Chemosphere 204 (2018) 227e234

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

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, **
a
State Key Laboratory of Heavy Oil Processing, State Key Laboratory of Petroleum Pollution Control, China University of Petroleum, Beijing 102249, China
b
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA
c
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: wangqhqh@163.com (Q. Wang), byoza@hawaii.edu
successfully applied in an activated sludge system (Cozma et al.,
(B.A. Yoza).

https://doi.org/10.1016/j.chemosphere.2018.04.040
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
removal efficiency (above 95%) (Bajaj et al., 2008). Anaerobic bio-
logical processes are preferred as they require no aeration,
consume low energy, exhibit a high operational efficiency and are
strongly resistant to loading impacts (Fang et al., 1996). Many
studies for the treatment of phenolic wastewater using anaerobic
bioreactors have been performed (Suidan et al., 1983; Chou and
Huang, 2005; Scully et al., 2006; Subramanyam and Mishra,
2007; Chen et al., 2009; Wang et al., 2017a,b).
Up-flow anaerobic sludge blanket (UASB) reactors have favor-
able performance characteristics when compared with other re-
actors. UASB reactors are able to accept high volume loads, have
favorable granule settling, and are more resistant to impact shock.
An existing problem for UASB reactors are long start-up times and
slow granule formation. Anaerobes would be sensitive to phenols at
the initial stages. The addition of support materials has been shown
to promote biological productivity and reduce start-up time
(Schmidt and Ahring, 1996; Pol et al., 2004). Support substrates that
have been successfully used to facilitate the formation of biologi-
cally active granular sludge include; zeolite, oyster shell, activated
carbon, ceramsite and fly ash (Schmidt and Ahring, 1996; Mila
n
et al., 2010).
In this study, turf soil was investigated for use as a novel support
material in a UASB reactor for the treatment of phenolic waste-
water. Turf soil is a widely available natural material that has an
irregular surface area. This irregular surface provides a nucleation
point promoting sludge granulation. Furthermore, humic sub-
stances contained in turf soil can promote biodegradation process.
The utilization of turf soil as a substrate in a USAB reactor and its
effect on sludge granulation and strength, organic removal,
methane production, and system stability were investigated.
Changes in microbial community structure within the granules was
also determined.
2. Materials and methods
2.1. Turf soil and seed sludge
Turf soil was purchased from Xingtong Trading Company
(Baishan, Jilin, China). It was oven dried at 105  C and ground in a
quartz mortar. The ground turf soil was then sieved through a 60-m
mesh and then a 200-m mesh sieve to collect particles having di-
ameters 0.076e0.25 mm. Total organic carbon (TOC) and the
chemical oxygen demand (COD) from the turf soil was shown in
Fig. s1 (supplementary material).
Sludge used for seeding was obtained from a hydrolytic acidi-
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
(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-
COD/gVSS∙d suggested low methanogenic activity.
2.2. Synthetic phenolic wastewater
Phenolic wastewater was prepared by dissolving different
amounts of phenol in water. The salts NH4Cl and KH2PO4 were used
to prepare solutions having ratios of COD: N: P range from 350:5:1
to 500:5:1. Trace elements were added to a final concentration of
FeCl2, 1275 mg/L; CoCl2, 1091 mg/L; EDTA, 994 mg/L; MnSO4, 888 mg/
L; ZnSO4, 60 mg/L; NiCl2, 32 mg/L; (NH4)6Mo7O24, 47 mg/L; H2BO3,
49 mg/L; CuCl2, 19 mg/L. Finally, NaHCO3 was added to maintain
CaCO3 alkalinity between 0 and 1000 mg/L. The initial influent pH
was adjusted to 6.5e8.0.
2.3. UASB configuration and experimental procedure
Two identical UASB reactors were constructed using Plexiglass,
having an internal diameter of 74 mm, a height of 1195 mm, and a
total volume of 4.715 L. The experimental reactor contained 0.15 L
of turf soil (R1), and the control reactor (R0) did not include any
support materials. The phenolic wastewater was introduced
through the bottom of each reactor using peristaltic pumps and
flow moved upward through the granular sludge. Samples of pro-
duced biogas were collected from the top of the reactor using a
three-phase separator and a 15 L gas collection bag. The biogas
volume was measured using a 150 mL syringe. The experiment was
performed using thermostatic conditions (36 ± 2  C) maintained
with a circulating water jacket. A schematic diagram of the UASB
reactor was shown in Supplementary Fig. s2.
