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Chemosphere 204 (2018) 227e234

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Turf soil enhances treatment efficiency and performance of phenolic

wastewater in an up-flow anaerobic sludge blanket reactor
Chunmao Chen a, Xianyang Yao a, Qing X. Li b, Qinghong Wang a, *, Jiahao Liang a,
Simin Zhang a, Jie Ming a, Zhiyuan Liu a, Jingmin Deng a, Brandon A. Yoza c, **
State Key Laboratory of Heavy Oil Processing, State Key Laboratory of Petroleum Pollution Control, China University of Petroleum, Beijing 102249, China
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA
Hawaii Natural Energy Institute, University of Hawaii at Manoa, Honolulu, HI 96822, USA

h i g h l i g h t s

 Turf soil was novel used in a UASB reactor for phenolic wastewater treatment.
 Turf soil improved performance and resilience to impact loading.
 Bigger granules were formed with better settling property with turf soil.
 Humic substances in turf soil facilitated phenol biodegradation.
 Rough surface and mesopores provided habitat for bacteria growth.

a r t i c l e i n f o a b s t r a c t

Article history: Phenols are industrially generated intermediate chemicals found in wastewaters that are considered a
Received 5 December 2017 class of environmental priority pollutants. Up-flow anaerobic sludge blanket (UASB) reactors are used for
Received in revised form phenolic wastewater treatment and exhibit high volume loading capability, favorable granule settling,
27 March 2018
and tolerance to impact loads. Use of support materials can promote biological productivity and accel-
Accepted 7 April 2018
Available online 10 April 2018
erate start-up period of UASB. In the present study, turf soil was used as a support material in a mes-
ophilic UASB reactor for the removal of phenols in wastewater. During sludge acclimatization (45e96
Handling Editor: A Adalberto Noyola days), COD and phenols in the treatments were both reduced by 97%, whereas these contents in the
controls were decreased by 81% and 75%, respectively. The phenol load threshold for the turf soil UASB
Keywords: reactor was greater (1200 mg/L, the equivalent of COD 3000 mg/L) in comparison with the control UASB
UASB reactor (900 mg/L, the equivalent of COD 2250 mg/L) and the turf soil UASB reactor was also more
Phenolic wastewater resistant to shock loading. Improved sludge settling, shear resistance, and higher biological activity
Turf soil occurred with the turf soil UASB reactor due to the formation of large granular sludge (0.6 mm or larger)
Sludge granulation
in higher relative percentages. Granular sludge size was further enhanced by the colonization of fila-
Microbial community
mentous bacteria on the irregular surface of the turf soil.
© 2018 Published by Elsevier Ltd.

1. Introduction Environmental Protection Agency (Office of the Federal

Registration, 1982). Physical, chemical and biological methods
Phenols are intermediate chemicals found in contaminated and combinations of them are used for the treatment of phenolic
wastewater generated from industrial processes, e.g., coking, oil wastewaters (Jiang et al., 2016; Khaksar et al., 2017). Physical and
refining, and plastic and textile manufacturing (Veeresh et al., chemical processes using solvent extraction and activated carbon
2005). Phenols are considered priority pollutants by the US adsorption are especially attractive for the treatment of wastewater
having high phenol concentrations. Environmentally friendly and
cost effective biological treatment processes are feasible for con-
* Corresponding author. centrations of phenol having a range between 5 and 500 mg/L
** Corresponding author. (Patterson, 1975). Aerobic biological removal of phenols have been
E-mail addresses: (Q. Wang),
successfully applied in an activated sludge system (Cozma et al.,
(B.A. Yoza).
0045-6535/© 2018 Published by Elsevier Ltd.
228 C. Chen et al. / Chemosphere 204 (2018) 227e234

