Characterization of co-metabolic biodegradation of methyl: Tert -butyl ether by a Acinetobacter sp. strain

Article abstract of DOI:10.1039/c9ra09507a

Co-metabolic bioremediation is a promising approach for the elimination of methyl tert-butyl ether (MTBE), which is a common pollutant found worldwide in ground water. In this paper, a bacterial strain able to co-metabolically degrade MTBE was isolated and named as Acinetobacter sp. SL3 based on 16S rRNA gene sequencing analysis. Strain SL3 could grow on n-alkanes (C₅-C₈) accompanied with the co-metabolic degradation of MTBE. The number of carbons present in the n-alkane substrate significantly influenced the degradation rate of MTBE and accumulation of tert-butyl alcohol (TBA), with n-octane resulting in a higher MTBE degradation rate (V_max = 36.7 nmol min^-1 mg_protein^-1, K_s = 6.4 mmol L^-1) and lower TBA accumulation rate. A degradation experiment in a fed-batch reactor revealed that the efficiency of MTBE degradation by Acinetobacter sp. strain SL3 did not show an obvious decrease after nine rounds of MTBE replenishment ranging from 0.1-0.5 mmol L^-1. The results of this paper reveal the preferable properties of Acinetobacter sp. SL3 for the bioremediation of MTBE via co-metabolism and leads towards the development of new MTBE elimination technologies.

Full text of DOI:10.1039/c9ra09507a

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Characterization of co-metabolic biodegradation
of methyl tert-butyl ether by a Acinetobacter sp.
strain
Cite this: RSC Adv., 2019, 9, 38962
*
Shanshan Li,
Dan Wang, Dan Du, Keke Qian and Wei Yan
Co-metabolic bioremediation is a promising approach for the elimination of methyl tert-butyl ether (MTBE),
which is a common pollutant found worldwide in ground water. In this paper, a bacterial strain able to co-
metabolically degrade MTBE was isolated and named as Acinetobacter sp. SL3 based on 16S rRNA gene
sequencing analysis. Strain SL3 could grow on n-alkanes (C5–C8) accompanied with the co-metabolic
degradation of MTBE. The number of carbons present in the n-alkane substrate significantly influenced
the degradation rate of MTBE and accumulation of tert-butyl alcohol (TBA), with n-octane resulting in
a higher MTBE degradation rate (Vmax ¼ 36.7 nmol minꢀ1 mgproteinꢀ1, Ks ¼ 6.4 mmol Lꢀ1) and lower TBA
accumulation rate. A degradation experiment in a fed-batch reactor revealed that the efficiency of MTBE
degradation by Acinetobacter sp. strain SL3 did not show an obvious decrease after nine rounds of MTBE
replenishment ranging from 0.1–0.5 mmol Lꢀ1. The results of this paper reveal the preferable properties
of Acinetobacter sp. SL3 for the bioremediation of MTBE via co-metabolism and leads towards the
development of new MTBE elimination technologies.
Received 14th November 2019
Accepted 20th November 2019
DOI: 10.1039/c9ra09507a
rsc.li/rsc-advances
In contrast, various microorganisms are able to oxidize MTBE in
the presence of another growth substrate through co-
1. Introduction
metabolism. Various compounds present in gasoline could be
used as co-metabolic growth substrates for MTBE degradation,
such as normal and branched alkanes and aromatics.7,11–13
Smith et al.14 reported that Pseudomonas mendocina KR-1 is able
to co-metabolically degrade MTBEꢀw1ith an average degradation
rate of 61.1 nmol minꢀ1 mgprotein with n-alkanes (C5–C8) as
carbon sources. Although MTBE was eliminated effectively,
stoichiometric accumulation of tert-butyl alcohol (TBA) was
observed. Two strains, Pseudomonas aeruginosa BM-B-450 and
Pseudomonas citronellolis BM-B-447, with different kinetic
parameters for MTBE degradation were isolated from an n-
pentane-adapted consortium, suggesting the presence of
cooperative degradation in the consortium.7
Though a variety of microorganisms able to degrade MTBE
by co-metabolism have been discovered, our understanding of
the process of co-metabolism in a pure culture remains
limited.15,16 Some studies have indicated that microorganisms
utilize the primary, growth-supporting substrate for cell growth,
inducing an enzyme that also catalyzes the conversion of the
non-growth substrate. Smith et al.17 characterized the initial
reaction of the co-metabolic MTBE degradation pathway of
Mycobacterium vaccae JOB5 grown on n-propone and found that
the alkane hydroxylase that catalyzed the transformation of n-
octane was also responsible for MTBE oxidation. Numerous
studies have indicated that non-specic monooxygenase
enzymes are responsible for the initial oxidation of MTBE to
TBA, including cytochrome P450 monooxygenases18,19 and
Co-metabolism is dened as the ability of a microorganism to
convert a non-growth substrate in the presence of either
a growth substrate or another biodegradable substrate.1 It
represents a potential alternative for the elimination of pollut-
ants, as it separates the process of pollutant biodegradation
from the growth of microorganisms, resulting in a shortened
period of adaption and propagation. Co-metabolism has been
conrmed to be effective in the elimination of alkanes, as well
as aromatic and chlorinated compounds in natural environ-
ments, including methyl tert-butyl ether (MTBE).2–4
MTBE is used as an effective gasoline oxygenate because of
its favorable properties, such as low production cost, high
octane rating capability, and miscibility with other gasoline
components. With widespread use of MTBE and increasing
reports of groundwater contamination, the development of
technology to eliminate MTBE contamination has become
a priority.5,6 Due to the presence of a stable ether bond and
sterically hindered carbon in its molecular structure, MTBE is
relatively difficult for microorganisms to assimilate.7 Currently,
only a few microorganisms are known to be able to grow using
MTBE as the sole carbon source, including Methylibium petro-
leiphilum PM1,8 Aquincola tertiaricarbonis L108,9 and Hydro-
genophaga ava ENV735.10 Growth of these strains on MTBE is
characterized, however, by low growth rate and biomass yield.
