Biodegradation (2019) 30:467–479

https://doi.org/10.1007/s10532-019-09888-5 (0123456789().,-volV)
( 01234567
89().,-volV)

ORIGINAL PAPER

Enrichment and characterization of a highly efficient

tetrahydrofuran-degrading bacterial culture
Hui Huang . Haixia Yu . Minbo Qi . Zubi Liu . Haixia Wang .
Zhenmei Lu

Received: 20 January 2019 / Accepted: 14 August 2019 / Published online: 28 August 2019
Ó Springer Nature B.V. 2019

Abstract Tetrahydrofuran (THF) is a ubiquitous
toxic and carcinogenic pollutant. Screening for pure or
mixed-culture microorganisms that can efficiently
degrade THF is difficult due to its chemical stability.
In this study, an enrichment culture, H-1, with a
stable THF-degrading ability and microbial commu-
nity structure was enriched from activated sludge and
could efficiently degrade 95% of 40 mM THF within
6 days. The optimal THF degradation conditions for
H-1 were an initial pH of 7.0–8.0 and a temperature of
30 °C. The substrate tolerance concentration of H-1
reached 200 mM. Heavy metals tolerance concentra-
tions of Cu2?, Cd2? and Pb2? of H-1 was 0.5 mM,
0.4 mM and 0.03 mM, and 4 mM Mn2? did not
significantly influence the THF degradation ratio or
biomass of H-1. H-1 might be a good material for
actual wastewater treatment because of its efficient
THF degradation performance and ability to resist
various stressful conditions. In addition, the THF-
degrading efficiency of H-1 was enhanced by the
addition of moderate carbon sources. High-throughput
sequencing of the 16S rRNA gene showed that
Rhodococcus sp. (a potential THF-degrading strain)
and Hydrogenophaga sp. (a potential non-THF-de-
grading strain) were the dominant microorganisms in
the H-1 culture. These results indicate the potential
coexistence of cooperation and competition between
THF-degrading bacteria and nondegrading bacteria in
this enrichment culture.
Keywords Tetrahydrofuran  Biodegradation 
Enrichment culture  Stress  Interactions
Introduction

Tetrahydrofuran (THF) is a type of heterocyclic

Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10532-019-09888-5) con-
tains supplementary material, which is available to authorized
users.
H. Huang  M. Qi  Z. Liu  H. Wang  Z. Lu (&)
MOE Laboratory of Biosystem Homeostasis and
Protection, College of Life Sciences, Zhejiang University,
Hangzhou, Zhejiang 310058, China
e-mail: lzhenmei@zju.edu.cn
H. Yu
Hangzhou Environmental Monitoring Center Station,
Hangzhou, China
organic compound and one of the most polar ethers.
In recent decades, THF has been widely used as a
solvent, chemical synthesis intermediate and analyt-
ical reagent due to its physical and chemical charac-
teristics. The emissions of THF have increased rapidly
with the rapid development of the chemical and
pharmaceutical industries, and the environmental
pollution of THF is currently an extremely acute
problem. The THF content in untreated wastewater
from pharmaceutical plants can reach 10 wt% (Xu and

123
468
Chen 2002), and THF has even been detected in
groundwater (Isaacson et al. 2006). THF may increase
the risk of kidney and liver cancer, and DNA-THF
adducts may contribute to the toxicological effects of
THF exposure (Gamer et al. 2002; Hermida et al.
2006). In addition, THF inhibits cytochrome P450 and
microbial degradation and causes cytotoxicity and
carcinogenesis (Moody 1991; Malley et al. 2001; Yao
et al. 2010, 2012). However, due to its chemical
stability, ordinary chemical degradation of THF is not
just expensive, the toxic catalysts used usually
produces harmful secondary pollutants (Metsger and
Bittner 2000). As an ecofriendly and cost-effective
strategy for pollutant removal, biodegradation can
achieve a high degradation rate under normal temper-
ature and pressure conditions without generating
harmful secondary pollutants (Oh et al. 2010).
To date, 6 genera of THF-degrading microorgan-
isms have been isolated, including Rhodococcus
(Bernhardt and Diekmann 1991; Daye et al. 2003;
Yao et al. 2009; Tajima et al. 2012), Pseudonocardia
(Parales et al. 1994; Kohlweyer et al. 2000; Daye et al.
2003; Masuda et al. 2012; Sei et al. 2013), Mycobac-
terium (Kim et al. 2009; Lan et al. 2013; Sei et al.
2013), Pseudomonas (Chen et al. 2010), Flavobac-
terium (Sun et al. 2011) and Afipia (Sei et al. 2013).
However, only a few of these microorganisms can
degrade high concentrations of THF (Bernhardt and
Diekmann 1991; Kohlweyer et al. 2000; Skinner et al.
2009; Yao et al. 2009). Among the THF-degrading
bacteria reported previously, Rhodococcus ruber YYL
had the highest THF tolerance (up to 200 mM) (Yao
et al. 2009) but could not degrade 20 mM THF
completely within 7 days even under optimal condi-
tions (Liu et al. 2017). Pseudomonas oleovorans DT4
had a better THF degradation performance (it
degraded 20 mM THF completely within 60 h) but
could not degrade THF completely when its concen-
tration was higher than 30 mM (Chen et al. 2010).
Thus, pure or mixed-culture microorganisms with
both a high THF-degrading efficiency and high THF
tolerance should be isolated for application in practical
situations, such as treatment of wastewater with a high
THF concentration.
Bacteria are found everywhere in nature but rarely
grow alone. Most nonpure cultured bacteria need
metabolites, signaling molecules and electrons provided
by other bacteria for survival (Stolyar et al. 2007;
McCutcheon and von Dohlen 2011; Hug et al. 2012).
123
Biodegradation (2019) 30:467–479
Therefore, researchers have proposed that mixed flora
may represent the general survival pattern of bacteria.
Compared with those of an individual bacterium, mixed
flora have richer metabolic patterns, a broader growth
environment and more efficient biological functions
(Zhang et al. 2013; Boon et al. 2014; Zhou et al. 2015).