2012) and biofilm reactors to achieve a high phenol and COD 2.3. UASB configuration and experimental procedure
removal efficiency (above 95%) (Bajaj et al., 2008). Anaerobic bio-
logical processes are preferred as they require no aeration, Two identical UASB reactors were constructed using Plexiglass,
consume low energy, exhibit a high operational efficiency and are having an internal diameter of 74 mm, a height of 1195 mm, and a
strongly resistant to loading impacts (Fang et al., 1996). Many total volume of 4.715 L. The experimental reactor contained 0.15 L
studies for the treatment of phenolic wastewater using anaerobic of turf soil (R1), and the control reactor (R0) did not include any
bioreactors have been performed (Suidan et al., 1983; Chou and support materials. The phenolic wastewater was introduced
Huang, 2005; Scully et al., 2006; Subramanyam and Mishra, through the bottom of each reactor using peristaltic pumps and
2007; Chen et al., 2009; Wang et al., 2017a,b). flow moved upward through the granular sludge. Samples of pro-
Up-flow anaerobic sludge blanket (UASB) reactors have favor- duced biogas were collected from the top of the reactor using a
able performance characteristics when compared with other re- three-phase separator and a 15 L gas collection bag. The biogas
actors. UASB reactors are able to accept high volume loads, have volume was measured using a 150 mL syringe. The experiment was
favorable granule settling, and are more resistant to impact shock. performed using thermostatic conditions (36 ± 2  C) maintained
An existing problem for UASB reactors are long start-up times and with a circulating water jacket. A schematic diagram of the UASB
slow granule formation. Anaerobes would be sensitive to phenols at reactor was shown in Supplementary Fig. s2.
the initial stages. The addition of support materials has been shown The UASB reactors were continuously operated for 180 days. The
to promote biological productivity and reduce start-up time influent composition and COD concentrations were shown in Fig. 1.
(Schmidt and Ahring, 1996; Pol et al., 2004). Support substrates that To initially enhance heterotrophic bioactivity, glucose (Beijing
have been successfully used to facilitate the formation of biologi- Chemical Reagents Co., China) was added with newly introduced
cally active granular sludge include; zeolite, oyster shell, activated sludge during the reactor startup. The experimental timeline pe-
carbon, ceramsite and fly ash (Schmidt and Ahring, 1996; Mila n riods are defined as; start-up (the first 45 days), acclimatization
et al., 2010). (day 45e96), steady state (day 96e127) and shock and recovery
In this study, turf soil was investigated for use as a novel support (day 127e180). During the start-up period, the influent COD was
material in a UASB reactor for the treatment of phenolic waste- gradually increased to 2000 mg/L and the volume load (OLR) to
water. Turf soil is a widely available natural material that has an 1.8 kg-COD/m3,day. During the acclimatization period, the influent
irregular surface area. This irregular surface provides a nucleation glucose concentration was reduced to zero, and the phenol con-
point promoting sludge granulation. Furthermore, humic sub- centration simultaneously increased to 800 mg/L. After 70 days
stances contained in turf soil can promote biodegradation process. additional turf soil (1.5% of the total volume) was added to reactor
The utilization of turf soil as a substrate in a USAB reactor and its R1. During the steady-state period, influent phenol concentration
effect on sludge granulation and strength, organic removal, was gradually increased until the COD removal rates decreased.