Department of Environmental Science & Engineering, Xi'an Jiaotong University, Xi'an,
Shaanxi 710049, China. E-mail: yanwei@xjtu.edu.cn
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alkane hydroxylases.17,20 Furthermore, alkane hydroxylases and analysis. Genomic DNA was extracted with the Ezup Column
their homologs appear to be widely distributed, especially Bacteria Genomic DNA Purication Kit (Sangon Biotech,
among Gram-negative organisms that use n-alkanes as growth Shanghai, China) according to the manufacturer's instructions.
substrates. This may help explain why alkanes are the preferred A partial 16S rRNA and rpoB sequence was amplied from the
co-metabolic substrates for the degradation of MTBE.
genomic extraction with universal primers 27F (50-AGAGTTT-
The objective of this work was to examine the co-metabolic GATCCTGGCTCAG-30), 1492R (50-GGTTACCTTGTTACGACTT-30)
degradation of MTBE by a newly isolated Acinetobacter sp. and AcF (50-GTGATAARATGGCBGGTCGT-30), AcR (50-
strain grown on n-octane. We characterized the co-metabolism CGBGCRTGCATYTTGTCRT-30), respectively. The amplication
of MTBE by this strain, including the range of co-metabolic reaction mixtures (50 mL) contained 1 mL of template, 1.5 U of
substrates, the kinetic parameters of the reaction, and the Takara PrimeSTAR HS DNA Polymerase (Takara Biotech,
ability of the strain to provide continuous degradation of MTBE. Dalian, China), 10 mL of 5ꢂ PrimeSTAR buffer, 2 mL each of
To our knowledge, it is the rst report on the co-metabolism of forward and reverse primers (10 mM), and 5 mL of dNTP mixture
MTBE by a Acinetobacter sp. strain. These results provide a basis (2.5 mM). Amplications were carried out on a T100 Thermal
for further development of technologies to eliminate MTBE Cycler (Bio-Rad, Hercules, CA, USA) with the following condi-
from the environment.
tions: 98 ꢁC for 5 min; 30 cycles of 98 ꢁC for 10 s, 55 ꢁC for 5 s,
and 72 ꢁC for 90 s; and a nal extension at 72 ꢁC for 7 min. Aer
purication, the PCR product was Sanger-sequenced by Sangon
Biotech.
The DNA sequence was used to identify homologous
sequences in GenBank with BLAST. Phylogenetic analysis of the
16S rRNA and rpoB sequence was performed with MEGA 5.1
soware.23 Multiple alignment with sequences from GenBank
was performed using the Clustal method.24 The phylogenetic
trees were constructed using the neighbor-joining method. The
16S rRNA sequence was deposited in the National Center for
Biotechnology Information (NCBI) GenBank database under
accession number KX639781.
2. Materials and methods
2.1 Chemicals and media
All gaseous alkanes (methane, ethane, propane, n-butane) were
purchased from Xi'an Standard Gas Station. MTBE (99%, r ¼
0.74 g cmꢀ3), TBA (99%, r ¼ 0.77 g cmꢀ3), n-pentane (99%, r ¼
0.63 g cmꢀ3), n-hexane (99%, r ¼ 0.69 g cmꢀ3), n-heptane (99%,
r ¼ 0.68 g cmꢀ3) and n-octane (99%, r ¼ 0.70 g cmꢀ3) were
obtained from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China).
Mineral salt medium (MSM) was used for growth of micro-
organism and degradation of MTBE and contained the following
(g Lꢀ1): 0.9 KH2PO4, 6.5 Na2HPO4$12H2O, 0.4 (NH4)2SO4, 0.2
MgSO4$7H2O, 0.01 CaCl2$2H2O, 0.001 FeSO4$7H2O. In addition,
MSM contained 1 mL of trace element solution, which was
composed of the following in 1 L of deionized water: 0.1 g H3BO3,
0.4 g CoCl$6H2O, 0.25 g ZnCl2, 1 g MnSO4$H2O, 0.25 g Na2-
MoO4$2H2O, 0.1 g NiSO4$6H2O, and 0.25 g CuCl2$2H2O. The pH
value of the medium was adjusted to 7.0, and then the medium
was autoclaved at 115 ꢁC for 15 min. The carbon source n-alkanes
and MTBE were then added at concentrations of 50 mg Lꢀ1 and
10 mg Lꢀ1, respectively.
2.4 Kinetic analysis
The kinetic parameters of MTBE oxidation by SL3 were
determined following the MTBE degradation experiment. SL3
was incubated in LB broth rst overnight at 30 ꢁC and 150 rpm.
Aer washing, 0.4 mL of SL3 culture was then inoculated into
20 mL of MSM in 125 mL serum bottles sealed with poly-
tetrauoroethylene (PTFE) stoppers. Initial cell suspension
was standardized to an optical density at 600 nm (OD600) of
0.1. The carbon source (C5 or C8 substrates) at a concentration
of 4.4 mM was added to the serum bottles through the PTFE
stoppers with syringes in the presence or absence of MTBE
ꢁ
(0.88 mM). Aer incubated at 30 C with shaking at 150 rpm
2.2 Isolation of MTBE-degrading strain
for 48 h, the cultures were harvested by centrifugation at
5000 rpm for 10 min in a Sorvall ST 40 Centrifuge (Thermo-
Fisher Scientic, Waltham MA, USA). Following washing and
sonication, one part of the supernatant was used to determine
the concentration of total protein using the Folin phenol
method. Bovine serum albumin (BSA) was used as a standard.