For instance, consortium-GR can decolorize and biode-
grade the textile azo dye Scarlet R better than individual
strains (Saratale et al. 2009). Microbial consortia can
enhance the production of natural products through
mutual cooperation (Zhou et al. 2015). Additionally,
many types of stress-tolerant (i.e., high salt, heavy
metals, low temperature, and other organics) bacterial
consortia have been isolated to degrade different
compounds (Thijs et al. 2014; Thavamani et al. 2015;
Adam 2016). Recently, more researchers have started to
pay attention to mixed culture (Wanapaisan et al. 2018;
Zhao et al. 2018a). However, few studies have focused
on THF degradation via microbial flora derived from
enriched cultures. THF contamination is frequently
accompanied by various harsh conditions (high con-
centrations of THF, inappropriate temperatures and the
presence of heavy metals and other organic matter)
(Babel and Dacera 2006; Ikehata et al. 2016), which
increases the difficulty of treating THF pollution. Using
an enrichment culture to degrade THF might enhance
the THF-degrading function of strains in practical
applications. Therefore, studies on THF degradation
by an enrichment culture have suitable application
prospects and clear research value.
This study aimed to enrich an efficient THF-
degrading bacterial culture and evaluate its potential
to remove the THF from water by testing the (1) THF
degradation performance; (2) influence of tempera-
ture, initial pH and external carbon sources; (3)
tolerance of heavy metals; and (4) microbial commu-
nity structure of the enrichment culture.
Materials and methods
Media and enrichment of the culture
The mixed THF-degrading bacterial culture was
enriched from activated sludge collected from a
chemical plant with THF emissions in Huzhou,
Zhejiang Province, China, using a procedure similar
to that described in a previous study (He et al. 2013).
Activated sludge was enriched for one month under a
Biodegradation (2019) 30:467–479
THF concentration gradients ranging from 5 to 40 mM
in a 500-mL Erlenmeyer flask at 30 °C and 160 rpm.
Sterile basal salt medium (BSM) (Parales et al. 1994)
was added to the domesticated liquid to a total liquid
volume of 100 mL every 3 days. To characterize the
THF-degrading bacterial culture obtained from the
enrichment procedure, two milliliters of culture was
subsequently transferred from the enrichment solution
one month later into BSM supplemented with 40 mM
THF as a carbon source in a 500-mL Erlenmeyer flask
as the first generation and cultured at 30 °C and
160 rpm. The enrichment culture H-1 was subcultured
every 3 days for 50 generations.
THF degradation under different environmental
conditions using the enrichment culture
THF degradation by H-1 was determined in BSM with
THF (40 mM) as a carbon source. Batch experiments
were performed in a 500-mL Erlenmeyer flask con-
taining 100 mL of BSM. The 50th generation of H-1
was cultured in BSM after 72 h, harvested by
centrifugation (70009g, 10 min) and washed thrice
with BSM. The resulting cells were resuspended
(OD600 = 6) and 1 mL of the cell suspension was
dispensed into the culture medium to perform THF
tolerance experiment. H-1 was subcultured sequen-
tially, and the 51th–54th generations of H-1 were used
in experiments of the influence of different factors
(temperature, initial pH, additional carbon sources and
addition of Cu2?, Mn2?, Cd2? and Pb2?) on THF
degradation by H-1. The method of cell suspension
preparation was the same as that described above.
The substrate tolerance of H-1 was studied at
various THF concentrations (20, 30, 40, 50, 60, 80,
Table 1 Growth profiles of H-1 in the presence of other organic contaminants (10 mM) as carbon and energy sources after 5 days of
cultivation
Substrate OD600
Negative control 0.049 ± 0.001g
3-Hydroxytetrahydrofuran 0.057 ± 0.001fg
1,4-Butyrolactone 0.681 ± 0.018c
1,4-Dioxane 0.051 ± 0.001g
Trichloromethane 0.050 ± 0.000g
Dichloromethane 0.050 ± 0.002g
Benzol 0.051 ± 0.001g
Methylbenzene 0.336 ± 0.016d
Table entries marked with difference lowercase letters in each column are significantly different (p \ 0.05). SD from three replicates
469
100, and 120 mM) with degradation and growth
conditions of 30 °C and 160 rpm. Samples were
collected every 24 h for analysis of the biomass and
residual THF during the degradation process. We also
tested the tolerance of H-1 at high THF concentrations
(140, 150, 200, 250, and 300 mM) (Table S1), and the
samples were collected after 10 days of cultivation.
The influence of environmental factors on THF
degradation by H-1 was investigated in BSM contain-
ing 40 mM THF at 160 rpm. The environmental
factors assessed were the incubation temperature (20,
25, 30, 35, and 40 °C), initial pH of BSM (4.0, 5.0, 6.0,
7.0, 8.0, 9.0, and 10.0), additional carbon sources
(sodium acetate, peptone, yeast, sucrose, and glucose)
at three concentrations (0.5, 1.5, and 3 g/L), addition
of Cd2? and Pb2? (0.05, 0.1, 0.2, 0.4, and 0.8 mM),
and addition of Cu2? and Mn2? (0.5, 1, 2, 4, and
8 mM). After 108 h of incubation, samples were
collected to determine the THF concentration and
biomass. The experimental conditions listed above
were obtained from previous studies on THF concen-
trations (Chen et al. 2010), heavy metals (Wang et al.
2017), pH and temperature (He et al. 2013).
Growth on other organic contaminants
To assess the ability of H-1 to utilize other compounds
as carbon sources, 15 types of organic contaminants
were investigated in this study, including derivatives
and putative metabolites of THF, aromatics, and other
organic contaminants (Table 1). The 55th generation
of H-1 was inoculated in BSM supplemented with
other compounds (10 mM each) and cultured at 30 °C
and 160 rpm. The biomass of H-1 was monitored by
Substrate OD600
O-xylene 0.082 ± 0.013fg
2,5-Dimethylfuran 0.059 ± 0.001fg
Ethyl acetate 0.724 ± 0.010b
Pyridine 0.074 ± 0.024fg
Acetone 0.056 ± 0.001fg
Methanol 0.185 ± 0.002e
Hexane 0.091 ± 0.006f
1,4-Butylene glycol 1.217 ± 0.051a
123
470
recording the optical density at 600 nm (OD600) after
5 days of incubation.
