource Technology 192 (2015) 507–513 511
stress, thereby decreasing the pollutants removal when externalcirculation ratio increased to 4. Additionally, too high external cir-culation ratio caused more toxic and harmful metabolic substancesrecycling to the reactor, and this was an important reason whichcould not be neglected.
As shown in Fig. 3a, concentrations of proteins and polysaccha-rides increased from 2 and 8 mg/L (R of 1) to 12 and 17 mg/L (R of4), respectively, revealing more transformation from bound EPS onthe AGS surface to free EPS in the liquid owing to enhanced frictionstrength. Besides, increasing external circulation ratio significantlyaffected the PSD of the AGS (Fig. 3b), thereby influencing the biore-actor performance. Slight variation was observed for the PSD of2.5–3.0 mm when external circulation ratio increased from 1 to2, ranging from 10% to 9%, while it decreased to 2% when the exter-nal circulation ratio further increased to 4; in addition, the PSD of2.0–2.5 mm at R of 4 was 15% lower than R of 3, these results indi-cated the disruption of AGS varied as external circulation ratioincreased. From Fig. 3b, it was speculated that the middle sizeAGS (1.0–2.5 mm) dominanted the main part, PSD percent increasefrom 72% to 82%, relying on the acceleration of biomass growth topromote the AGS formation, while bigger and smaller particle size(2.5–3.0 and 0–1.0 mm) acutely decreased due to the strongcollision.
3.2.3. Variations of STPh-UR, SVP-UR and SMATable 2 presents the variations of STPh-UR, SVP-UR and SMA
with increasing external circulation ratio. As observed, theSTPh-UR was 103 ± 16 mgTPh/(g VSS d) at R of 1 and increased to135 ± 29 mgTPh/(g VSS d) at R of 3. Noticeably, the STPh-UR at R
of 2 was the lowest due to the sudden hydraulic condition change(external circulation ratio ranging from 1 to 2), however, decreas-ing STPh-UR did not dramatically affect the methanogens activity,slight SMA increment has been observed, even the methanogenswere expected to be more sensitive to the phenolic compounds.
While R of 4 decreased STPh-UR to 105 ± 8 mgTPh/(g VSS d) inrelation with the inhibitory effect of metabolic product in the cir-culation liquid (mainly the accumulation of alkylphenols). It wasobserved that SVP-UR with higher external circulation ratios werein a low and stable level, decreasing about 30% at R of 4, comparedwith R of 1. Higher SMA values were recorded at R of 3 with valuesof 118 ± 15 mg COD-CH4/(g VSS d), displaying the highest valuesfor the reactor, yet lower than the study of Wang et al. (2011).This might be due to the thermophilic temperature condition inthis study, resulting in higher ammonia toxicity to methanogens(Gallert and Winter, 1997). To some extent, STPh-UR would influ-ence SMA. Generally, TPh in terms of the polyhydric phenol inCGW were inhibitory to methanogenic bacteria (Wang et al.,2011), but as the external circulation ratio increased, decrease ofTPh removal was observed which did not significantly affect theSMA, revealing the high methanogenic activity after long timeoperation.