methane production, and system stability were investigated. This point was defined as the phenol load threshold. The R1 reactor
Changes in microbial community structure within the granules was was operated at an influent COD of around 3000 mg/L (completed
also determined. phenolic wastewater, equivalent to 1200 mg/L of phenol), while the
R0 reactor had a maximum of 2250 mg/L (phenol 900 mg/L). After
the reactor was again stabilized, as indicated by its COD removal
2. Materials and methods
rates. Shock exposure included changes in temperature, pH and the
addition of high concentrations of phenol. To test for low-
2.1. Turf soil and seed sludge
temperature resilience, at day 127 the temperature was
decreased to 17  C for 24 h. To determine how the influx of high
Turf soil was purchased from Xingtong Trading Company
concentrations of phenol impacts its productivity, influent having
(Baishan, Jilin, China). It was oven dried at 105  C and ground in a
concentrations of 1500 mg/L and 2000 mg/L were added to R0 and
quartz mortar. The ground turf soil was then sieved through a 60-m
R1, respectively, at day 133. At day 139, the pH was decreased to 2.2
mesh and then a 200-m mesh sieve to collect particles having di-
and then increased to 9.45 at day 146 to investigate the impacts of
ameters 0.076e0.25 mm. Total organic carbon (TOC) and the
exposure to acid/alkaline conditions. The recovery time and the
chemical oxygen demand (COD) from the turf soil was shown in
performance of the two reactors were determined after exposure to
Fig. s1 (supplementary material).
the variables. The pH, COD, biogas production and composition
Sludge used for seeding was obtained from a hydrolytic acidi-
were measured throughout the experiment.
fication tank in the wastewater treatment plant at Liaohe Petro-
chemical Company (Panjin, Liaoning, China). The total suspended
solids (TSS), volatile suspended solids (VSS), sludge settling ratio 2.4. Analytical methods
(SV), and sludge volume index (SVI) of the seed sludge was 57.07 g/
L, 98 and 12.78 mL/g, respectively. The low SMA of 0.0642 g CH4- Standard methods of the American Public Health Association
COD/gVSS∙d suggested low methanogenic activity. were used to determine COD, total suspended solids (TSS), volatile
suspended solids (VSS), mixed liquid suspended solids (MLSS),
2.2. Synthetic phenolic wastewater concentration of phenol and volatile fatty acid (VFA) (APHA, 2005).
Sludge settling ratio (SV30) was the sludge volume after 30 min of
Phenolic wastewater was prepared by dissolving different settling in a 100 mL graduated cylinder. The SVI was calculated as
30 10
amounts of phenol in water. The salts NH4Cl and KH2PO4 were used follows: SVI ¼ SVMLSS .
to prepare solutions having ratios of COD: N: P range from 350:5:1 The pH was measured with a Mettler Toledo MP220 m. Humic
to 500:5:1. Trace elements were added to a final concentration of substances (HS) from soil, including humic acid and fulvic acid
FeCl2, 1275 mg/L; CoCl2, 1091 mg/L; EDTA, 994 mg/L; MnSO4, 888 mg/ contents, were determined according to a previously described
L; ZnSO4, 60 mg/L; NiCl2, 32 mg/L; (NH4)6Mo7O24, 47 mg/L; H2BO3, method (Aivalioti et al., 2012).
49 mg/L; CuCl2, 19 mg/L. Finally, NaHCO3 was added to maintain The composition of biogas was analyzed on a gas chromato-
CaCO3 alkalinity between 0 and 1000 mg/L. The initial influent pH graph (GC 7806, Wenling, China) having a thermal conductivity
was adjusted to 6.5e8.0. detector (TCD) and a Porapak Q packing column. The inlet, analyzer
C. Chen et al. / Chemosphere 204 (2018) 227e234 229