Sample collection, DNA extraction and 16S rRNA
gene sequencing
H-1 was cultured in BSM supplemented with 40 mM
THF. Samples from every ten generations during the
50 generations of H-1 subculture were collected at the
logarithmic growth phase (OD600 = 2.0–2.5). Samples
were also collected from the 50th generation of H-1 at
24, 72 and 144 h. Cell samples were obtained by
centrifugation (12,0009g, 1 min) and stored at
- 20 °C prior to use.
Total genomic DNA was extracted using the
E.Z.N.A.Ò Bacterial DNA Kit (Omega Biotek, Nor-
cross, GA, USA) according to the manufacturer’s
instructions. The 16S rRNA high-throughput sequenc-
ing was performed by Realbio Genomics Institute
(Shanghai, China) on the Illumina HiSeq PE250
platform. Polymerase chain reaction amplification of
the 16S rRNA gene V3-V4 region was conducted
using the universal bacterial primers F341 50 -
ACTCCTACGGGRSGCAGCAG-30 and R806 50 -
GGACTACVVGGGTATCTAAT-30 . The amplicons
were sequenced to obtain 425-bp paired-end reads.
The raw data were subjected to a quality control
procedure using UPARSE (Edgar 2013). Chimeras
were filtered with USEARCH (Edgar 2010), and the
remaining sequences were clustered into operational
taxonomic units (OTUs) at 97% identity. The RDP
classifier (Maidak et al. 1997) was used to assign a
representative sequence for each OTU to the taxo-
nomic level in the RDP database. Sequencing data
were deposited into the NCBI Sequence Read Archive
(SRA) database under accession number SRP190367.
Kinetic model
The Haldane–Andrew model was modified for high
concentrations of substrate, which might inhibit the
growth of cells. The equation was published as follows
(Kim et al. 2005):
qmax S
q¼ ð1Þ
S þ Ks þ SKi
2
where qmax is the maximum specific degradation rate
(h-1), Ks is the half-saturation constant (mM), and Ki
is an inhibition constant (mM).
123
Biodegradation (2019) 30:467–479
Analytical methods and statistical analysis
The cultures were centrifuged at 10,0009g for
10 min, and 500 lL of the supernatant was analyzed
by a GC-2014C gas chromatograph equipped with a
flame ionization detector and an AOC-20i autoinjector
(SHIMADZU, Japan) to assess the THF concentra-
tion. The temperatures of the injector, oven, and
detector were set to 250, 160, and 250 °C, respec-
tively. The THF peak was observed at a retention time
of 1.7 min. The degradation ratio was calculated using
the following equation:
C0  Ct
Degradation ratio (%) ¼  100 ð2Þ
C0
where C0 is the initial concentration of THF and Ct is
the instant concentration in the sample. The degrada-
tion ratio was used to predict the kinetic performance
of H-1. The biomass of H-1 was monitored by
recording the OD600 using a UV spectrometer.
The Cu2? and Pb2? concentrations were deter-
mined by atomic absorption spectrophotometry Spectr
AA 240 FS (Varian, Australia) under the following
conditions: slit width: 0.5 nm, lamp current: 4 mA,
and wavelength: 324.8 nm for Cu2? and slit width:
1.0 nm, lamp current: 10 mA, and wavelength:
217 nm for Pb2?. The metal concentrations were
calculated using appropriate standards.
Each experiment was performed in triplicate.
p values for all assays were determined using a two-
tailed Student’s t test. Variance analysis was per-
formed (ANOVA, least significant difference test,
p \ 0.05), and Spearman’s correlation coefficients
were calculated using PASW Statistics 19.0.
Results
Culture enrichment and THF degradation
Beginning with the 20th to 30th subculture genera-
tions, the enrichment culture could degrade 40 mM
THF within 6 days steadily, suggesting that a mixed
culture with a stable THF-degrading ability generated
from the previous domesticated activated sludge. We
designated this mixed culture as H-1. Figure 1 shows
the THF degradation and growth characteristics of H-1
(50th generation) in BSM. At 40 mM THF, the
degradation ratio of H-1 exceeded 95% within 6 days,
Biodegradation (2019) 30:467–479
Fig. 1 Impacts of different THF concentrations on THF degradation (a) and growth curves (b) by H-1. Error bars: SD from three
replicates and may be smaller than the marker
the maximum growth rate was 1.083 day-1, and the
degradation rate was 3.271 g THF d-1 g-1 dry weight
at a yield (YX/C) up to 51.7% (dry weight/substrate
carbon). To test the growth and degradation of H-1 at
low THF concentrations, we assessed the ability of
H-1 to degrade 6 mM THF (Fig. S1a). The maximum
growth rate and maximum THF degradation rate were
0.143 h-1 and 208.522 mg THF h-1 g-1 dry weight
for H-1 and 0.0892 h-1 and 137.60 mg THF h-1 g-1
dry weight for the pure strain YYL, respectively (Yao
et al. 2009), indicating that H-1 also had a better
degradation ability than the pure strain YYL at low
THF concentrations. The change in the culture
medium pH during THF degradation was also mon-
itored (Fig. S1b). Although the pH decreased from
neutral (7.170 ± 0.016) to weakly acidic
Fig. 2 Effects of different initial pH values at 30 °C (a) and
different temperatures at pH 7.0 (b) on THF degradation by H-1.
The bars of each group (THF degradation ratio) marked with
471
(6.230 ± 0.056), the decrease was not obvious
throughout the process.
Effects of the initial pH and temperature on THF
degradation
The effects of the initial pH of BSM (4.0, 5.0, 6.0, 7.0,
8.0, 9.0, and 10.0) on THF degradation by H-1 are
shown in Fig. 2a. The THF degradation ratio
decreased rapidly from 62.64% to 27.99% when the
pH changed from 7.0 to 6.0. However, when the pH
value was 8.0, the degradation ratio was 68.93%,
which was similar to that obtained at pH 7.0,
suggesting that the optimal pH for THF degradation
by H-1 was between 7.0 and 8.0 (Fig. 2a). Further-
more, a high pH (9.0 and 10.0) resulted in precipitation
different lowercase letters differ significantly from one another
(p \ 0.05). Error bars: SD from three replicates
123
472
in the medium, which was not conducive to the growth
and THF degradation ability of the enrichment culture.