The remainder of the supernatant was incubated with various
initial concentrations of MTBE for 25 min before the nal TBA
concentration was determined by GC-MS (see below). The rate
of MTBE oxidation was calculated from the nal TBA
concentration, assuming that the rate of oxidation of MTBE
remained constant for the rst 30 min and that no further
oxidation of TBA occurred during this period. Kinetic
constants were derived by computer-tting the data to a single
substrate-binding model, y ¼ Vmax [x/(Ks + x)], using Origin
A single strain that can co-metabolically degrade MTBE with n-
octane as the growth substrate was isolated from mixed
culture.21,22 The isolation experiment was conducted in 100 mL
Erlenmeyer asks sealed with silica gel stoppers. Microbial
consortium was incubated in 20 mL MSM with n-octane as the
carbon source at 30 ꢁC and 150 rpm for 10 d. The mixed culture
was then diluted and spread on solid MSM plates supplemented
with n-octane and MTBE for screening single colonies. Aer-
wards, various single colonies were re-streaked onto MSM agar
plates with n-octane until pure culture was obtained. Finally,
a single strain (SL3) that grew rapidly on n-octane was selected
and preserved for further analysis.
2.3 Phylogenetic identication
Strain SL3 was classied by 16S rRNA sequence analysis version 6.1 (Northampton, MA, USA). All experiments were
together with rpoB (RNA polymerase subunit) sequence performed in triplicate.
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2.5 Fed-batch reactor
3. Results and discussion
Fed-batch reactor was operated for MTBE degradation. Briey,
the reactors were closed with tight Teon-coated caps. Diffusers
and magnetic stirrers coated with Teon were used to aerate and
mix the content of the reactors. Air was passed via columns
containing activated carbon to supply oxygen to the reactors. SL3
was rst incubated in MSM supplemented with n-octane (4.4
3.1 Isolation and identication of co-metabolic MTBE-
degrading strain
One of the key factors constraining the application of MTBE
bioremediation is the extremely low biomass of microorgan-
isms grown on MTBE. Co-metabolism, which offers the benets
of higher biomass yield and simultaneous elimination of at
least two pollutants, represents a potential alternative for MTBE
elimination. In this study, a single strain SL3 of Gram-negative
and short, rod-shaped cells has been screened from mixed
culture grown on n-octane in the presence of MTBE. Strain SL3
grew rapidly on solid MSM plates in the presence of n-octane
and formed circular yellow colonies. Molecular identication of
the isolates was performed by sequencing the 16S rRNA gene
and comparing the sequences with known 16S rRNA sequences
in the GenBank database (Fig. 1a). Though SL3 strain showed
higher sequence identity to Acinetobacter pittii ATCC 19004
(GenBank accession number NR_117621), it could not be clas-
sied into Acinetobacter pittii species simply without any other
evidence. Besides 16S rRNA sequence analysis, housekeeping
genes are developed in identication of environmental isolates.
Among them, the rpoB gene, encoding the b-subunit of RNA
polymerase, has emerged as a core gene candidate for phylo-
genetic analyses allowing discrimination of closely related
isolates.25,26 Therefore, we carried out further identication of
SL3 strain by the rpoB gene analysis. As shown in Fig. 1b, SL3
strain displayed an exceptionally lower similarity with A. pittii.
Notably, SL3 strain did not fall into the cluster of pathogenic
Acinetobacter calcoaceticus-baumannii (ACB) complex, including
Acinetobacter pittii, Acinetobacter baumannii, Acinetobacter sei-
fertii and Acinetobacter nosocomialis. Furthermore, SL3 strain
showed similarity with non-ACB complex species Acinetobacter
calcoaceticus in rpoB gene, which indicated that SL3 may belong
to non-pathogenic Acinetobacter species. It was therefore
designated as Acinetobacter sp. SL3.
According to previous reports, species in the genus Acineto-
bacter occupy an important niche in nature, as they are ubiq-
uitously distributed in numerous environments, including soil,
sediments, aquatic environments, and contaminated sites.27,28
Moreover, Acinetobacter populations have the ability to miner-
alize versatile pollutants, such as various long-chain dicarbox-
ylic acids, as well as aromatic and hydroxylated aromatic
compounds.29,30 The various desirable proprieties of Acineto-
bacter strains make them promising alternatives to Escherichia
coli (a traditional model organism) for metabolism research.
One of these desirable properties is the simplicity of genetic
manipulation by homology-directed recombination with linear
DNA fragments.31 The broad scope of available substrates and
easy accessibility of genetic transformations facilitate the use of
Acinetobacter in biotechnology and bioremediation.
ꢁ
mM) for 24 h at 30 C and 150 rpm, and then inoculated into
2.0 L of MSM in reactor vessel with the initial OD600 value of 0.1.
Subsequently MTBE and n-octane were added to the reactor with
a ratio of 1 : 2 (mol : mol). At timed intervals, samples were
collected to determine the residual MTBE. When MTBE
concentration had decreased by 90% of the initial concentration,
MTBE was replenished from the stock solution. The MTBE
concentrations for each dosing ranged from 0.1 to 0.5 mM. The
reactors were operated under the following conditions: dissolved
oxygen was controlled at 3 ꢃ 1 mg Lꢀ1, pH was 7.0 ꢃ 0.2, and the
ꢁ
operating temperature was 30 ꢃ 2 C. The reactor without SL3
inoculation was operated as the control group. Both experimental
and control groups were conducted in triplicate.
2.6 Analytical methods
MTBE and TBA concentrations were analyzed by headspace
solid-phase dynamic extraction-gas chromatography-mass
spectrometry (HS-SPDE-GC/MS). Liquid culture was collected
and centrifuged at 6000 rpm for 10 min. Next, 2 mL of super-
natant was added to a 10 mL glass vial sealed with a screw cap
and silicone-polyperuoroethylene gaskets and immediately
underwent HS-SPDE analysis on a CTC CombiPAL-xt autosam-
pler (Chromtech, Idstein, Germany) equipped with a polar,
polyethylene glycol-coated WAX syringe, as described by Li
et al.21 The sample absorbed on the SPDE syringe was auto-
matically injected into a TraceGC ULTRA (ThermoFinnigan,
Milano, Italy) gas chromatograph equipped with a HP-5MS
capillary column (30 m length, 0.25 mm ID, 0.25 mm lm) and
a TraceISQ (ThermoFinnigan) mass spectrometric detector. The
injection port was set in splitless mode and held at 260 ꢁC. The
oven temperature was held at 40 ꢁC for 2 min and was then
ramped to 120 ꢁC at 5 ꢁC minꢀ1. The ow rate of carrier gas
(helium 5.0) was 1.0 mL minꢀ1. The interface and ion source
temperatures were maintained at 280 ꢁC and 230 ꢁC, respec-
tively. The mass spectrometer was operated in electron impact
mode at 70 eV in selected ion monitoring (SIM) mode at 73 and
59 m/z for MTBE and TBA, respectively. MTBE and TBA were
quantied using standards of known concentrations.