Fig. 1. COD and phenol measurements of the influent feed water for the UASB reactors over the operational period.

and detector temperatures were set at 80  C, 150  C, and 100  C, pH, VFA, volumetric biogas production (VBP) and methane content
respectively. (Fig. 2). During the start-up period, the influent had COD contents
Samples (30 mL) from the sludge blanket were analyzed and of 1000 mg/L and OLR of 1 kg-COD/m3$day. The influent COD was
granular size distributions determined according to a wet-sieve gradually increased until the OLR reached 1.8 kg-COD/m3$day. The
method (Laguna et al., 1999). The morphology of the soil and rate of COD removal for both reactors was over 80%. Biogas pro-
anaerobic sludge was observed under a scanning electron micro- duction reached 0.6 m3/m3$day and had a 60% methane content.
scope (S-3000N SEM, Hitachi, Japan). The granules were left to dry These results show that by supplementing the reactor with glucose,
at ambient temperature before gold sputter coating. The elemental the start-up period for the reactor was 45 days. In a previous study
composition of the soil was determined by energy dispersive X-ray that used only phenol as the sole carbon source, a 3 month start up
spectroscopy (Genesis XM4 EDX, EDAX, USA). The strength of period was reported (Tay et al., 2000).
granules was evaluated according to an ultrasonic method During the acclimatization period (45e96 days), influent phenol
described by Wan et al. (2013) with some modifications. Granular concentrations were gradually increased. After progressively
sludge was harvested and gently rinsed three times with distilled increasing the addition of phenol a decrease in COD removal effi-
water to remove loosely flocculent sludge. The compact granular ciency for both reactors was observed (The removal rate decreased
sludge was collected by centrifugation at 5000 rpm for 5 min to to 60% in R0 and 65% for R1 at day 65). The maximum thresholds
remove the supernatant, and then the equivalent of 25 mg dry were determined to be 1200 mg/L of phenol for R1 and 900 mg/L
biomass was re-suspended into 10 mL centrifugal tube with 5 mL of for R0. Gas production also decreased to 0.15 m3/m3$d as did the
deionized water. The collected granular sludge was gently proportion of total methane content (65.9% for R1 and 55.4% for
dispersed with an ultrasonic homogenizer in an ice bath. Sonication R0). At 70 days, additional turf soil was added into R1. Sixteen days
was intermittently applied having a 3 s (on)-2s (off) cycle. The after the addition both COD and phenol removal rates increased to
sonicated samples were collected and intermittently measured 97% compared with 81% and 75%, respectively, for R0. Biogas pro-
spectrophotometrically at 600 nm, until readings were stable. The duction also increased to 0.65 m3/m3$d in R1 and was 20% higher
rate of A600 change was representative of the relative strength of when compared with R0. The increased VBP is indicative of stim-
granules. ulated biological activity, suggesting that the observed results for
The specific methanogenic activity (SMA) measurements were phenol measurements in the effluent were not entirely due to non-
determined according to Erguder and Demirer (2008). specific interactions with the additional turf material. Acclimati-
zation was achieved at day 96, at this time phenol was the sole
influent carbon source used in both reactors. Stable effluent COD
2.5. Microbial community structure analysis
(30 mg/L) and VBP (0.52 m3/m3$d) was achieved for R1. These re-
sults were significantly better when compared with R0, effluent
The microorganism community structure was investigated us-
COD (250 mg/L) was more than 8 times higher and VBP production
ing next gen pyrosequencing. DNA was extracted using a PowerSoil
(0.27 m3/m3$d) nearly 50% lower.
DNA Isolation kit (OMEGA-soil, Omega Bio-Tek, USA). The V4 region
Adsorption isotherms of phenol were determined to better
of 16S rDNA gene were targeted for PCR amplification using the
understand the interactions between turf soil and phenol. The
515FmodF (50 -GTGYCAGCMGCCGCGGTAA-30 ) and 806RmodR(50 -
adsorption behaviors of phenol followed an equation of
GGACTACNVGGGTWTCTAAT-30 )primer pair. The amplification
lgQe ¼ 1.22lgCe-3.53, where Qe is the equilibrium adsorption ca-
protocol utilized an initial 30 s exposure at 95  C, followed by 27
pacity and Ce is the equilibrium concentration of phenol in water.
cycles at 95  C for 30 s, 55  C for 30 s, and 72  C for 45 s, and a final
The correlation coefficient (R2) was 0.924 and the calculated 1/n
extension at 72  C for 10 min. The PCR products were sent to
value (1.22) was greater than 1. It was determined that the
Majorbio (Shanghai, China) and sequenced using an Illumina MiS-
adsorption behaviors obey the Freundlich adsorption isotherm
ieq platform. The microbial community structure and diversity
model. At the initial phenol concentration of 1000 mg/L, the
were analyzed using the Quantitative Insights into Microbial
adsorption capacity of phenol on the turf soil was only 1.124 mg/g.
Ecology (QIIME) program.
The turf soil did not significantly adsorb phenol, and did not
directly have any significant impact on the elimination of phenol
3. Results and discussion nonspecifically.
During the steady state period (96e127 days), the influent
3.1. Start-up and long-term operation of UASB organic loading rate was increased by phenol addition. The
maximum phenol threshold was determined by a sudden decrease
The reactors performance were determined by changes in COD, in COD removal rate. The threshold for R0 was 900 mg/L (equivalent
230 C. Chen et al. / Chemosphere 204 (2018) 227e234