The effects of several different temperatures (20,
25, 30, 35, and 40 °C) on THF degradation by H-1
were tested. As shown in Fig. 2b, temperature had a
significant effect on THF degradation. The optimal
temperature for THF degradation by H-1 was 30 °C, in
which 63.90% of THF was degraded within 108 h.
Only 50.53% and 31.70% of THF was degraded by
H-1 at 35 and 40 °C, respectively, and less than 10%
of THF was degraded by H-1 at low temperatures.
Overall, H-1 was able to tolerate high temperatures
better than low temperatures (Fig. 2b).
THF tolerance of H-1
The tolerance of H-1 (50th generation, same as in the
other experiments) to different THF concentrations
(20, 30, 40, 50, 60, 80, 100, 120, and 140 mM) was
studied (Fig. 1). The mixed culture took only 3 days to
completely degrade 20 mM THF and took 5, 6 and
10 days to degrade 30, 40 and 50 mM THF, respec-
tively. Moreover, H-1 grew well in BSM with 80 mM
THF, and approximately 70% of THF could be
degraded within 10 days. H-1 exhibited an excellent
substrate tolerance even in the presence of 140 mM
THF. The highest THF concentration tolerated by H-1
was 200 mM, as described previously for R. ruber
Fig. 3 Effects of additional carbon sources (0.5 g/L, 1.5 g/L,
and 3.0 g/L) on THF degradation (a) and the H-1 biomass (b).
CK means without additional carbon sources, and the initial
THF concentration of BSM was 40 mM. The P value indicates
123
Biodegradation (2019) 30:467–479
YYL and P. oleovorans DT4 (Yao et al. 2009; Chen
et al. 2010) (Table S1). To investigate the kinetics of
THF degradation by H-1, different initial THF
concentrations (Fig. 1a) were used. The THF degra-
dation rate decreased with increasing THF concentra-
tion, indicating that THF might act as an inhibitor to
H-1. Therefore, a substrate inhibition model (Eq. (1))
was used to fit the specific degradation rates (q) for
different initial concentrations. The kinetic parameters
qmax, Ks, and Ki were determined to be 0.171 h-1,
102 mM, 116 mM, respectively.
Effects of additional carbon sources on THF
degradation
Cosubstrates play a vital role in the biodegradation of
organic pollutants and may affect the metabolism of
the target substrate. In this study, the effects of five
different carbon sources (sodium acetate, peptone,
yeast, sucrose, and glucose) on THF degradation by
H-1 were examined. Each carbon source was assessed
at three concentrations (0.5, 1.5 and 3.0 g/L) to
explore the most efficient THF-degrading conditions.
As shown in Fig. 3a, low concentrations (0.5 g/L) of
additional carbon sources (except glucose) signifi-
cantly enhanced THF degradation, whereas high
concentrations (3.0 g/L) inhibited THF degradation.
The addition of glucose had inhibitory effects even at
statistical significance between the control and experimental
groups determined using Student’s t test (n = 3, *p \ 0.05,
**p \ 0.01, ***p \ 0.001)
Biodegradation (2019) 30:467–479
the initial concentration of 0.5 g/L, although the effect
was more obvious at the higher concentrations
(Fig. 3a). In fact, most of the additional carbon
sources could promote the growth of H-1 (Fig. 3b)
but enhanced THF degradation at only low
concentrations.
Effects of heavy metal ions on THF degradation
The effects of four different heavy metal ions (Cu2?,
Mn2?, Cd2?, and Pb2?) on the degradation of THF by
H-1 were studied. Data analysis using ANOVA
revealed that H-1 had different tolerances for the
tested heavy metals (Fig. 4). The THF degradation
performance of H-1 with 0.1 or 0.5 mM Cu2? was
improved compared to that of the control. However,
Cu2? sharply inhibited the degradation activity, with a
THF degradation ratio less than 10% after 108 h, when
Fig. 4 Effects of various concentrations of heavy metal ions on
THF biodegradation by H-1. a Cu2?, b Mn2?, c Cd2?, and
d Pb2?. The bars of each group (THF degradation ratio) marked
473
its concentration increased to 1 mM. The actual
concentrations of heavy metals in BSM were detected,
0.5 mM Cu2? was fully dissolved in BSM, but 1, 2 and
4 mM Cu2? could be only partially dissolved
(0.63–0.65 mM); the results are shown in Table S2.
However, Mn2? did not significantly influence the
THF degradation ratio or biomass of H-1. The THF
degradation ratio remained above 60% after 108 h
when the Mn2? concentration was increased to 4 mM,
and Mn2? could be fully dissolved in BSM. The THF
degradation ratio was maintained at 25% with the
addition of 0.8 mM Cd2? and Pb2?. Besides, the
actual concentrations of Pb2? were much lower than
the added concentrations (Table S2), and the maxi-
mum dissolved concentration was 0.06 mM in this
experiment.
with different lowercase letters differ significantly from one
another (p \ 0.05). Error bars: SD from three replicates
123
474
Substrate utilization of H-1
BSM was supplemented with fifteen types of com-
pounds, including derivatives and putative intermedi-
ate metabolites (Bernhardt and Diekmann 1991) of
THF and aromatics, as carbon sources (10 mM each)
for the H-1 culture. The results showed that in addition
to THF, H-1 can also utilize methylbenzene, ethyl
acetate and methanol (Table 1). In addition, 1,4-
butylene glycol and c-butyrolactone, which are spec-
ulated to be intermediate metabolites of THF degra-
dation, were utilized by H-1.
Structural analysis of the H-1 microbial
community
The microbial community diversity and structure
dynamics of H-1 during THF degradation were
Fig. 5 Microbial
community structure
analysis of H-1 during 50
generations of passages
(a) and during THF
degradation within 6 days of
cultivation (b)
123
Biodegradation (2019) 30:467–479
investigated by 16S rRNA gene high-throughput
sequencing. As shown in Fig. 5, Rhodococcus sp.,
which belongs to the phylum Actinobacteria, was the
predominant genus in H-1, with a relative abundance
of 58.78 ± 12.81%. Based on identification of the
pure cultures isolated from H-1, Rhodococcus sp. may
be responsible for the THF degradation ability of H-1
(Table S3). We tested the THF degradation ability of
H-1 and pure strain Rhodococcus sp. with the same
initial total cell concentrations (Fig. S1c), revealing
that H-1 exhibited more excellent growth and degra-
dation abilities than individual strains during THF
biodegradation. Furthermore, the relative abundance
of Hydrogenophaga sp. was outstanding both during
the passaging (Fig. 5a) and THF degradation (Fig. 5b)
processes, increasing from 2.87 ± 1.41% at D1 to
12.04 ± 3.43% at D6 for H-1. In addition, the relative
abundances of Sphingopyxis sp., Aquamicrobium sp.