3.2.4. Bacterial community shiftPyrosequencing of 16S rRNA gene revealed slight biodiversity
variations in ECAR along with the increasing external circulationratio (data not shown but the biodiversity indices of OTUs andShannon’s diversity index were approximately same). However,results showed that external circulation ratio significantly affected
512 S. Jia et al. / Bioresource Technology 192 (2015) 507–513
the abundance of the most genera present in the bioreactor. Asshown in Fig. 4, among the genera, Saccharofermentans,Comamonas, Bradyrhizobium, Oligotropha, Vulcanibacillus,Brachymonas, Diaphorobacter and Thauera were the eight predom-inant genera (abundance > 2% in any sample) in the bioreactor,with the increasing external circulation ratio. Particularly, genusSaccharofermentans had increased abundance at R of 4, reaching25.85%, which was 2.5 times R of 1. This genus has been reportedas a novelty genus isolated from anaerobic sludge treating brewerywastewater, a representative of this genus, Saccharofermentansacetigenes produces acetate, lactate, fumarate, and trace amountsof molecular hydrogen from several sugars (Chen et al., 2010).Additionally, this genus has also been detected in abiogas-generating microbial community utilizing agriculturalwastes (Ziganshina et al., 2014). Up to date, this is the first timethat the Saccharofermentans was detected as predominant genusin anaerobic bioreactor treating the CGW. Oligotropha carboxidovo-rans is the sole species represented by the genus Oligotropha, char-acterized by the ability to grow with carbon monoxide as a solesource of carbon and energy under denitrifying conditions(Meyer et al., 1993). Genus Desulfacinum was isolated from an oilfield, representing biodegradability of the long chain hydrocarbons(Beeder et al., 1995). And Defluviicoccus has been investigated todetermine the metabolic pathways involved in the anaerobic for-mation of polyhydroxyalkanoates and carbon storage polymers,which was important for the proliferation of microorganisms inenhanced biological phosphorus removal processes (Burow et al.,2009). It has been suggested that genus Afipia represented highability to degrade dioxane in the drainage area of a chemical fac-tory (Sei et al., 2013). Huang et al. (2014) showed thatNovosphingobium was identified as potential tetracycline resistant
1 2 3R
Fig. 4. Abundance of the major bacterial genera (>0.5% in any sample) in the anaerobic ac
bacteria in the sludge cultured with different concentrations oftetracycline. Bacterial genus Sphingomonas, which has been sug-gested to be one of the major polycyclic aromatic hydrocarbons(PAHs) degraders in the environment, was observed in this biore-actor, additionally, bacterial genera Comamonas was able todegrade PAHs (Muangchinda et al., 2014; Zhang et al., 2011).
In this study, pyrosequencing demonstrated that a total of 7genera of potential denitrifiers were detectable in the four sludgesamples, including Comamonas, Bradyrhizobium, Thauera,Hyphomicrobium, Paracoccus, Mesorhizobium and Pseudolabrys(Miao et al., 2015), among which, except for Hyphomicrobium, con-tinuously increasing trend in abundance was observed along withthe increasing external circulation ratio. L’Haridon et al. (2006)showed that Vulcanibacillus could use nitrate as the sole electronacceptor, which was reduced to nitrite and not further reducedto ammonia or N2. Recently, González-Martínez et al. (2013)showed that genus Diaphorobacter was in close relation withpartial-SHARON process and dominanted the bacterial biodiver-sity. Furthermore, the total abundance of these genera increasedfrom 30.59% (R of 1) to 40.65% (R of 4) (average values), this mayexplain the improved NH4
+-N biodegradation along with theincreasing external circulation ratio, but the specific metabolicpathways needed further investigation.
To ascertain if the anaerobic ammonium oxidation contributedfor NH4
+-N removal, the bacteria were identified according to previ-ous literature (Li et al., 2009), and result showed 1.16% of the geneaffiliated to order Planctomycetales, in which, bacteria were closelyrelated to the anaerobic ammonium oxidation, however, typicalanammox bacterium Candidatus Brocadia anammoxidans,Candidatus Kuenenia stuttgartiensis and Nitrosomonas eutrophawere not detected in this study, therefore the anaerobic
4
-2.500
-2.000
-1.500
-1.000
-0.5000
RudaeaBeijerinckiaPetrimonasSphingomonasMeniscusPlanctomycesLevilineaSyntrophusAquicellaMethylocystisParachlamydiaSyntrophomonasSphingopyxisLongilineaNovosphingobiumSyntrophorhabdusDesulfacinumAlkaliflexusDefluviicoccusRhodoplanesAfipiaPseudolabrysMesorhizobiumParacoccusNeochlamydiaHyphomicrobiumThaueraDiaphorobacterBrachymonasVulcanibacillusOligotrophaBradyrhizobiumComamonasSaccharofermentans
lg( Relative Abundance )
tivated sludge samples of the bioreactor with different external circulation ratio (R).