Fig. 2. Performance of the control (R0) and sample (R1) reactors over the operational period. Organic Loading Rate (OLR) and temperature (a1, b1), Chemical Oxygen Demand (COD)
(a2, b2), pH and Volatile Fatty Acids (VFA) (a3, b3), and Volumetric Biogas Production (VBP) and methane content (a4, b4).

to COD 2250 mg/L) and the OLR 2.0 kg-COD/m3$day. VBP was stably was tolerant of lower influent concentrations but exhibited favor-
produced at a rate of 0.7 m3/m3$day and had a methane content of able HRT (24 h) and phenol removal efficiency.
56%. The turf soil resulted in a higher phenol threshold of 1200 mg/ A 24 h exposure of the reactors to 17  C did not affect the activity
L (equivalent to COD 3000 mg/L) and also results in a higher OLR of of the reactor containing turf soil. The activity of the control how-
2.5 kg-COD/m3∙day. The VBP and methane contents also increased ever decreased and required 3 days for recovery to baseline. These
to 1.1 m3/m3$day and 67%. Using the maximum influent threshold, results indicate that the addition of turf soil provided the reactor
phenol was entirely removed from the R1 reactor and only organic with resiliency against brief low temperature shock, having no
acids remained in the effluents. Residual phenol was still detected impact on its performance.
in the R0 effluent (almost 19.54% of total effluent organics). From a Increasing the influent phenol concentration at day 133
more efficient anaerobic metabolism, 20% greater amounts of (maximum; R0-1500 mg/L and R1- 2000 mg/L) decreased the
phenolic compounds can be rapidly degraded and also 300 mg/L metabolic activity for both reactors. The R0 reactor required double
higher influent concentrations sustained through the use of turf the amount of time for recovery compared with R1 (48 h). During
soil. The highest previously reported influent phenol concentration the recovery period, the effluent VFA content was variable for R0
that was sustained using a UASB reactor was 1260 mg/L (3000 mg (between 100 mg/L and 300 mg/L) and was stable for R1 (120 mg/
COD/L) having an OLR of 6 g-COD/L∙day (Tay et al., 2000). The L). These results indicated that the biological communities were
achieved phenol degradation, and COD removal efficiencies were better maintained and better able to recover from chemical stress
88 and 86%. When compared with the present study, the use of turf exposure.
soil required a lower OLR (2.5 kg-COD/m3∙day), however a 10% Decreasing the reactors pH to 2.2 at day 139 reduced the per-
higher treatment performance was obtained (97% of phenol formance for R0, requiring 5 days for recovery, whereas R1 was
degradation and COD removal efficiency) at similar influent phenol unaffected. Increasing pH to 9.45 on day 146 had a dramatic effect
concentrations. In another study, it was reported that 90% of phenol on UASB stability, biogas production decreased to nearly zero for
was removed from influent having a phenol concentration of both reactors. The effluent VFA increased to 334 mg/L for R0 and
1400 mg/L at HRT of 60 h (Chang et al., 1995). The UASB in our study 223 mg/L for R1. While a recovery period was needed for both
C. Chen et al. / Chemosphere 204 (2018) 227e234 231