Biodegradation (2019) 30:467–479
and Devosia sp. were 2.04 ± 0.10%, 1.54 ± 0.03%
and 1.16 ± 0.26%, respectively, in the 50th genera-
tion (Fig. 5a). According to Spearman’s correlation
analysis, Pseudomonas and Flavobacterium were
positively correlated with Rhodococcus sp. In contrast,
Stenotrophomonas sp., Hydrogenophaga sp., Devosia
sp., and Ochrobactrum sp., among others, were
negatively correlated with Rhodococcus sp.
(Fig. S1d), which had relatively high abundances
during passaging (Fig. 5a). In addition, 8 other pure
strains were isolated from H-1 (see supporting meth-
ods), and their abilities to degrade THF were deter-
mined (Table S3). Unfortunately, these strains could
not degrade THF independently. However, uncultured
strains or strains that were not isolated might also
degrade THF in the enriched culture H-1.
Discussion
The microbial community is the main unit of bacteria
and allows the group to survive and exert various
physiological and biochemical functions. Some bac-
teria perform biological functions, resist complex
environmental stress and restore the degradation
abilities of contaminants by communicating with their
cooperators (Benomar et al. 2015; Liu et al. 2017). The
THF degradation abilities of the enriched culture H-1
under different environmental conditions were
explored in this study, and H-1 demonstrated different
tolerances to various environmental stresses (Figs. 2,
4). Mixed cultures may exhibit more excellent growth
and degradation abilities than individual strains. Based
on our analysis of the degradation efficiency under
high concentrations of THF, H-1 showed better
substrate degradation performance than individual
bacterial strains (Fig. S1c), which is consistent with
previous studies (Kılıç et al. 2007; Sathishkumar et al.
2008). However, research on THF biodegradation by
mixed-culture microorganisms is limited, and our
study might provide a reference for this area.
THF was previously classified as a relatively
refractory organic (King and Painter 1983). In con-
trast, the enrichment culture H-1 completely degraded
20 and 40 mM THF within approximately 3 and
6 days, respectively. The previously reported fastest
strain R. ruber YYL degraded 20 mM THF com-
pletely in more than 6 days (Yao et al. 2009); thus, the
advantages of using H-1 to degrade THF are obvious.
475
The pH value is an important physiochemical index in
microbial remediation, and low or high pH values may
induce a low bioremediation efficiency (Liu et al.
2017; Zagrodnik and Łaniecki 2017). pH stress (initial
pH of 7.0) inhibited THF degradation by the YYL
strain, but this phenomenon did not occur with H-1
(Fig. 2a). THF degradation is an acid-producing
process (Bernhardt and Diekmann 1991; Skinner
et al. 2009), and the pH value of the system declines
dramatically from 8.3 to less than 4.0 during the THF
degradation process (Yao et al. 2009, 2010; Liu et al.
2017). The enrichment culture H-1 maintained the
system pH value at an appropriate level. The medium
buffer is an important reason for maintaining the pH of
the system, but the utilization of acidic substances by
non-THF-degrading bacteria cannot be ignored. Thus,
H-1 may be an available system for exploring
interaction mechanisms.
During the actual wastewater treatment process,
THF-degrading strains often encounter various stres-
ses, such as high-concentration substrates, that might
inhibit the growth and metabolism of the strains,
resulting in a low biodegradation efficiency. The
growth of the Pseudonocardia sp. strain K1 was
obviously inhibited when the THF concentration
reached 60 mM (Kohlweyer et al. 2000). However,
the enrichment culture H-1 showed excellent substrate
tolerance even during degradation of 200 mM THF
(Table S1). Furthermore, the growth rate of H-1 was
not inhibited at THF concentrations as high as 80 mM,
indicating that H-1 could maintain a high degradation
efficiency under high-substrate concentration stress.
As stated above, H-1 had a much higher degradation
efficiency for different THF concentrations than most
pure strains, perhaps due to the inhibition of interme-
diate metabolite release (acidic metabolites) and gain
of growth factors by the non-THF-degrading strains.
The biodegradation of substrates is inhibited by
coexistence with heavy metals (Arjoon et al. 2015;
Zhao et al. 2018b); thus, studies on the tolerance of
microorganisms to heavy metals are particularly
important. The maximum permissible concentrations
of heavy metals in water, as stated by Environmental
Protection Agency (EPA) regulations based on the
Comprehensive Environmental Response Compensa-
tion and Liability Act, USA, are 2.046 9
10-2, 9.101 9 10-4, 4.448 9 10-5, and 7.239 9
10-5 mM for Cu, Mn, Cd, and Pb, respectively
(Chaturvedi et al. 2015), and we determined the
123
476
tolerance of H-1 to these heavy metals to be
200–20,000 times higher than the standard concentra-
tions. H-1 exhibited different tolerances to heavy
metals (Fig. 4). For instance, H-1 had better tolerance
for Mn2? than Cu2? at the same concentration, and
Cu2? was more toxic to H-1 than Mn2?. THF
degradation by the H-1 culture was not inhibited even
in the presence of 0.2 mM Cd2? and 0.1 mM Pb2?,
concentrations that are more than 1000 times higher
than the standards stated by the EPA (Fig. 4c, d). Yu
and Cheng found that the amount of microbial
biological carbon decreased significantly with the
addition of heavy metals Cd2? and Pb2? in brown soil
(Yu and Cheng 2015). Similarly, both the biomass and
THF degradation rate of H-1 were inhibited when
concentrations exceeded 0.2 mM and 0.1 mM, respec-
tively (Fig. 4c, d). The toxicity of contaminants in the
biota depends on the physicochemical properties of
the receptor environment (Luo et al. 2017). The use of
multiple species consortia has proved advantageous
for high metal scavenging and provides more stability
against environmental fluctuations, and resistance
thresholds of heavy metals might be higher in the
consortium than in pure culture (Sprocati et al. 2006),
which might be one important reason for its good
heavy metal tolerance.