S. Jia et al. / Bioresource Technology 192 (2015) 507–513 513
ammonium oxidation process might not take place in the bioreac-tor (Kuenen, 2008).
The genera of Methanosaeta, Methanobacterium and Methano-spirillum (not shown in Fig. 4) were detected in low abundanceat R of 4, for 0.25%, 0.02% and 0.04% (average values), respectively,and these were of Methanomicrobiales order with the ability to pro-duce methane (Demirel and Scherer, 2008). Low abundancerevealed low performance of the methanogenic bacteria, mainlydue to the toxicity of high concentrations ammonia (Demirel andScherer, 2008), thereby resulting in low specific methanogenicactivity. In addition, it was found that the TPh and VP removal effi-ciencies both decreased when external circulation ratio increasedfrom 3 to 4 (Fig. 2b and c), therefore, aggravated inhibition bythe recycling phenolic compounds to methanogenic bacteria repro-duction could be another reason for the low methane productionwhich could not be neglected.
4. Conclusions
The study showed external circulation ratio played significanteffects on the ECAR performance with HRT of 50 h and pH higherthan 8.0. At R of 3, COD, TPh, VP and NH4
+-N removal efficienciesreached 10 ± 2%, 22 ± 5%, 18 ± 1%, and �1 ± 2%, respectively,besides, BOD/COD ratios stayed in a high and stable range of0.64–0.74, revealing enhanced biodegradability. As external circu-lation ratio increased, the free EPS increased and PSD of AGS accu-mulated in the middle size range, and variations of STPh-UR,SVP-UR and SMA were observed. Additionally, results demon-strated the pollutants removal was in close relation with the mainbacterial community shift.
Acknowledgement
This work was supported by State Key Laboratory of UrbanWater Resource and Environment, Harbin Institute of Technology(No. 2015DX02).
References
AHPA, 1998. Standard methods for the examination of water and wastewater, 20thed. American Public Health Association, American Water Works Association,Water Environment Federation, Washington, DC.
Asadi, A., Zinatizadeh, A.A.L., Sumathi, S., 2012. Simultaneous removal of carbon andnutrients from an industrial estate wastewater in a single up-flow aerobic/anoxic sludge bed (UAASB) bioreactor. Water Res. 46, 4587–4598.
Beeder, J., Torsvik, T., Lien, T., 1995. Thermodesulforhabdus norvegicus gen. nov., sp.nov., a novel thermophilic sulfate-reducing bacterium from oil field water. Arch.Microbiol. 164, 331–336.
Burow, L.C., Mabbett, A.N., Borrás, L., Blackall, L.L., 2009. Anaerobic centralmetabolic pathways active during polyhydroxyalkanoate production inuncultured cluster 1 Defluviicoccus enriched in activated sludgecommunities. Fems. Microbiol. Lett. 298, 79–84.
Chan, Y.J., Chong, M.F., Law, C.L., Hassell, D.G., 2009. A review on anaerobic–aerobictreatment of industrial and municipal wastewater. Chem. Eng. J. 155, 1–18.
Chen, S., Niu, L., Zhang, Y., 2010. Saccharofermentans acetigenes gen. nov., sp. nov.,an anaerobic bacterium isolated from sludge treating brewery wastewater. Int.J. Syst. Evol. Micr. 60, 2735–2738.
Costa, J.C., Abreu, A.A., Ferreira, E.C., Alves, M.M., 2007. Quantitative image analysisas a diagnostic tool for monitoring structural changes of anaerobic granularsludge during detergent shock loads. Biotechnol. Bioeng. 98, 60–68.
Demirel, B., Scherer, P., 2008. The roles of acetotrophic and hydrogenotrophicmethanogens during anaerobic conversion of biomass to methane: a review.Rev. Environ. Sci. Bio/Technol 7, 173–190.