Gas-phase samples were removed from the sealed reaction
bottles through the PTFE valves with syringes and immediately
injected into an Agilent 6890 gas chromatograph equipped with
a thermal conductivity detector (TCD) and a TDX-01 column
(2 mm ꢂ 2 m). The column was operated at 60 ꢁC, and the
injector and detector were maintained at 110 ꢁC and 160 ꢁC,
respectively.
3.2 MTBE co-metabolism with n-alkanes as growth substrate
All computer-based statistical analyses were performed with
SPSS 16.0 soware for Windows (SPSS Inc., Chicago, IL, USA). As the important components of rened gasoline, the detection
One-way ANOVA was used to assess differences between treat- of MTBE oen accompanies with the presence of n-alkanes and
ments at a signicance level of 0.01 or 0.05.
polycyclic aromatic hydrocarbons (PAH) in the pollution sites.
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Fig. 1 Phylogenetic tree of Acinetobacter sp. SL3 based on 16S rRNA gene (A) and rpoB gene (B) sequences. The trees were constructed using
the neighbor-joining method with 1000 bootstrap replicates. The trees were drawn to scale, with branch lengths in the same units as the
evolutionary distances used to infer the phylogenetic tree. All positions containing gaps and missing data were eliminated from the dataset
(complete deletion option).
Furthermore, the co-metabolism has been established micro-
bial degradation of MTBE in the presence of alkanes.32 Though
Table 1 Growth of Acinetobacter sp. SL3 on various growth substrates
strain SL3 was isolated from a mixed culture with n-octane as
Substratea
Culture turbidityb (OD600
)
the sole carbon source, other n-alkanes and aromatics were also
employed to test their ability to support its growth (Table 1). In
addition to n-octane, strain SL3 grew well on C5 to C7 straight-
chain liquid alkanes. In contrast, none of the tested gaseous
alkanes (C1 to C4) supported growth of strain SL3. Furthermore,
little or no growth was observed with tested aromatic
compounds, including benzene, toluene, and xylene. Neither
MTBE nor TBA could be utilized as growth substrates for Aci-
netobacter sp. SL3. At current, only a few single strains have
been demonstrated to mineralize MTBE as the sole carbon
source. Moreover, most microorganisms grow poorly on MTBE
producing extremely low biomass even aer long incubation
times.33 Therefore, co-metabolism has been considered an
potential alternative because it uncouples contaminant
biodegradation from growth allowing shorter adaptation/
propagation periods for biotreatment systems.32,34
n-Alkanes
Methane
Ethane
Propane
n-Butane
n-Pentane
n-Hexane
n-Heptane
n-Octane
<0.02
<0.02
<0.02
<0.02
0.215 (0.018)
0.266 (0.010)
0.243 (0.012)
0.489 (0.022)
Aromatics
Benzene
Toluene
o-Xylene
p-Xylene
m-Xylene
0.035 (0.008)
0.043 (0.007)
0.054 (0.006)
0.038 (0.012)
0.042 (0.013)
The potential substrates for the co-metabolism of MTBE by
strain SL3 were determined (Table 2). Though SL3 grew slightly
with benzene, toluene, and xylene as sole carbon sources and
degraded these at low rates (7.43–9.37 nmolsubstrate minꢀ1
mgproteinꢀ1), no MTBE oxidation or TBA production was
observed in cultures with the used BTEX. All short chain n-
alkanes tested (C5–C8) were able to support the growth of
MTBE and metabolites
MTBE
TBA
<0.02
<0.02
a
Triplicate cultures of SL3 were grown for 5 days in the presence of each
substrate respectively at an initial substrate concentration of 0.44 mM
b
(the initial value of OD600 was #0.02). Optical densities are reported
as means (SE).
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Table 2 Specific degradation rates and products of co-metabolism of Acinetobacter sp. SL3 on diverse substrates
Aromatics
n-Alkanes
Others
Parametersa
Benzene Xylene
Toluene n-Pentane n-Hexane n-Heptane
n-Octane
MTBE TBA
Biomass (mgprotein dꢀ1
)
2.3 (0.1) 4.3 (0.4) 3.5 (0.5) 11.2 (1.4) 13.6 (1.1) 12.2 (0.7)
8.25 (0.17) 7.43 (0.46) 9.37 (1.19) 88.98 (3.49) 88.32 (7.04) 103.47 (3.94) 125.71 (3.62) —
16.9 (0.3)
—
—
—
Degradation rate of substrateꢀs1
(nmolsubstrate minꢀ1 mgprotein
)
Degradation rate (nmolMTBE minꢀ1
NDc
NDc
NDc
11.06 (1.59) 11.40 (1.74) 13.69 (0.97) 17.29 (1.50)
—
—
ꢀ1
mgprotein
)
CCb (mgMTBE mgsubstrate
)
—
—
—
—
—
—
0.62 (0.08) 0.56 (0.1) 0.74 (0.10) 0.86 (0.07)
0.14 (0.03) 0.16 (0.02) 0.17 (0.02) 0.11 (0.01)
—
—
—
ꢀ1
Residual TBA (mmol)
19.8 (0.1)
a
Triplicate cultures of each single strains were cultivated in 20 mL of MSM in 125 mL serum bottles sealed with polytetrauoroethylene (PTFE)
stoppers for 5 days in the presence of carbon substrate (4.4 mM) and MTBE (0.88 mM) (the initial value of OD600 was #0.02); all data are
b
reported as means (SE). The co-metabolic coefficient (CC) is the ratio of degraded oxygenated compound (MTBE) to the consumed n-alkane.
c
ND means not detect.