The UASB reactors were continuously operated for 180 days. The
influent composition and COD concentrations were shown in Fig. 1.
To initially enhance heterotrophic bioactivity, glucose (Beijing
Chemical Reagents Co., China) was added with newly introduced
sludge during the reactor startup. The experimental timeline pe-
riods are defined as; start-up (the first 45 days), acclimatization
(day 45e96), steady state (day 96e127) and shock and recovery
(day 127e180). During the start-up period, the influent COD was
gradually increased to 2000 mg/L and the volume load (OLR) to
1.8 kg-COD/m3,day. During the acclimatization period, the influent
glucose concentration was reduced to zero, and the phenol con-
centration simultaneously increased to 800 mg/L. After 70 days
additional turf soil (1.5% of the total volume) was added to reactor
R1. During the steady-state period, influent phenol concentration
was gradually increased until the COD removal rates decreased.
This point was defined as the phenol load threshold. The R1 reactor
was operated at an influent COD of around 3000 mg/L (completed
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
rates. Shock exposure included changes in temperature, pH and the
addition of high concentrations of phenol. To test for low-
temperature resilience, at day 127 the temperature was
decreased to 17  C for 24 h. To determine how the influx of high
concentrations of phenol impacts its productivity, influent having
concentrations of 1500 mg/L and 2000 mg/L were added to R0 and
R1, respectively, at day 133. At day 139, the pH was decreased to 2.2
and then increased to 9.45 at day 146 to investigate the impacts of
exposure to acid/alkaline conditions. The recovery time and the
performance of the two reactors were determined after exposure to
the variables. The pH, COD, biogas production and composition
were measured throughout the experiment.
2.4. Analytical methods
Standard methods of the American Public Health Association
were used to determine COD, total suspended solids (TSS), volatile
suspended solids (VSS), mixed liquid suspended solids (MLSS),
concentration of phenol and volatile fatty acid (VFA) (APHA, 2005).
Sludge settling ratio (SV30) was the sludge volume after 30 min of
settling in a 100 mL graduated cylinder. The SVI was calculated as
30 10
follows: SVI ¼ SVMLSS .
The pH was measured with a Mettler Toledo MP220 m. Humic
substances (HS) from soil, including humic acid and fulvic acid
contents, were determined according to a previously described
method (Aivalioti et al., 2012).
The composition of biogas was analyzed on a gas chromato-
graph (GC 7806, Wenling, China) having a thermal conductivity
detector (TCD) and a Porapak Q packing column. The inlet, analyzer
 C. Chen et al. / Chemosphere 204 (2018) 227e234
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,
respectively.
Samples (30 mL) from the sludge blanket were analyzed and
granular size distributions determined according to a wet-sieve
method (Laguna et al., 1999). The morphology of the soil and
anaerobic sludge was observed under a scanning electron micro-
scope (S-3000N SEM, Hitachi, Japan). The granules were left to dry
at ambient temperature before gold sputter coating. The elemental
composition of the soil was determined by energy dispersive X-ray
spectroscopy (Genesis XM4 EDX, EDAX, USA). The strength of
granules was evaluated according to an ultrasonic method
described by Wan et al. (2013) with some modifications. Granular
sludge was harvested and gently rinsed three times with distilled
water to remove loosely flocculent sludge. The compact granular
sludge was collected by centrifugation at 5000 rpm for 5 min to
remove the supernatant, and then the equivalent of 25 mg dry
biomass was re-suspended into 10 mL centrifugal tube with 5 mL of
deionized water. The collected granular sludge was gently
dispersed with an ultrasonic homogenizer in an ice bath. Sonication
was intermittently applied having a 3 s (on)-2s (off) cycle. The
sonicated samples were collected and intermittently measured
spectrophotometrically at 600 nm, until readings were stable. The
rate of A600 change was representative of the relative strength of
granules.
The specific methanogenic activity (SMA) measurements were
determined according to Erguder and Demirer (2008).