Refractory organic compounds are not directly or
easily degraded by microorganisms, and cosubstrates
could expand the degradation spectrum of degraders
and enhance the biodegradation of refractory organics.
Indeed, replenishment of the appropriate carbon
sources could improve the THF degradation by H-1.
When a cosubstrate is not suitable, the microbial
degradation of organics might be inhibited. Low
concentrations of additional carbon sources enhanced
THF degradation (Fig. 3a), whereas high concentra-
tions had a severe inhibitory effect. We speculated that
the addition of low concentrations of additional carbon
sources satisfies the growth of microorganisms and
thus promotes THF degradation. However, high
concentrations of these additional carbon sources
may inhibit the biodegradation of THF and result in
diauxic growth (Lee et al. 2003). The addition of
glucose led to a more significant inhibition of THF
degradation than that achieved with other carbon
sources (Fig. 3a). Previous study confirmed that
glucose inhibited the decolorization activity due to
the low pH caused by the glucose consumption or
123
Biodegradation (2019) 30:467–479
converted organic acids (Chen et al. 2003). H-1 was
sensitive to low pH (Fig. 2a) during THF degradation
that might be a reason for inhibiting the THF
degradation efficiency with additional glucose. Fur-
thermore, bacteria might utilize glucose preferentially,
thus resulting in the ‘‘latent period’’ or lower THF
degradation efficiency. Therefore, in the practical
application of sewage treatment, an appropriate and
inexpensive carbon source, such as sodium acetate,
could be used to improve the THF degradation rate.
A total of 17 single THF-degrading strains have
been reported to date, 13 of which are in the phylum
Actinobacteria and 4 that are in the genus Rhodococ-
cus (Bernhardt and Diekmann 1991; Daye et al. 2003;
Yao et al. 2009; Tajima et al. 2012). In the enrichment
culture H-1, Rhodococcus sp. and Hydrogenophaga
sp. were the dominant species. Rhodococci have
outstanding potential for the biodegradation of envi-
ronmental pollutants due to their broad degradation
spectrum, efficient degradability (Martı́nková et al.
2009), and compatibility with foreign genes (Larkin
et al. 2006). Thus, the THF-degrading strains of
Rhodococcus sp. (Table S3) may play a crucial role in
efficient biodegradation. The bacteria of non-THF-
degrading strains, such as Hydrogenophaga sp. and
Stenotrophomonas sp. (Table S3), are indispensable in
the stable H-1 system. Cooperators may maintain the
stability of microbial communities through the
exchange of metabolites (Phelan et al. 2012; Zelezniak
et al. 2015), such as nutrients and growth factors. In
H-1, the non-THF-degrading bacteria need to utilize
metabolic intermediates of THF as carbon sources and
energy, which may relieve metabolic inhibition and
promote THF degradation. In turn, the non-THF-
degrading strains may provide some trace micronutri-
ents, such as growth factors, to help the degrading
bacteria, especially auxotrophs. Interestingly, Steno-
trophomonas sp., Hydrogenophaga sp. and most of the
non-THF-degrading strains were negatively correlated
with Rhodococcus sp. (Fig. S1d), indicating there are
other unexplored relationships especially competition
between degrading and non-degrading bacteria. One
view is that competition, not cooperation, dominates
interactions and plays an important role in maintaining
community homeostasis (Foster and Bell 2012). In
H-1, interactions in communities may promote com-
munity stability during THF degradation with envi-
ronmental perturbations.
Biodegradation (2019) 30:467–479
Conclusions
H-1 is a mixed culture enriched from activated sludge
that completely degrades high concentrations of THF
under pH conditions (initial pH of 7.0). The enrich-
ment culture H-1 was demonstrated to have both high
THF-degrading efficiency and high THF tolerance
even under generally harsh environmental conditions.
Moreover, the replenishment of appropriate carbon
sources could improve THF degradation by H-1. The
results showed that H-1 is a valuable material for
sewage treatment cocontaminated with high concen-
trations of THF, heavy metals, etc. The 16S rRNA
sequencing results indicated that Rhodococcus sp. and
Hydrogenophaga sp. were the dominant species in
H-1. Notably, these species may cooperate with each
other to degrade THF in a sustainable and effective
manner. Some pure cultures from H-1 were isolated,
and their roles in the system during THF degradation
and the mechanisms underlying the interactions in the
microbial community need to be explored further.
Acknowledgements This work was financially supported by
the National Natural Science Foundation of China (Nos.
41630637 and 41721001).
References
Adam M (2016) Biodegradation of marine crude oil pollution
using a salt-tolerant bacterial consortium isolated from
Bohai Bay, China. Mar Pollut Bull 105:43–50
Arjoon A, Olaniran AO, Pillay B (2015) Kinetics of heavy metal
inhibition of 1,2-dichloroethane biodegradation in co-
contaminated water. J Basic Microbiol 55:277–284. https://
doi.org/10.1002/jobm.201200776
Babel S, Dacera DDM (2006) Heavy metal removal from con-
taminated sludge for land application: a review. Waste
Manage 26:988–1004
Benomar S et al (2015) Nutritional stress induces exchange of
cell material and energetic coupling between bacterial
species. Nat Commun 6:6283
Bernhardt D, Diekmann H (1991) Degradation of dioxane,
tetrahydrofuran and other cyclic ethers by an environ-
mental Rhodococcus strain. Appl Microbiol Biotechnol
36:120–123
Boon E, Meehan CJ, Whidden C, Wong DH-J, Langille MG,
Beiko RG (2014) Interactions in the microbiome: com-
munities of organisms and communities of genes. FEMS
Microbiol Rev 38:90–118
Chaturvedi AD, Pal D, Penta S, Kumar A (2015) Ecotoxic heavy
metals transformation by bacteria and fungi in aquatic
ecosystem. World J Microbiol Biotechnol 31:1595–1603.
https://doi.org/10.1007/s11274-015-1911-5
477
Chen K-C, Wu J-Y, Liou D-J, Hwang S-CJ (2003) Decol-
orization of the textile dyes by newly isolated bacterial
strains. J Biotechnol 101:57–68
Chen J-M, Zhou Y-Y, Chen D-Z, Jin X-J (2010) A newly iso-
lated strain capable of effectively degrading tetrahydrofu-
ran and its performance in a continuous flow system.