Dos Reis, C.M., Silva, E.L., 2011. Effect of upflow velocity and hydraulic retentiontime in anaerobic fluidized-bed reactors used for hydrogen production. Chem.Eng. J. 172, 28–36.
Gallert, C., Winter, J., 1997. Mesophilic and thermophilic anaerobic digestion ofsource-sorted organic wastes: effect of ammonia on glucose degradation andmethane production. Appl. Microbiol. Biot. 48, 405–410.
González-Martínez, A., Calderón, K., Albuquerque, A., Hontoria, E., González-López,J., Guisado, I.M., Osorio, F., 2013. Biological and technical study of a partial-
SHARON reactor at laboratory scale: effect of hydraulic retention time. Bioproc.Biosyst. Eng. 36, 173–184.
Huang, K.L., Tang, J.Y., Zhang, X.X., Xu, K., Ren, H.Q., 2014. A comprehensive insightinto tetracycline resistant bacteria and antibiotic resistance genes in activatedsludge using next-generation sequencing. Int. J. Mol. Sci. 15, 10083–10100.
Kuenen, J.G., 2008. Anammox bacteria: from discovery to application. Nat. Rev.Microbiol. 6, 320–326.
L’Haridon, S., Miroshnichenko, M.L., Kostrikina, N.A., Tindall, B.J., Spring, S.,Schumann, P., Stackebrandt, E., Bonch-Osmolovskaya, E.A., Jeanthon, C., 2006.Vulcanibacillus modesticaldus gen. nov., sp. nov., a strictly anaerobic, nitrate-reducing bacterium from deep-sea hydrothermal vents. Int. J. Syst. Evol. Micr.56, 1047–1053.
Li, X.R., Du, B., Fu, H.X., Wang, R.F., Shi, J.H., Wang, Y., Jetten, M.S.M., Quan, Z.X.,2009. The bacterial diversity in an anaerobic ammonium-oxidizing (anammox)reactor community. Syst. Appl. Microbiol. 32, 278–289.
Liang, F., Xiao, Y., Zhao, F., 2013. Effect of pH on sulfate removal from wastewaterusing a bioelectrochemical system. Chem. Eng. J. 218, 147–153.
Ma, Q., Qu, Y.Y., Shen, W.L., Zhang, Z.J., Wang, J.W., Liu, Z.R., Li, D.X., Li, H.J., Zhou, J.T.,2015. Bacterial community compositions of coking wastewater treatmentplants in steel industry revealed by Illumina high-throughput sequencing.Bioresour. Technol. 179, 436–443.
Meyer, O., Stackebrandt, E., Auling, G., 1993. Reclassification of Ubiquinone Q-10Containing Carboxidotrophic Bacteria: Transfer of ‘‘[Pseudomonas]carboxydovorans’’ OM5T to Oligotropha, gen. nov., as Oligotrophacarboxidovorans, comb. nov., Transfer of ‘‘[Alcaligenes] carboxydus’’ DSM1086T to Carbophilus, gen. nov., as Carbophilus carboxidus, comb. nov.,Transfer of ‘‘[Pseudomonas] compransoris’’ DSM 1231T to Zavarzinia, gen.nov., as Zavarzinia compransoris, comb. nov., and Amended Descriptions of theNew Genera. Syst. Appl. Microbiol. 16, 390–395.
Miao, Y., Liao, R.H., Zhang, X.X., Wang, Y., Wang, Z., Shi, P., Liu, B., Li, A.M., 2015.Metagenomic insights into Cr(VI) effect on microbial communities andfunctional genes of an expanded granular sludge bed reactor treating high-nitrate wastewater. Water Res. 76, 43–52.
Mu, H., Zheng, X., Chen, Y.G., Chen, H., Liu, K., 2012. Response of anaerobic granularsludge to a shock load of zinc oxide nanoparticles during biological wastewatertreatment. Environ. Sci. Technol. 46, 5997–6003.