2.5. Microbial community structure analysis
The microorganism community structure was investigated us-
ing next gen pyrosequencing. DNA was extracted using a PowerSoil
DNA Isolation kit (OMEGA-soil, Omega Bio-Tek, USA). The V4 region
of 16S rDNA gene were targeted for PCR amplification using the
515FmodF (50 -GTGYCAGCMGCCGCGGTAA-30 ) and 806RmodR(50 -
GGACTACNVGGGTWTCTAAT-30 )primer pair. The amplification
protocol utilized an initial 30 s exposure at 95  C, followed by 27
cycles at 95  C for 30 s, 55  C for 30 s, and 72  C for 45 s, and a final
extension at 72  C for 10 min. The PCR products were sent to
Majorbio (Shanghai, China) and sequenced using an Illumina MiS-
ieq platform. The microbial community structure and diversity
were analyzed using the Quantitative Insights into Microbial
Ecology (QIIME) program.
3. Results and discussion
3.1. Start-up and long-term operation of UASB
The reactors performance were determined by changes in COD,
229
pH, VFA, volumetric biogas production (VBP) and methane content
(Fig. 2). During the start-up period, the influent had COD contents
of 1000 mg/L and OLR of 1 kg-COD/m3$day. The influent COD was
gradually increased until the OLR reached 1.8 kg-COD/m3$day. The
rate of COD removal for both reactors was over 80%. Biogas pro-
duction reached 0.6 m3/m3$day and had a 60% methane content.
These results show that by supplementing the reactor with glucose,
the start-up period for the reactor was 45 days. In a previous study
that used only phenol as the sole carbon source, a 3 month start up
period was reported (Tay et al., 2000).
During the acclimatization period (45e96 days), influent phenol
concentrations were gradually increased. After progressively
increasing the addition of phenol a decrease in COD removal effi-
ciency for both reactors was observed (The removal rate decreased
to 60% in R0 and 65% for R1 at day 65). The maximum thresholds
were determined to be 1200 mg/L of phenol for R1 and 900 mg/L
for R0. Gas production also decreased to 0.15 m3/m3$d as did the
proportion of total methane content (65.9% for R1 and 55.4% for
R0). At 70 days, additional turf soil was added into R1. Sixteen days
after the addition both COD and phenol removal rates increased to
97% compared with 81% and 75%, respectively, for R0. Biogas pro-
duction also increased to 0.65 m3/m3$d in R1 and was 20% higher
when compared with R0. The increased VBP is indicative of stim-
ulated biological activity, suggesting that the observed results for
phenol measurements in the effluent were not entirely due to non-
specific interactions with the additional turf material. Acclimati-
zation was achieved at day 96, at this time phenol was the sole
influent carbon source used in both reactors. Stable effluent COD
(30 mg/L) and VBP (0.52 m3/m3$d) was achieved for R1. These re-
sults were significantly better when compared with R0, effluent
COD (250 mg/L) was more than 8 times higher and VBP production
(0.27 m3/m3$d) nearly 50% lower.
Adsorption isotherms of phenol were determined to better
understand the interactions between turf soil and phenol. The
adsorption behaviors of phenol followed an equation of
lgQe ¼ 1.22lgCe-3.53, where Qe is the equilibrium adsorption ca-
pacity and Ce is the equilibrium concentration of phenol in water.
The correlation coefficient (R2) was 0.924 and the calculated 1/n
value (1.22) was greater than 1. It was determined that the
adsorption behaviors obey the Freundlich adsorption isotherm
model. At the initial phenol concentration of 1000 mg/L, the
adsorption capacity of phenol on the turf soil was only 1.124 mg/g.
The turf soil did not significantly adsorb phenol, and did not
directly have any significant impact on the elimination of phenol
nonspecifically.