Bioresour Technol 101:6461–6467
Daye K, Groff J, Kirpekar A, Mazumder R (2003) High effi-
ciency degradation of tetrahydrofuran (THF) using a
membrane bioreactor: identification of THF-degrading
cultures of Pseudonocardia sp. strain M1 and Rhodococcus
ruber isolate M2. J Ind Microbiol Biotechnol 30:705–714
Edgar RC (2010) Search and clustering orders of magnitude
faster than BLAST. Bioinformatics 26:2460–2461
Edgar RC (2013) UPARSE: highly accurate OTU sequences
from microbial amplicon reads. Nat Methods 10:996–998.
https://doi.org/10.1038/nmeth.2604
Foster KR, Bell T (2012) Competition, not cooperation, domi-
nates interactions among culturable microbial species. Curr
Biol 22:1845–1850. https://doi.org/10.1016/j.cub.2012.08.
005
Gamer A, Jaeckh R, Leibold E, Kaufmann W, Gembardt C,
Bahnemann R, Van Ravenzwaay B (2002) Investigations
on cell proliferation and enzyme induction in male rat
kidney and female mouse liver caused by tetrahydrofuran.
Toxicol Sci 70:140–149
He Z, Xiao H, Tang L, Min H, Lu Z (2013) Biodegradation of di-
n-butyl phthalate by a stable bacterial consortium, HD-1,
enriched from activated sludge. Bioresour Technol
128:526–532
Hermida SA et al (2006) 20 -Deoxyguanosine, 20 -deoxycytidine,
and 20 -deoxyadenosine adducts resulting from the reaction
of tetrahydrofuran with DNA bases. Chem Res Toxicol
19:927–936
Hug LA, Beiko RG, Rowe AR, Richardson RE, Edwards EA
(2012) Comparative metagenomics of three Dehalococ-
coides-containing enrichment cultures: the role of the non-
dechlorinating community. BMC Genomics 13:327
Ikehata K, Wang-Staley L, Qu X, Li Y (2016) Treatment of
groundwater contaminated with 1, 4-dioxane, tetrahydro-
furan, and chlorinated volatile organic compounds using
advanced oxidation processes. Ozone Sci Eng 38:413–424
Isaacson C, Mohr TK, Field JA (2006) Quantitative determi-
nation of 1,4-dioxane and tetrahydrofuran in groundwater
by solid phase extraction GC/MS/MS. Environ Sci Technol
40:7305–7311
Kılıç NK, Nielsen JL, Yüce M, Dönmez G (2007) Characteri-
zation of a simple bacterial consortium for effective
treatment of wastewaters with reactive dyes and Cr(VI).
Chemosphere 67:826–831
Kim D-J, Choi J-W, Choi N-C, Mahendran B, Lee C-E (2005)
Modeling of growth kinetics for Pseudomonas spp. during
benzene degradation. Appl Microbiol Biotechnol
69:456–462
Kim Y-M, Jeon J-R, Murugesan K, Kim E-J, Chang Y-S (2009)
Biodegradation of 1,4-dioxane and transformation of
related cyclic compounds by a newly isolated Mycobac-
terium sp. PH-06. Biodegradation 20:511
King E, Painter H (1983) Assessment of biodegradability of
chemicals in water by manometric respirometry. Comm
Eur Communities EUR 8631:31
123
478
Kohlweyer U, Thiemer B, Schräder T, Andreesen JR (2000)
Tetrahydrofuran degradation by a newly isolated culture of
Pseudonocardia sp. strain K1. FEMS Microbiol Lett
186:301–306
Lan RS, Smith CA, Hyman MR (2013) Oxidation of cyclic
ethers by alkane-grown Mycobacterium vaccae JOB5.
Remediat J 23:23–42
Larkin MJ, Kulakov LA, Allen CC (2006) Biodegradation by
members of the genus Rhodococcus: biochemistry, physi-
ology, and genetic adaptation. Adv Appl Microbiol
59:1–29
Lee K, Park J-W, Ahn I-S (2003) Effect of additional carbon
source on naphthalene biodegradation by Pseudomonas
putida G7. J Hazard Mater 105:157–167
Liu Z, He Z, Huang H, Ran X, Oluwafunmilayo AO, Lu Z
(2017) pH Stress-induced cooperation between
Rhodococcus ruber YYL and Bacillus cereus MLY1 in
biodegradation of tetrahydrofuran. Front Microbiol 8:2297
Luo J, Hein C, Mücklich F, Solioz M (2017) Killing of bacteria
by copper, cadmium, and silver surfaces reveals relevant
physicochemical parameters. Biointerphases 12:020301.
https://doi.org/10.1116/1.4980127
Maidak BL, Olsen GJ, Larsen N, Overbeek R, McCaughey MJ,
Woese CR (1997) The RDP (ribosomal database project).
Nucleic Acids Res 25:109–110
Malley LA, Christoph GR, Stadler JC, Hansen JF, Biesemeier
JA, Jasti SL (2001) Acute and subchronic neurotoxico-
logical evaluation of tetrahydrofuran by inhalation in rats.
Drug Chem Toxicol 24:201–219
Martı́nková L, Uhnáková B, Pátek M, Nešvera J, Křen V (2009)
Biodegradation potential of the genus Rhodococcus.