Muangchinda, C., Chavanich, S., Viyakarn, V., Watanabe, K., Imura, S., Vangnai, A.S.,Pinyakong, O., 2014. Abundance and diversity of functional genes involved inthe degradation of aromatic hydrocarbons in Antarctic soils and sedimentsaround Syowa Station. Environ. Sci. Pollut. R. 22, 4725–4735.
Prakash, S.M., Gupta, S.K., 2000. Biodegradation of tetrachloroethylene in upflowanaerobic sludge blanket reactor. Bioresour. Technol. 72, 47–54.
Ramakrishnan, A., Gupta, S.K., 2008. Effect of hydraulic retention time on thebiodegradation of complex phenolic mixture from simulated coal wastewater inhybrid UASB reactors. J. Hazard. Mater. 153, 843–851.
Rosman, N.H., Nor Anuar, A., Chelliapan, S., Md Din, M.F., Ujang, Z., 2014.Characteristics and performance of aerobic granular sludge treating rubberwastewater at different hydraulic retention time. Bioresour. Technol. 161, 155–161.
Sei, K., Miyagaki, K., Kakinoki, T., Fukugasako, K., Inoue, D., Ike, M., 2013. Isolationand characterization of bacterial strains that have high ability to degrade 1,4-dioxane as a sole carbon and energy source. Biodegradation 24, 665–674.
Taimur Khan, M.M., Takizawa, S., Lewandowski, Z., Habibur Rahman, M., Komatsu,K., Nelson, S.E., Kurisu, F., Camper, A.K., Katayama, H., Ohgaki, S., 2013.Combined effects of EPS and HRT enhanced biofouling on a submerged andhybrid PAC-MF membrane bioreactor. Water Res. 47, 747–757.
Vázquez, I., Rodríguez, J., Marañón, E., Castrillón, L., Fernández, Y., 2006.Simultaneous removal of phenol, ammonium and thiocyanate from cokewastewater by aerobic biodegradation. J. Hazard. Mater. 137, 1773–1780.
Wang, W., Han, H.J., 2012. Recovery strategies for tackling the impact of phenoliccompounds in a UASB reactor treating coal gasification wastewater. Bioresour.Technol. 103, 95–100.
Wang, W., Han, H.J., Yuan, M., Li, H.Q., 2010. Enhanced anaerobic biodegradability ofreal coal gasification wastewater with methanol addition. J. Environ. Sci. 22,1868–1874.
Wang, W., Han, H.J., Yuan, M., Li, H.Q., Fang, F., Wang, K., 2011. Treatment of coalgasification wastewater by a two-continuous UASB system with step-feed forCOD and phenols removal. Bioresour. Technol. 102, 5454–5460.
Wang, W., Zhang, J., Wang, S., Shen, J., Pan, S.L., 2014. Oxygen-limited aeration forrelieving the impact of phenolic compounds in anaerobic treatment of coalgasification wastewater. Int. Biodeter. Biodegr. 95 (Part A), 110–116.
Yu, Y., Lu, X., 2014. Start-up performance and granular sludge features of animproved external circulating anaerobic reactor for algae-laden watertreatment. Saudi J. Biol. Sci. http://dx.doi.org/10.1016/j.sjbs.2014.09.011.
Zhang, J.X., Zhang, Y.B., Quan, X., Chen, S., 2015. Enhancement of anaerobicacidogenesis by integrating an electrochemical system into an acidogenicreactor: effect of hydraulic retention times (HRT) and role of bacteria andacidophilic methanogenic Archaea. Bioresour. Technol. 179, 43–49.
Zhang, S.Y., Wan, R., Wang, Q.F., Xie, S.G., 2011. Identification of anthracenedegraders in leachate-contaminated aquifer using stable isotope probing. Int.Biodeter. Biodegr. 65, 1224–1228.
Ziganshina, E.E., Bagmanova, A.R., Khilyas, I.V., Ziganshin, A.M., 2014. Assessment ofa biogas-generating microbial community in a pilot-scale anaerobic reactor. J.Biosci. Bioeng. 117, 730–736.