During the steady state period (96e127 days), the influent
organic loading rate was increased by phenol addition. The
maximum phenol threshold was determined by a sudden decrease
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$day. VBP was stably
produced at a rate of 0.7 m3/m3$day and had a methane content of
56%. The turf soil resulted in a higher phenol threshold of 1200 mg/
L (equivalent to COD 3000 mg/L) and also results in a higher OLR of
2.5 kg-COD/m3∙day. The VBP and methane contents also increased
to 1.1 m3/m3$day and 67%. Using the maximum influent threshold,
phenol was entirely removed from the R1 reactor and only organic
acids remained in the effluents. Residual phenol was still detected
in the R0 effluent (almost 19.54% of total effluent organics). From a
more efficient anaerobic metabolism, 20% greater amounts of
phenolic compounds can be rapidly degraded and also 300 mg/L
higher influent concentrations sustained through the use of turf
soil. The highest previously reported influent phenol concentration
that was sustained using a UASB reactor was 1260 mg/L (3000 mg
COD/L) having an OLR of 6 g-COD/L∙day (Tay et al., 2000). The
achieved phenol degradation, and COD removal efficiencies were
88 and 86%. When compared with the present study, the use of turf
soil required a lower OLR (2.5 kg-COD/m3∙day), however a 10%
higher treatment performance was obtained (97% of phenol
degradation and COD removal efficiency) at similar influent phenol
concentrations. In another study, it was reported that 90% of phenol
was removed from influent having a phenol concentration of
1400 mg/L at HRT of 60 h (Chang et al., 1995). The UASB in our study
was tolerant of lower influent concentrations but exhibited favor-
able HRT (24 h) and phenol removal efficiency.
A 24 h exposure of the reactors to 17  C did not affect the activity
of the reactor containing turf soil. The activity of the control how-
ever decreased and required 3 days for recovery to baseline. These
results indicate that the addition of turf soil provided the reactor
with resiliency against brief low temperature shock, having no
impact on its performance.
Increasing the influent phenol concentration at day 133
(maximum; R0-1500 mg/L and R1- 2000 mg/L) decreased the
metabolic activity for both reactors. The R0 reactor required double
the amount of time for recovery compared with R1 (48 h). During
the recovery period, the effluent VFA content was variable for R0
(between 100 mg/L and 300 mg/L) and was stable for R1 (120 mg/
L). These results indicated that the biological communities were
better maintained and better able to recover from chemical stress
exposure.
Decreasing the reactors pH to 2.2 at day 139 reduced the per-
formance for R0, requiring 5 days for recovery, whereas R1 was
unaffected. Increasing pH to 9.45 on day 146 had a dramatic effect
on UASB stability, biogas production decreased to nearly zero for
both reactors. The effluent VFA increased to 334 mg/L for R0 and
223 mg/L for R1. While a recovery period was needed for both
 C. Chen et al. / Chemosphere 204 (2018) 227e234
reactors, R1 was 4 times shorter. After 4 days, the COD removal
efficiency for R1 was 90% of baseline while R0 required 16 days.
Many studies suggest that low temperature shock or exposure to
high OLR have a negative impact on the UASB performance when
treating phenolic wastewater. Fang et al. (1996) found that shock
exposure to high OLR, required a subsequent recovery period of 2
weeks before COD removal rates returned to baseline levels. Re-
ported exposure to reduced temperature, from 37 to 21  C for 48 h,
results in decreased in gas productivity and COD removal (Tay et al.,
2001). Furthermore, 22 days was required to recover to baseline
productivity. In the present study, no changes in productivity were
observed after transient exposure to low temperatures and also low
influent pH. Faster recovery times were found after exposure to
alkaline influents and also after increased phenol concentrations.
The lower VFA in effluents and higher methane production for R1
suggest that hydrolytic-acidification bacteria and methanogenic
communities were better maintained. These results indicate a
benefit for turf soil use in a UASB reactor.
3.2. Sludge granulation and morphology of granular sludge
The size, morphology, SVI and strength of the granule from the
two reactors was characterized to study the influence of turf soil.
Changes in particle size distribution over the operational period
were shown in Fig. 3. The flocculent seed sludge had an average size
of less than 0.067 mm. Granules are defined as particles having
diameters >0.125 mm (Mieczkowski et al., 2016). The proportion of
granules and also particle size increased with operating time. After
60 days of operation, the total granule content was determined to
be 55% for R1 and 16% R0. The proportion of granular sludge con-
tents having diameters >0.25 mm was significantly higher in R1,
40% compared with 7%. These larger particles increased settling
velocity and reduced sludge loss. The SVI for R0 was 64.8 mL/g and
40.3 mL/g for R1. The turf soil accelerated the formation of large
anaerobic granules during the start-up period, improving sludge
settling.
At day 120, granular particles comprised 80% of the sludge for
both reactors. Granular sludge contents that have diameters
>0.25 mm was 67% in R1 and 63% in R0. The SVI for R0 and R1 was
37.4 mL/g and 22.7 mL/g. After 180 days the average granular size
further increased for both reactors. Granules having diameters
>1 mm was 19% in R1 and 16% in R0. The proportion of large
granules contributed to better settling that results in lower SVI
values.