reactors, R1 was 4 times shorter. After 4 days, the COD removal Turbidity measurement after the ultrasonic dispersion of the
efficiency for R1 was 90% of baseline while R0 required 16 days. granules was utilized to determine granular strength (Wan et al.,
Many studies suggest that low temperature shock or exposure to 2013; Li et al., 2014). The A600 value increases with increased ul-
high OLR have a negative impact on the UASB performance when trasonic exposure time. Using identical conditions, the A600 values
treating phenolic wastewater. Fang et al. (1996) found that shock for granule containing liquid suspensions from R1 and R0 were
exposure to high OLR, required a subsequent recovery period of 2 stable after 25 min and 15 min of sonication. The longer time
weeks before COD removal rates returned to baseline levels. Re- required to reach stable A600 values for R1 suggests that the
ported exposure to reduced temperature, from 37 to 21  C for 48 h, granules were more difficult to disrupt, implying greater structural
results in decreased in gas productivity and COD removal (Tay et al., integrity. The turf soil may have acted as a nucleation core for
2001). Furthermore, 22 days was required to recover to baseline microorganism attachment, promoting the formation of a more
productivity. In the present study, no changes in productivity were dense granular structure.
observed after transient exposure to low temperatures and also low SEM was used to study the granular sludge morphology
influent pH. Faster recovery times were found after exposure to (Supplementary Fig. s3). The surface of the granular sludge from
alkaline influents and also after increased phenol concentrations. both reactors is rough and uneven. The rough surface provides a
The lower VFA in effluents and higher methane production for R1 suitable surface substrate for microorganism cultivation. The sur-
suggest that hydrolytic-acidification bacteria and methanogenic face appears to include a community of tightly packed microor-
communities were better maintained. These results indicate a ganisms having different morphologies. Bacillus, coccus and
benefit for turf soil use in a UASB reactor. filamentous bacteria are observed along with extracellular poly-
meric substances covering the surface of bacteria. Bacteria can
3.2. Sludge granulation and morphology of granular sludge easily adhere to the rough surface, and filamentous bacteria can
grow within the porous structure. Visually, cross-sectional images
The size, morphology, SVI and strength of the granule from the suggest that there are differences in microbial populations between
two reactors was characterized to study the influence of turf soil. the reactors. Proportionally, a higher content of filamentous bac-
Changes in particle size distribution over the operational period teria was observed with R1 granules. The growth of filamentous
were shown in Fig. 3. The flocculent seed sludge had an average size bacteria can contribute toward the formation of more prominent
of less than 0.067 mm. Granules are defined as particles having granules adding to the higher granule strength that was found in
diameters >0.125 mm (Mieczkowski et al., 2016). The proportion of the R1 reactor.
granules and also particle size increased with operating time. After
60 days of operation, the total granule content was determined to
3.3. Microbial community analysis
be 55% for R1 and 16% R0. The proportion of granular sludge con-
tents having diameters >0.25 mm was significantly higher in R1,
The elemental composition and surface morphology of turf soil
40% compared with 7%. These larger particles increased settling
were further studied using EDX and SEM (Fig. 4). The EDX results
velocity and reduced sludge loss. The SVI for R0 was 64.8 mL/g and
showed that turf soil contained carbon, silicon, oxygen, nitrogen,
40.3 mL/g for R1. The turf soil accelerated the formation of large
sodium, calcium, iron, and aluminum. The turf soil was principally
anaerobic granules during the start-up period, improving sludge
organic, and its primary elemental composition included O (62.41%
wt.), C (17.25% wt.) and Si (16.98% wt.). Further analysis revealed
At day 120, granular particles comprised 80% of the sludge for
that the organic component was comprised of mostly humic
both reactors. Granular sludge contents that have diameters
compounds (48.49%), humic acid (61.5 mg/g) and fulvic acid
>0.25 mm was 67% in R1 and 63% in R0. The SVI for R0 and R1 was
(49.6 mg/g).
37.4 mL/g and 22.7 mL/g. After 180 days the average granular size
Microbes exhibit high rates of respiration in the presence of
further increased for both reactors. Granules having diameters
humic substances. It is reported that only a few milligram of humus
>1 mm was 19% in R1 and 16% in R0. The proportion of large
in the natural water can stimulate the bioconversion of pollutants
granules contributed to better settling that results in lower SVI
(Lovley et al., 1999). Stimulation could potentially be due to the
presence of quinonoid compounds as natural redox mediators that