Acinetobacter sp. SL3 with relatively high biomass yield (11.2– decreasing degradation rate of MTBE. Furthermore, TBA pres-
16.9 mgprotein dꢀ1) and n-alkane degradation rates (88.32– ence in groundwater is also of concern and some states in the
125.71 nmolsubstrate minꢀ1 mgproteinꢀ1). As expected, the oxida- U.S. have established maximum concentration levels in water.32
tion rateꢀo1f MTBE increased from 11.06 to 17.29 nmol minꢀ1 Diverse n-alkane-oxidizing microbes are reported to degrade
mgprotein
with increasing degradation of n-alkanes. It is MTBE through co-metabolism, but there are few reports of
interesting to observe that TBA, a stable metabolite of MTBE strains able to degrade both MTBE and TBA. In most reported
oxidation, was consumed and did not accumulate in the culture microorganisms, MTBE was converted to TBA, which could not
of strain SL3 when n-alkanes (C5–C8) are used as growth be degraded further and would accumulate in the system.14,32 P.
substrates. These results highlight the potential capacity of mendocina KR-1 grows well on toluene, n-alkanes (C5–C8), and
Acinetobacter sp. SL3 to degrade both MTBE and its metabolite, alcohols and oxidizes MTBE to TBA in the presence of n-alkanes
TBA. The n-alkane growth substrates for these organisms (C5–C8) at an average rate of 61.1 nmol minꢀ1 mgprotein
.
ꢀ1
include n-octane and some of the other shorter-chain (C # 10) n- However, in this system, TBA accumulates and cannot be
alkanes that are abundant in gasoline, the principal source of degraded further.14 Nevertheless, an signicant highlight of our
most MTBE found in the environment. As the majority of MTBE study which should be noted is that complete mineralization of
enters the environment as part of gasoline, this range of MTBE was attained and TBA did not accumulated aer metab-
cosubstrates raises the possibility that organisms involved in olism completed. In our work, high TBA degradation efficiencies
gasoline biodegradation could contribute to MTBE degradation by strain SL3 on the growth of C5–C8 n-alkanes were observed,
in gasoline-impacted environments. This may help interpret in which help overcome the problem of TBA accumulation during
terms of why shorter-chain n-alkanes were cosubstrates for MTBE elimination. In this sense, the application of co-
most MTBE-degrading microorganisms by co-metabolism.
metabolism of MTBE by SL3 strain is apparently attractive and
To further characterize the co-metabolism of MTBE and the economical, because compounds used as growth source (n-
its intermediate TBA, time courses of TBA production and octane) are non-expensive and available, TBA did not accumu-
consumption in relation to MTBE and n-octane degradation by lated aer metabolism completed.
Acinetobacter sp. SL3 were determined (Fig. 2). The addition of
Like many other n-alkane-utilizing microorganisms, Acine-
MTBE did not elicit any obvious negative effects on the rate of tobacter sp. SL3 appears to initiate the oxidation of MTBE with
consumption of n-octane. Time courses of the consumption of n- a monooxygenase catalyst, resulting in TBA as the metabolite.
octane and MTBE were similar in pattern. In SL3 cultures con- Previous studies have demonstrated that aerobic co-
taining both n-octane and MTBE, n-octane was consumed within metabolism of MTBE by microorganisms using n-alkanes as
60 h. Consumption of MTBE and production of TBA were growth substrates is initiated by n-alkane monooxygenase-
detected aer n-octane was added. Oxidation of MTBE did not catalyzed reactions.35,36 The n-alkane monooxygenase inserts
continue past 60 h. At this time point, the accumulation of TBA as one oxygen atom from an oxygen molecule into the substrate
an intermediate also reached its maximum, but TBA was subse- and reduces the other to water.37 This is in agreement with
quently degraded, with complete degradation occurring at 120 h. several previous studies on the co-metabolism of MTBE with n-
Associated to the hydrocarbon degradation, maximum CO2 alkanes as growth substrates.35
production was aer 80 h. Consistent with the results descripted
above, the degradation curve (Fig. 2) by strain SL3 conrmed the
mineralization of TBA previously suggested. A major concern in
MTBE co-metabolism is that TBA accumulation may result in an
incomplete site cleanup. The accumulated TBA could inhibit the
enzyme activity responsible for MTBE oxidation resulting in the
3.3 Kinetic characteristics of MTBE co-metabolism by
Acinetobacter sp. SL3 on n-alkanes
The co-metabolic MTBE degradation pathway of most previ-
ously studied microorganisms is initiated with the oxidation of
MTBE to TBA, which is believed to be the limiting step in the
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Fig. 2 Time courses of n-octane and MTBE consumption during growth of Acinetobacter sp. SL3. Acinetobacter sp. SL3 was grown in the
presence or absence of n-octane and MTBE. Time courses (A) presented include n-octane consumption and MTBE consumption for cultures
grown with MTBE only (open violet diamonds), n-octane only (open pink squares), or MTBE plus n-octane (filled violet diamonds and filled pink
squares, respectively). In addition, the accumulated amount of TBA (filled orange circles) generated from MTBE and CO2 production (filled green
triangles) are shown for cultures grown with n-octane plus MTBE. Symbols represent averages of three replicates, with error bars indicating
ranges of individual data points. The mass spectrum of MTBE and TBA detected during the process were shown (B).