Fig. 3. Changes in granular sludge size distribution over the operational period.
231
Turbidity measurement after the ultrasonic dispersion of the
granules was utilized to determine granular strength (Wan et al.,
2013; Li et al., 2014). The A600 value increases with increased ul-
trasonic exposure time. Using identical conditions, the A600 values
for granule containing liquid suspensions from R1 and R0 were
stable after 25 min and 15 min of sonication. The longer time
required to reach stable A600 values for R1 suggests that the
granules were more difficult to disrupt, implying greater structural
integrity. The turf soil may have acted as a nucleation core for
microorganism attachment, promoting the formation of a more
dense granular structure.
SEM was used to study the granular sludge morphology
(Supplementary Fig. s3). The surface of the granular sludge from
both reactors is rough and uneven. The rough surface provides a
suitable surface substrate for microorganism cultivation. The sur-
face appears to include a community of tightly packed microor-
ganisms having different morphologies. Bacillus, coccus and
filamentous bacteria are observed along with extracellular poly-
meric substances covering the surface of bacteria. Bacteria can
easily adhere to the rough surface, and filamentous bacteria can
grow within the porous structure. Visually, cross-sectional images
suggest that there are differences in microbial populations between
the reactors. Proportionally, a higher content of filamentous bac-
teria was observed with R1 granules. The growth of filamentous
bacteria can contribute toward the formation of more prominent
granules adding to the higher granule strength that was found in
the R1 reactor.
3.3. Microbial community analysis
The elemental composition and surface morphology of turf soil
were further studied using EDX and SEM (Fig. 4). The EDX results
showed that turf soil contained carbon, silicon, oxygen, nitrogen,
sodium, calcium, iron, and aluminum. The turf soil was principally
organic, and its primary elemental composition included O (62.41%
wt.), C (17.25% wt.) and Si (16.98% wt.). Further analysis revealed
that the organic component was comprised of mostly humic
compounds (48.49%), humic acid (61.5 mg/g) and fulvic acid
(49.6 mg/g).
Microbes exhibit high rates of respiration in the presence of
humic substances. It is reported that only a few milligram of humus
in the natural water can stimulate the bioconversion of pollutants
(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
(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
chain (Jiang and Kappler, 2008). Through this mechanism, the
addition of turf soil could contribute to the rapid proliferation of
anaerobes resulting in the accelerated biodegradation of phenol.
Humic and fulvic acid can assist in the regulation of alkalinity,
which might explain for the faster recovery times of R1 after
alkaline exposure. Studies have further investigated the use of
humus as quinonoid redox mediators for the biodegradation of
refractory wastewater. Studies include its immobilization on anion
exchange resins or nanoparticles (Cervantes et al., 2011, 2013; 2015;
Dos Santos et al., 2005). Turf soil is an economical source that is rich
in humus and humic acids. Furthermore, it is readily available and
convenient for use in industrial anaerobic bioreactors.
Community DNA was sequenced using extracted DNA samples
from the initial inoculum, and samples from both reactors during
the acclimation and stable operation periods. The subsequent
metagenomic analysis was used to estimate the presence of po-
tential metabolisms present within the biological community. The
presence of many community members from both bacteria and
archaea suggest that the formation of granules as well as the
enhanced degradation of phenol can be attributed to specific
groups. A total of 510 OTUs were assigned from 188,055 sequences
with a coverage rate >99% for each sample.
The microbial diversity of the different samples were shown in
Fig. 5. There was 11 phylum having a relative abundance >2%. The
Acetothermia, Euryarchaeota, Chloroflexi, Firmicutes, and Proteo-
bacteria were the most dominant and represented 80% of the total
community.