Fig. 4. Energy-dispersive X-ray (EDX) analysis and scanning electron micrograph

Fig. 3. Changes in granular sludge size distribution over the operational period. (SEM) image of the turf soil.
232 C. Chen et al. / Chemosphere 204 (2018) 227e234

can accelerate the transfer of electrons in the electron transport the stable operation period, portions of bacteria belonging to the
chain (Jiang and Kappler, 2008). Through this mechanism, the Proteobacteria and Firmicutes increased. Proteobacteria accounted
addition of turf soil could contribute to the rapid proliferation of for 7.61% of the total population in the seed sludge and increased to
anaerobes resulting in the accelerated biodegradation of phenol. 28.21% in R0-2 and 17.94% in R1-2. Firmicutes followed a similar
Humic and fulvic acid can assist in the regulation of alkalinity, trend and initially accounted for only 3.31% of the population that
which might explain for the faster recovery times of R1 after increased to 11.32% in R0-2 and 18.57% in R1-2. An abundance of
alkaline exposure. Studies have further investigated the use of phenol-degrading bacteria is known to be Proteobacteria and Fir-
humus as quinonoid redox mediators for the biodegradation of micutes members (Wang et al., 2015).
refractory wastewater. Studies include its immobilization on anion The microbial community distributions for the different samples
exchange resins or nanoparticles (Cervantes et al., 2011, 2013; 2015; were shown in Fig. 5. During the startup period, fermentative
Dos Santos et al., 2005). Turf soil is an economical source that is rich bacteria such as Gastranaerophilales which are capable of convert-
in humus and humic acids. Furthermore, it is readily available and ing glucose, starch, or glycogen into lactate, ethanol, and formate
convenient for use in industrial anaerobic bioreactors. were found in a high proportion (Soo et al., 2014).
Community DNA was sequenced using extracted DNA samples The community distribution changed significantly for both re-
from the initial inoculum, and samples from both reactors during actors during the acclimation stage. There was an increase in the
the acclimation and stable operation periods. The subsequent proportions of detected methanogenic archaea. Methanosaeta and
metagenomic analysis was used to estimate the presence of po- Longilinea which are beneficial for accelerating granular sludge
tential metabolisms present within the biological community. The formation were also present in a high proportion (Li et al., 2015;
presence of many community members from both bacteria and Yamada et al., 2007). Other phyla that have been implicated in
archaea suggest that the formation of granules as well as the phenol degradation include Syntrophus and Trichococcus (Wang
enhanced degradation of phenol can be attributed to specific et al., 2017a,b; Garcia-Mancha et al., 2012). Trichococcus has been
groups. A total of 510 OTUs were assigned from 188,055 sequences detected in an expanded granular sludge bed used for the treat-
with a coverage rate >99% for each sample. ment of industrial wastewater containing phenolic compounds
The microbial diversity of the different samples were shown in (Garcia-Mancha et al., 2012).
Fig. 5. There was 11 phylum having a relative abundance >2%. The The relative abundances of the archaeal populations were
Acetothermia, Euryarchaeota, Chloroflexi, Firmicutes, and Proteo- shown in Table 1. All classified archaea from the three samples were
bacteria were the most dominant and represented 80% of the total found in three genera, one acetoclastic methanogen (Methanosaeta)
community. and two hydrogenotrophic methanogens (Methanobacterium, and
The initial seed sludge was obtained from a hydrolytic- Methanolinea) (Na et al., 2016). The majority of methanogens in the
acidification tank that was determined to have a high proportion seed sludge was principally acetoclastic; Methanosaeta accounted
of hydrolytic-acidification bacteria, including Acetothermia. During for 19.86% of the total archaea population. Methanosaeta was found
to promote granulation, contributing to a rapid and stable UASB
reactor community (Demirel and Scherer, 2008). On the 60th day,
the diversity of the archaea community increased significantly for
both reactors. After introducing phenol into the reactor, the pro-
portion of hydrogenotrophic methanogens increased. Meth-
anobacterium and Methanolinea are often found in biological
communities during anaerobic digestion processes for the treat-
ment of organic wastes, and also in phenol-degrading enrichment
culture (Chen et al., 2008; Zhang et al., 2005). The relative abun-
dance of Methanobacterium increased from 6.02% to 8.15%. Meth-
anolinea rose from 2.61% to 3.02% in R1, whereas Methanobacterium
and Methanolinea decreased in R0.
The humic content of the turf soil could further promote
anaerobic phenol digestion through the provision of redox medi-
ators that can facilitate electron transfer (Piepenbrock et al., 2014).
This anaerobic pathway for the degradation of phenol includes
decyclization, acidification, and methanation. The dominant genera
involved in this process would include Syntrophorhabdus, Tricho-
coccus, Methanosaeta, and Methanobacterium. These groups carry

Table 1
Changes in the relative percent abundance of the dominant Archaea species within
the control reactor (R0) and the turf soil reactor (R1).