ꢀ1
mineralization of MTBE. Apparent TBA accumulation during nmol minꢀ1 mgprotein
) and P. mendocina KR-1 (61.1
the initial period of MTBE oxidation by cells growing on n- nmol minꢀ1 mgproteinꢀ1) respectively on the growth of propane
alkanes indicates that the kinetics of MTBE oxidation may be and short chain n-alkanes (C5–C8), but the intermediate TBA
directly assessed by measuring the rate of accumulation of TBA
(Fig. 2). Values of Ks and Vmax for MTBE oxidation were deter-
mined for pure SL3 culture grown on n-pentane and n-octane in
the presence of MTBE (Fig. 3). Kinetic parameters varied when
Acinetobacter sp. SL3 was grown with equivalent concentrations
of MTBE and n-alkanes with different carbon numbers (C5 and
C8). Ks values of 6.4 and 15.7 mꢀm1ol Lꢀ1 and Vmax values of 36.7
and 17.9 nmol minꢀ1 mgprotein were determined for Acineto-
bacter sp. SL3 grown on n-octane and n-pentane, respectively.
The differences on kinetic parameters indicated that higher
MTBE affinity and degradation rate were achieved with the
growth on n-octane than those on n-pentane. Indeed, the
degradation efficiency of n-octane by SL3 was considerably
higher than that of n-pentane (Table 2), suggesting a larger
quantity or greater activity of the alkane monooxygenase
induced by n-octane and resulting in a higher MTBE degrada-
tion rate. Morales et al. examined the kinetics of MTBE co-
metabolism on n-alkanes (C3 and C7), and obtained the
contrary results. Apparent higher MTBE degradation rate was
observed with longer n-alkanes.7
Fig. 3 Kinetics of MTBE oxidation by Acinetobacter sp. SL3 with n-
Table 3 summarized the main kinetic results of co-metabolic pentane or n-octane as the co-metabolic substrate. Average rates of
MTBE oxidation on n-octane (filled circles) or n-pentane (filled
squares) are shown. All experiments were performed in triplicate. Error
bars show ranges of values around the mean.
degradation of MTBE by different pure culture on n-alkanes.
Notably, higher Vmax were reported with Xanthobacter sp. (69.96
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Table 3 Co-metabolic characteristic of MTBE by microorganisms
Growth
substrate
Vmax
Ks
aꢁ
TBA
degradation Reference
MTBE
(nmol minꢀ1 mgprotein
)
(mmol Lꢀ1
)
(L gprotein
hꢀ1
)
ꢀ1
ꢀ1
Microorganism
P. mendocina KR-1
Pseudomonas sp. DZ13
Pseudomonas aeruginosa BM-B-450
Pseudomonas citronellolis BM-B-447 n-Pentane
Arthrobacter sp. ATCC 27778
Rhodococcus rubber ENV425
Mycobacterium vaccae JOB5
Xanthobacter sp.
n-Alkanes (C5–C8) 61.1 (average)
0.013
6.8
0.28
0.34
4.39
0.15
16.95
20.76
1.08
1.75
0.34
ꢀ
+
+
14
16
7
7
38
39
13
32
n-Pentane
n-Pentane
38.1
19.13
34.47
6.78
4.6
0.26
6.21
0.024
0.013
1.36
2.4
+
Butane
ꢀ
ꢀ
ꢀ
ꢀ
+
n-Propane
n-Propane
n-Propane
n-Octane
13
69.96
36.7
Acinetobacter sp. SL3
6.4
This study
accumulation was observed in both strains. Comparing with culture could degrade MTBE continuously. Moreover, the
those of previously discovered microorganisms able to co- degradation rate of MTBE did not decrease apparently aer the
metabolically degrade MTBE on n-alkanes, the Ks value for the ninth round of substrate replenishment. Then the apparent
degradation of MTBE by strain SL3 in this study was much decrease in the MTBE degradation rate in the nal round may
higher. For instance, the Ks value for MTBE degradation by be attributed to the production of potential toxic oxidation
Arthrobacter sp. ATCC 27778 (ref. 38) on butane is 0.024 mmol products from MTBE degradation. As reported by Fortin et al.,41
Lꢀ1 and that of P. mendocina KR-1 is 0.013 mmol Lꢀ1
.
One a mixed F-consortium was able to degrade MTBE following two
14
possible explanation for this result is that different n-alkane rounds of replenishment in the presence of or aer incubation
monooxygenases are responsible for the degradation of MTBE with diethyl-ether (DiEE). MTBE was only transformed,
in different strains. To characterize the specic affinity for however, aer the complete degradation of the co-metabolic
MTBE, as an index aꢁMTBE (aꢁ
¼ Vmax/Ks) is generally used to carbon source, indicating that MTBE biodegradation is
MTBE
reect substrate specicity. The aꢁ
value for Acinetobacter subject to some type of inhibition in this system. Volpe et al.42
MTBE
ꢀ1
sp. SL3 on n-octane was 0.34 L gprotein
hꢀ1, which is almost assessed a microbial consortium in a batch reactor by repeat-
four times higher than the aꢁ
value on n-pentane (0.07 L edly feeding it with MTBE and oxygen. Aer several rounds of
MTBE
ꢀ1
gprotein
hꢀ1); this difference is mainly due to the large feeding and depletion, a signicant decrease in the time to
difference between Ks values for MTBE on the different complete degradation of MTBE was observed which may be due
substrates. A similar aꢁMTBE value was obtained for P. mendocina to the accumulation of TBA.
KR-1 (0.28 L gprotein ꢀ1 hꢀ1), which also has a relatively high Ks
In most cases, MTBE degradation rates in a xed system
values.14 Though higher aꢁ
values were calculated for Rho- would decrease with time which could be attributed to the
MTBE
ꢀ1
hꢀ1
)
and Arthro- accumulation of TBA. In order to minimize the inhibition effect
39
dococcus rubber ENV425 (20.76 L gprotein
bacter sp. ATCC 27778 (16.95 L gprotein
ꢀ1
hꢀ1),38,40 TBA of TBA, Liu et al. centrifuged and resuspended Arthrobacter sp.
accumulation accompanied the MTBE degradation process of ATCC 27778 (ref. 38) in fresh medium before each additional
lower rate in these systems. Pseudomonas citronellolis BM-B-447 dosing of MTBE. Finally, the MTBE remediation rate of ATCC
could degrade MTBE in the presence of ꢀn-1Pentane with the Vmax 27778 did not decrease obviously aer six time of dosing. This
and Ks of 34.47 nmol minꢀ1 mgprotein and 6.21 mmol Lꢀ1 process prevented excessive accumulation of TBA in the culture
respectively,7 which was much similar with those of SL3 strain. medium effectively, but increase the labor and cost absolutely.