The initial seed sludge was obtained from a hydrolytic-
acidification tank that was determined to have a high proportion
of hydrolytic-acidification bacteria, including Acetothermia. During
Fig. 5. Changes in diversity of key bacteria acclimated to phenol by next generation
sequencing (R0-1 ¼ samples were taken at day 60 of R0, R1-1 ¼ samples were taken at
day 60 of R1, R0-2 ¼ samples were taken at day 120 of R0, R1-2 ¼ samples were taken
at day 120 of R1, R0-3 ¼ samples were taken at day 180 of R0, and R1-3 ¼ samples were
taken at day 180 of R1).
the stable operation period, portions of bacteria belonging to the
Proteobacteria and Firmicutes increased. Proteobacteria accounted
for 7.61% of the total population in the seed sludge and increased to
28.21% in R0-2 and 17.94% in R1-2. Firmicutes followed a similar
trend and initially accounted for only 3.31% of the population that
increased to 11.32% in R0-2 and 18.57% in R1-2. An abundance of
phenol-degrading bacteria is known to be Proteobacteria and Fir-
micutes members (Wang et al., 2015).
The microbial community distributions for the different samples
were shown in Fig. 5. During the startup period, fermentative
bacteria such as Gastranaerophilales which are capable of convert-
ing glucose, starch, or glycogen into lactate, ethanol, and formate
were found in a high proportion (Soo et al., 2014).
The community distribution changed significantly for both re-
actors during the acclimation stage. There was an increase in the
proportions of detected methanogenic archaea. Methanosaeta and
Longilinea which are beneficial for accelerating granular sludge
formation were also present in a high proportion (Li et al., 2015;
Yamada et al., 2007). Other phyla that have been implicated in
phenol degradation include Syntrophus and Trichococcus (Wang
et al., 2017a,b; Garcia-Mancha et al., 2012). Trichococcus has been
detected in an expanded granular sludge bed used for the treat-
ment of industrial wastewater containing phenolic compounds
(Garcia-Mancha et al., 2012).
The relative abundances of the archaeal populations were
shown in Table 1. All classified archaea from the three samples were
found in three genera, one acetoclastic methanogen (Methanosaeta)
and two hydrogenotrophic methanogens (Methanobacterium, and
Methanolinea) (Na et al., 2016). The majority of methanogens in the
seed sludge was principally acetoclastic; Methanosaeta accounted
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
120days 2.69 8.15
Methanolinea 0days 1.10 1.10
60days 1.87 2.61
120days 0.77 3.02
 C. Chen et al. / Chemosphere 204 (2018) 227e234
out necessary metabolisms to perform the conversions of [phenol
/ benzoate / acetate þ hydrogen], [acetate / methane], and
[hydrogen / methane] (Na et al., 2016). In this process, phenol is
first converted into small molecular fatty acids through a series of
oxidation reactions in the presence of NAD(P)þ/NAD(P)H. Hydro-
genotrophic methanogens can degrade phenol to benzoate (Qiu
et al., 2008; Tschech and Schink, 1986). Oxidized redox mediators
(RMoxidized HS) can obtain electron generated during methanation,
producing reduced redox mediators (RMreduced HS). RMreduced HS can
then supply electrons to NAD(P)þ that can be used to accelerate VFA
production through hydrolytic acidification of phenol. The transfer
of electrons from humic substances to donors such as H2 can be
then mediated through microbial oxidation (Garcia-Mancha et al.,
2012). Within this scheme, redox mediators facilitate electron
transfers, promoting the conversion of NAD(P)þ/NAD(P)H and ATP/
ADP, enhancing the anaerobic degradation of phenol (Gibson and
Harwood, 2002; Coates et al., 2002; Jiang and Kappler, 2008).
4. Conclusions
This study demonstrates that turf soil can be used as an
economical support material in UASB reactors for the improved
treatment of phenolic wastewaters. The turf soil provides a nucle-
ation point for the accelerated formation of larger and stronger
sludge granules. These larger and dense granules are formed by
providing a substrate for the fast cultivation of microorganisms and
also for the development of an anaerobic environment. The
developed biological community includes filamentous bacteria,
that increase granule strength, and other organisms that impart the
reactor with higher wastewater processing capabilities and better
resilience to impact loading. The humic component of turf soils can
also increase the degradation rates of phenol by facilitating the
metabolic transfer of electrons. The large volume of biogas having a
high percentage of methane could be reclaimed for use as an en-
ergy producing byproduct. Furthermore, turf soils are natural,
readily available and economical.
Acknowledgements
This work was supported in part by the National Science and
Technology Major Project of China (No. 2016ZX05040-001), the
National Natural Science Foundation of China (No. 21776307), and
the US Office of Naval Research (Grants N00014-09-1-0709 and
N00014-12-1-0496 284).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.chemosphere.2018.04.040.
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