Methanogen species Relative content (%)

R0 R1

Methanosaeta 0days 19.68 19.68

60days 23.56 20.92
120days 19.98 19.30
Methanobacterium 0days 2.36 2.36
60days 5.25 6.02
Fig. 5. Changes in diversity of key bacteria acclimated to phenol by next generation
120days 2.69 8.15
sequencing (R0-1 ¼ samples were taken at day 60 of R0, R1-1 ¼ samples were taken at
Methanolinea 0days 1.10 1.10
day 60 of R1, R0-2 ¼ samples were taken at day 120 of R0, R1-2 ¼ samples were taken
60days 1.87 2.61
at day 120 of R1, R0-3 ¼ samples were taken at day 180 of R0, and R1-3 ¼ samples were
120days 0.77 3.02
taken at day 180 of R1).
C. Chen et al. / Chemosphere 204 (2018) 227e234 233

out necessary metabolisms to perform the conversions of [phenol Kinetics during the redox biotransformation of pollutants mediated by immo-
bilized and soluble humic acids. App. Microbial. Biotechnol. 97, 2671e2679.
/ benzoate / acetate þ hydrogen], [acetate / methane], and
Chang, Y.J., Nishio, N., Nagai, S., 1995. Characteristics of granular methanogenic
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This study demonstrates that turf soil can be used as an
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economical support material in UASB reactors for the improved blanket reactors. J. Environ. Eng. 134 (4), 295e303.
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Garcia-Mancha, N., Puyol, D., Monsalvo, V.M., Rajhi, H., Mohedano, A.F.,
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that increase granule strength, and other organisms that impart the phenolic wastewater with high salinity using a novel integrated system of
reactor with higher wastewater processing capabilities and better magnetically immobilized cells coupling with electrodes. Bioresour. Technol.
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also increase the degradation rates of phenol by facilitating the substances: implications for electron shuttling. Environ. Sci. Technol. 42,
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high percentage of methane could be reclaimed for use as an en-
petrochemical wastewater considering turbidity factor by backlight cascade
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Acknowledgements sludge. Water Sci. Technol. 40 (8), 1e8.
Li, L., Zheng, M., Ma, H., Gong, S., Ai, G., Liu, X., Wang, K., Dong, X., 2015. Significant
This work was supported in part by the National Science and performance enhancement of a UASB reactor by using acyl homoserine lactones
to facilitate the long filaments of Methanosaeta harundinacea 6Ac. App. Mi-
Technology Major Project of China (No. 2016ZX05040-001), the crobial. Biotechnol. 99, 6471e6480.
National Natural Science Foundation of China (No. 21776307), and Li, Y., Hao, W., Lv, J., Wang, Y., Zhong, C., Zhu, J., 2014. The role of N-acyl homoserine
the US Office of Naval Research (Grants N00014-09-1-0709 and lactones in maintaining the stability of aerobic granules. Bioresour. Technol.
159, 305e310.
N00014-12-1-0496 284). Lovley, D.R., Fraga, J.L., Coates, J.D., Blunt-Harris, E.L., 1999. Humics as an electron
donor for anaerobic respiration. Environ. Microbiol. 1 (1), 89e98.
Appendix A. Supplementary data Mieczkowski, D., Cydzik-Kwiatkowska, A., Rusanowska, P., Swiatczak, P., 2016.
Temperature-induced changes in treatment efficiency and microbial structure
of aerobic granules treating landfill leachate. World J. Microbiol. Biotechnol. 32
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Borja, R., 2010. The impact of ammonia nitrogen concentration and zeolite
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