However, the aꢁMTBE value for Acinetobacter sp. SL3 on n-octane In the fed-batch reactor of this study, MTBE degradation rate of
was 0.34 L gprotein ꢀ1 hꢀ1, which is more than two times of that SL3 did not show apparent decline aer nine time of replen-
for P. citronellolis BM-B-447 which indicated the higher specic ishment. This maybe contribute to the degradation capability of
affinity to MTBE of SL3. In summary, strain SL3 showed the both MTBE and TBA by SL3. This result has implications for the
obvious advantages in MTBE elimination of comparable TBA decontamination of MTBE-contaminated sites, as it suggests
degradation capability, preferable MTBE affinity and degrada- that metabolism of both MTBE and its intermediate TBA will
tion rate, which highlight its potential application in MTBE occur in the presence of n-octane.
elimination process.
3.5 Potential MTBE degradation pathway by Acinetobacter
3.4 MTBE degradation by Acinetobacter sp. SL3 grown on n-
sp. SL3 grown on n-octane
octane in fed-batch reactor
In the co-metabolism of MTBE, the initial attack of the MTBE
An additional experiment was conducted to determine the molecule is performed by a monooxygenase, especially n-alkane
MTBE degradation capability of strain SL3. Acinetobacter sp. SL3 monooxygenase. Alkane monooxygenase and their close
on the growth of n-octane was cultivated in the presence of homologs appear to be widely distributed, especially among
MTBE, with both MTBE being replenished at timed intervals Gram-negative organisms that utilize shorter chain (C5 to C10) n-
(Fig. 4). The MTBE degradation results indicate that the SL3 alkanes as growth substrates.43,44 In our study, several
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Fig. 4 Co-metabolism of MTBE by Acinetobacter sp. SL3 in a fed-batch reactor. Acinetobacter sp. SL3 was grown in the presence of MTBE and
n-octane. When MTBE concentration had decreased by 90% of the initial concentration, MTBE was replenished ranging from 0.1 to 0.5 mM. Data
are presented as means of three replicate experiments, with error bars showing the standard error. Data points represent the residual
concentrations of MTBE.
observations suggest that the enzyme responsible for the rst possibility.35,45 M. vaccae JOB5 could oxidize MTBE and TBA with
reaction in the proposed sequence, the oxidation of MTBE, is the same n-alkane monooxygenase on the growth of propane.35
the n-alkane monooxygenase that initiates n-octane oxidation For comparing MTBE and TBA as substrates for n-alkane
by n-octane-grown cells of Acinetobacter sp. SL3. First, the monooxygenase in JOB5, MTBE (Vmax/Ks ¼ 1.79 ꢂ 10ꢀ5) can be
differences in MTBE-oxidizing activity in propane-grown cells seen to be a better overall substrate than TBA (Vmax/Ks ¼ 8.8 ꢂ
and cells grown on other substrates. If, as we suggest, the co- 10ꢀ6). This difference may help rationalize why MTBE appears
metabolism of MTBE is catalyzed by n-alkane monooxygenase, to be preferentially oxidized over TBA. Aer MTBE is degraded
MTBE should not be converted in the absence of n-alkane via the degradation intermediates TBF, TBA, HIBA catalyzed by
monooxygenase. It means that MTBE degradation could not the same n-alkane monooxygenase, HIBA could be further
occur on the growth of the substrate which cannot induce the degraded to pyruvic acid through a series of metabolic pathways
expression of n-alkane monooxygenase. This is the behavior as various publication reported, which will be completed
that we observed in this study for aromatics-grown cells (Table degraded to CO2 via tricarboxylic acid cycle. The proposed
2). Second, the immediate MTBE-oxidizing activity observed MTBE co-metabolic pathway by SL3 strain is shown in Fig. 5.
with n-octane-grown cells. In a co-metabolic process the trans-
Acinetobacter as common indigenous microorganisms have
formation of a non-growth substrate is oen catalyzed by the been reported the occurrence in heavy metal contaminated
enzyme that catalyzed the growth substrate degradation. It wastewater and its role in the reduction of hexavalent chro-
therefore follows that transformation of a non-growth substrate mium,46 and in degradation of crude oil and alkanes,
should commence immediately at the time that the cells are permethrin,47 benzene homologues48 and pharmaceutical
challenged with that substrate. In our study, the degradation of compounds.49 The extraordinary competence of Acinetobacter
MTBE began immediately in the presence of n-alkane (Fig. 2). baylyi strain ADP1 for natural transformation and its genetic
Our aforementioned results are fully compatible with the ex- and physiological properties made this strain as a model
pected suggestion that the transformation of MTBE is catalyzed organism for metabolic.50 Acinetobacter pittii, formerly known as
by n-alkane monooxygenase.