Environ Int 35:162–177
Masuda H, McClay K, Steffan RJ, Zylstra GJ (2012)
Biodegradation of tetrahydrofuran and 1, 4-dioxane by
soluble diiron monooxygenase in Pseudonocardia sp.
strain ENV478. J Mol Microbiol Biotechnol 22:312–316
McCutcheon JP, von Dohlen CD (2011) An interdependent
metabolic patchwork in the nested symbiosis of mealy-
bugs. Curr Biol 21:1366–1372
Metsger L, Bittner S (2000) Autocatalytic oxidation of ethers
with sodium bromate. Tetrahedron 56:1905–1910
Moody DE (1991) The effect of tetrahydrofuran on biological
systems: does a hepatotoxic potential exist? Drug Chem
Toxicol 14:319–342
Oh ST, Kim JR, Premier GC, Lee TH, Kim C, Sloan WT (2010)
Sustainable wastewater treatment: how might microbial
fuel cells contribute. Biotechnol Adv 28:871–881
Parales R, Adamus J, White N, May H (1994) Degradation of 1,
4-dioxane by an actinomycete in pure culture. Appl Envi-
ron Microbiol 60:4527–4530
Phelan VV, Liu W-T, Pogliano K, Dorrestein PC (2012)
Microbial metabolic exchange—the chemotype-to-pheno-
type link. Nat Chem Biol 8:26
Saratale R, Saratale G, Kalyani D, Chang J-S, Govindwar S
(2009) Enhanced decolorization and biodegradation of
textile azo dye Scarlet R by using developed microbial
consortium-GR. Bioresour Technol 100:2493–2500
Sathishkumar M, Binupriya AR, Baik SH, Yun SE (2008)
Biodegradation of crude oil by individual bacterial strains
and a mixed bacterial consortium isolated from hydrocar-
bon contaminated areas. Clean: Soil, Air, Water 36:92–96
123
Biodegradation (2019) 30:467–479
Sei K, Miyagaki K, Kakinoki T, Fukugasako K, Inoue D, Ike M
(2013) Isolation and characterization of bacterial strains
that have high ability to degrade 1, 4-dioxane as a sole
carbon and energy source. Biodegradation 24:665–674
Skinner K, Cuiffetti L, Hyman M (2009) Metabolism and
cometabolism of cyclic ethers by a filamentous fungus, a
Graphium sp. Appl Environ Microbiol 75:5514–5522
Sprocati AR, Alisi C, Segre L, Tasso F, Galletti M, Cremisini C
(2006) Investigating heavy metal resistance, bioaccumu-
lation and metabolic profile of a metallophile microbial
consortium native to an abandoned mine. Sci Total Environ
366:649–658
Stolyar S, Van Dien S, Hillesland KL, Pinel N, Lie TJ, Leigh JA,
Stahl DA (2007) Metabolic modeling of a mutualistic
microbial community. Mol Syst Biol 3:92
Sun B, Ko K, Ramsay JA (2011) Biodegradation of 1, 4-dioxane
by a Flavobacterium. Biodegradation 22:651–659
Tajima T, Hayashida N, Matsumura R, Omura A, Nakashimada
Y, Kato J (2012) Isolation and characterization of
tetrahydrofuran-degrading Rhodococcus aetherivorans
strain M8. Process Biochem 47:1665–1669
Thavamani P, Megharaj M, Naidu R (2015) Metal-tolerant
PAH-degrading bacteria: development of suitable test
medium and effect of cadmium and its availability on PAH
biodegradation. Environ Sci Pollut Res 22:8957–8968
Thijs S, Weyens N, Sillen W, Gkorezis P, Carleer R, Van-
gronsveld J (2014) Potential for plant growth promotion by
a consortium of stress-tolerant 2, 4-dinitrotoluene-de-
grading bacteria: isolation and characterization of a mili-
tary soil. Microb Biotechnol 7:294–306
Wanapaisan P et al (2018) Synergistic degradation of pyrene by
five culturable bacteria in a mangrove sediment-derived
bacterial consortium. J Hazard Mater 342:561–570
Wang Y et al (2017) Biodegradation of di-n-butyl phthalate by
bacterial consortium LV-1 enriched from river sludge.
PLoS ONE 12:e0178213
Xu Y, Chen X (2002) Recovering tetrahydrofuran from waste
pharmaceutical liquor using new separation methods.
Chem Eng (China) 30:17–19
Yao Y, Lv Z, Min H, Lv Z, Jiao H (2009) Isolation, identifica-
tion and characterization of a novel Rhodococcus sp. strain
in biodegradation of tetrahydrofuran and its medium opti-
mization using sequential statistics-based experimental
designs. Bioresour Technol 100:2762–2769
Yao Y et al (2010) Assessment of toxicity of tetrahydrofuran on
the microbial community in activated sludge. Bioresour
Technol 101:5213–5221
Yao Y, Lu Z, Min H, Gao H, Zhu F (2012) The effect of
tetrahydrofuran on the enzymatic activity and microbial
community in activated sludge from a sequencing batch
reactor. Ecotoxicology 21:56–65
Yu L, Cheng JM (2015) Effect of heavy metals Cu, Cd, Pb and
Zn on enzyme activity and microbial biomass carbon in
brown soil. Adv Mater Res 1073–1076:726–730
Zagrodnik R, Łaniecki M (2017) The effect of pH on coopera-
tion between dark-and photo-fermentative bacteria in a co-
culture process for hydrogen production from starch. Int J
Hydrog Energy 42:2878–2888
Zelezniak A, Andrejev S, Ponomarova O, Mende DR, Bork P,
Patil KR (2015) Metabolic dependencies drive species co-
Biodegradation (2019) 30:467–479
occurrence in diverse microbial communities. Proc Natl
Acad Sci U S A 112:6449–6454
Zhang L, Hu J, Zhu R, Zhou Q, Chen J (2013) Degradation of
paracetamol by pure bacterial cultures and their microbial
consortium. Appl Microbiol Biotechnol 97:3687–3698
Zhao H, Liu Q, Wang Y, Han Z, Chen Z, Mu Y (2018a) Biochar
enhanced biological nitrobenzene reduction with a mixed
culture in anaerobic systems: short-term and long-term
assessments. Chem Eng J 351:912–921
Zhao L et al (2018b) Co-contaminant effects on 1,4-dioxane
biodegradation in packed soil column flow-through
479
systems. Environ Pollut 243:573–581. https://doi.org/10.
1016/j.envpol.2018.09.018
Zhou K, Qiao K, Edgar S, Stephanopoulos G (2015) Distribut-
ing a metabolic pathway among a microbial consortium
enhances production of natural products. Nat Biotechnol
33:377
Publisher’s Note Springer Nature remains neutral with
regard to jurisdictional claims in published maps and
institutional affiliations.
123