Acinetobacter genomospecies 3, is an aerobic Gram-negative
Notably, our results suggest that the rst stable MTBE- bacillus that can be found in various settings, including soil,
degrading intermediate TBA is also further oxidized by the foods, sewage, and animals, as well as on human skin and
same n-alkane monooxygenase responsible for both MTBE and ora.51,52 As a nosocomial opportunistic pathogen which mainly
n-octane oxidation. Our evidence for the role of this enzyme in invades patients with immunosuppression and serious
this reaction is that oxidation of MTBE TBA subsequently diseases, A. pittii is commonly considered less virulent than
commence aer MTBE has been eliminated completely (Fig. 2). Acinetobacter baumannii within the Acinetobacter calcoaceticus-
One potential explanation of the observation should be that the baumannii complex.53
affinity of MTBE with n-alkane monooxygenase is higher than
Based on the results above, bioaugmentation of MTBE-
TBA. Several previous studies have also recognized this contaminated environment with strain SL3 signicantly
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Fig. 5 Proposed pathway for the co-metabolism of MTBE by n-octane-grown cells of Acinetobacter sp. SL3.
improved the removal rate of MTBE from the contaminated this study belong to Acinetobacter genus, which constitutes
water. However, this bioremediation application may increase a heterogeneous group of bacteria widespread in various
their occurrence in the environment which resulting in the settings, including soil, foods, sewage, and animals, and known
potential environmental risk concerns. The SL3 strain used in for its broad metabolic potential. Acinetobacter has been in the
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focus of many studies addressing fundamental biological
questions as well as its virulence potential. The strains from
Acinetobacter genus could degrade various kinds of pollutants,
including furthermore, SL3 was indigenous microorganisms
isolated from the MTBE-contaminated environment without
recombinant modication. Based of all, though the bio-
augmentation by strain SL3 will potentially increase its pres-
ence in natural environment, this increasing of indigenous wild
7 M. Morales, V. Nava, E. Velasquez, E. Razo-Flores and
S. Revah, Biodegradation, 2009, 20, 271.
8 S. R. Kane, A. Y. Chakicherla, P. S. Chain, R. Schmidt,
M. W. Shin, T. C. Legler, K. M. Scow, F. W. Larimer,
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9 M. Rosell, D. Barcelo, T. Rohwerder, U. Breuer, M. Gehre and
H. H. Richnow, Environ. Sci. Technol., 2007, 41, 2036.
SL3 strain may cause little effect on the microbial community. 10 S. H. Streger, S. Vainberg, H. Dong and P. B. Hatzinger, Appl.
Environ. Microbiol., 2002, 68, 5571.
11 M. Salazar, M. Morales and S. Revah, J. Environ. Sci. Health,
4. Conclusions
Part A: Toxic/Hazard. Subst. Environ. Eng., 2012, 47, 1017.
MTBE is one of the most common contaminants in gasoline- 12 R. J. Steffan, S. Vainberg, C. W. Condee, K. McClay and
polluted soil or groundwater and is oen accompanied by
other gasoline components, such as alkanes. Alkanes have been
P. B. Hatzinger, Biotreatment of MTBE with a new bacterial
isolate, Battelle press, Columbus, Ohio, USA, 2000.
shown to act as growth substrates for microorganisms capable 13 A. J. House and M. R. Hyman, Biodegradation, 2010, 21, 525.
of co-metabolically degrading MTBE. It is necessary therefore to 14 C. A. Smith, K. T. O'Reilly and M. R. Hyman, Appl. Environ.
investigate the development of methods to efficiently eliminate
MTBE contamination from the environment.
Microbiol., 2003, 69, 7385.
15 C. W. Lin and C. Y. Lin, Int. Biodeterior. Biodegrad., 2007, 59,
The results of this study indicate that a single strain of Aci-
97.
netobacter sp. SL3, possesses the ability to degrade MTBE co- 16 S. S. Li, S. Wang and W. Yan, Int. J. Environ. Res. Public
metabolically when grown on n-alkane (C5–C8) substrates. The Health, 2016, 13.
MTBE degradation rate of SL3 varied according to the carbon 17 C. A. Smith and M. R. Hyman, Appl. Environ. Microbiol., 2004,
number of the n-alkane growth substrate. Acinetobacter sp. SL3 70, 4544.
is able to co-metabolically degrade MTBE and its intermediate 18 C. Malandain, F. Fayolle-Guichard and T. M. Vogel, FEMS
TBA continuously, with no obvious reduction in degradation
Microbiol. Ecol., 2010, 72, 289.
rate aer nine rounds of n-octane replenishment. The results of 19 S. Chauvaux, F. Chevalier, C. Le Dantec, F. Fayolle, I. Miras,
this paper reveal the useful properties of Acinetobacter sp. SL3 F. Kunst and P. Beguin, J. Bacteriol., 2001, 183, 6551.
for the bioremediation of MTBE via co-metabolism and provide 20 E. L. Johnson and M. R. Hyman, Appl. Environ. Microbiol.,
a reference for the development of new MTBE elimination
technologies.
2006, 72, 950.
21 S. Li, D. Li and W. Yan, Environ. Sci. Pollut. Res. Int., 2015, 22,
10196.
22 S. Li, K. Qian, S. Wang, K. Liang and W. Yan, Int. J. Environ.
Res. Public Health, 2017, 14.
Funding
This work was supported by National Natural Science Founda- 23 S. Kumar, M. Nei, J. Dudley and K. Tamura, Briengs Bioinf.,
tion of China (Grant number 31670512), and Natural Science 2008, 9, 299.
Basic Research Plan in Shaanxi Province of China (Grant 24 M. A. Larkin, G. Blackshields, N. P. Brown, R. Chenna,
number 2018JM3039).
P. A. McGettigan, H. McWilliam, F. Valentin, I. M. Wallace,
A. Wilm, R. Lopez, J. D. Thompson, T. J. Gibson and
D. G. Higgins, Bioinformatics, 2007, 23, 2947.
Conflicts of interest
25 T. Adekambi, M. Drancourt and D. Raoult, Trends Microbiol.,
2009, 17, 37.
The authors declare that they have no conict of interest.
26 X. Wang, J. Li, X. Cao, W. Wang and Y. Luo, Antonie van
Leeuwenhoek, 2019, DOI: 10.1007/s10482-019-01312-5.
27 J. E. Kostka, O. Prakash, W. A. Overholt, S. J. Green, G. Freyer,
A. Canion, J. Delgardio, N. Norton, T. C. Hazen and
M. Huettel, Appl. Environ. Microbiol., 2011, 77, 7962.
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