A novel fluorescent long-chain fatty acid-substituted dye: labeling and biodegrading of Microthrix parvicella

Article abstract of DOI:10.1039/C8RA06986G

Microthrix parvicella (M. parvicella) is a filamentous bacterium that induces bulking in activated sludge. Here, we used the affinity of long-chain fatty acids (LCFA) for M. parvicella to create a novel fluorescent probe of carbazole modified by LCFA. The structure was characterized by ¹H NMR spectroscopy and mass spectrometry. The spectral properties, photostability, and hydrophobic properties of the probe were also characterized. Fluorescent-labeling results showed that it can label M. parvicella in situ and could be biodegraded via metabolism. The stable docking mode of carbazole probes with different fatty acid chains and lipases was also docked by the density functional tight-binding (DFTB) method.

Full text of DOI:10.1039/C8RA06986G

RSC Advances
View Article Online
View Journal | View Issue
PAPER
A novel fluorescent long-chain fatty acid-
substituted dye: labeling and biodegrading of
Cite this: RSC Adv., 2018, 8, 35855
Microthrix parvicella
a
Yingchun Gu,†a Dayong Lin,†b Xuening Fei,
Cuihong Wang,a Qi Yang,a
*
c
Yalin Tang
and Xueling Rend
*
Microthrix parvicella (M. parvicella) is a filamentous bacterium that induces bulking in activated sludge. Here,
we used the affinity of long-chain fatty acids (LCFA) for M. parvicella to create a novel fluorescent probe of
carbazole modified by LCFA. The structure was characterized by 1H NMR spectroscopy and mass
spectrometry. The spectral properties, photostability, and hydrophobic properties of the probe were also
characterized. Fluorescent-labeling results showed that it can label M. parvicella in situ and could be
biodegraded via metabolism. The stable docking mode of carbazole probes with different fatty acid
chains and lipases was also docked by the density functional tight-binding (DFTB) method.
Received 21st August 2018
Accepted 14th October 2018
DOI: 10.1039/c8ra06986g
rsc.li/rsc-advances
normal lipids. That is, M. parvicella has affinity for LCFAs via
lipases. Oleic acid and hexadecanoic acid are of two typical
LCFAs.1,15
Carbazole derivatives are highly conjugated and rigid struc-
tures which are easily functionalized and have excellent bio-
logical activity. They serve as a functional framework for the
fabrication of medicines, materials, and probes. Carbazole
derivatives in particular are excellent electron-donating chro-
mophores for creating uorescent probes with large Stokes
shis, excellent stability, and high quantum yields.16–18
1. Introduction
Activated sludge-bulking occurs in wastewater treatment plants
(WWTPs) with a wide range and frequency. This causes great
damage and is difficult to treat. The literature shows that
excessive proliferation of Microthrix parvicella (M. parvicella) is
responsible for 90% of sludge-bulking.1–5 Therefore, if M. par-
vicella can be targeted, then methods for early identication
and treatment could prevent or solve the issue of sludge-
bulking.6
Here, we created and characterized a carbazole uorescent
probe (FP1, Fig. 1) modied by LCFAs (hexadecanoic acid).
Ultraviolet-visible (UV-vis) absorption and uorescence spec-
troscopy studies characterized the photostability and thermo-
stability. M. parvicella from activated sludge was labeled in situ
without broken or lysozyme pretreatment. Two other probes
FP2 (ref. 13) and FP3 (ref. 19) were also used to study the impact
of side-chain length on the labeling efficiency, identication,
and metabolism of M. parvicella (Fig. 1).
Traditional sludge volume index (SVI) and morphological
recognition7,8 have been used with uorescence in situ hybrid-
ization (FISH) to identify M. parvicella,9,10 but this has many
disadvantages, including complex pretreatment, low cell
permeability, destructiveness, weak uorescence signal, long
turnaround times, complicated processing, narrow application
scope, and high expense. We recently reported some uorescent
probes with long-chain alkanes for labeling M. parvicella in
sludge samples.11–13
Nielsen14 reported that the lipases on M. parvicella can
catalyze the hydrolyzation of oils and lipid compounds into
long-chain fatty acids (LCFAs) and transport them into cells as
2. Experimental procedure
2.1 Chemicals and equipment
All reagents and solvents were from commercial sources and
were used without further purication. 16-Hydroxyhex-
aTianjin Engineering Technology Center of Chemical Wastewater Source Reduction and
Recycling, School of Science, Tianjin Chengjian University, Tianjin, 300384, China.
E-mail: xueningfei@126.com
adecanoic
acid
($98%)
and
9-ethyl-9H-carbazole-3-
bAnda Duosen New Material Technology CO., LTD, Heilongjiang Province, 151400,
carbaldehyde ($98%) were from Macklin. The FISH reagents
were from Takara. Other commonly used reagents were
purchased locally.
China
cBeijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese
Academy of Sciences (ICCAS), Beijing, 100190, China. E-mail: tangyl@iccas.ac.cn
dKey Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, School of
Pharmaceutical Sciences, Zhengzhou University, Henan Province, 450001, China
† These authors contributed equally to this work and should be considered
co-rst authors.
The 1H-NMR spectra were obtained in CDCl3 using a Bruker
AVANCE III 400 MHz spectrometer. The HPLC was used to
analyze the chemical purity on a Lab alliance PC4006A (USA).
The mobile phase was a solution of 95% CH3OH and 5% H2O
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 35855–35862 | 35855

View Article Online
RSC Advances
Paper
Scheme 1 Synthesis of FP1.
2.2.2 Preparation of stock solutions. For absorption or
uorescence measurements, FP1, FP2, and FP3 were dissolved
into DMSO in the dark and then stored at room temperature to
obtain the stock solution (1 ꢀ 10ꢂ3 mol Lꢂ1). These were diluted
with solutions to the desired concentration (6 ꢀ 10ꢂ6 mol Lꢂ1).
2.2.3 Fluorescence quantum yields. Fluorescence quantum
yields (F) were calculated according to the following eqn (1)
using Rhodamine B in EtOH (F ¼ 0.89):23,24
Fig. 1 Structure of FP1, FP2 and FP3.
ꢀ
ꢁ
2
Fx As nx
Fx ¼ Fs
(1)
Fs Ax ns
using a reversed-phase column C18 ODS (4.6 mm ꢀ 250 mm, 5
mm). Mass analysis was obtained on an electrospray ionization
(ESI) mass spectrometer. UV-vis and uorescence spectra were
carried out on a Perkin Elmer LAMBDA 35 UV-vis spectrometer
and HITACHIF-7000 uorescence spectrophotometer, respec-
tively. (The excitation and emission slits were both at 5 nm; PTM
voltage at 400 V.) Activated sludge sample and labeling images
were collected with a uorescence inverted microscope from
OLYMPUS-IX71 (10ꢀ objective).
here, Fx and Fs represent the F of the sample and reference,
respectively. Terms Fx and Fs are the integrated uorescence
spectra for the sample and reference, respectively. Terms Ax and
As are the absorbance for the sample and reference at the
excitation wavelength, respectively, and nx and ns are the
refractive indexes of the sample and reference, respectively.
2.2.4 Photostability and thermostability. The photo-
stability of FP1 was performed in EtOH at room temperature in
the dark. We used an iodine-tungsten lamp (500 W) as the light
source and 50.0 g Lꢂ1 NaNO2 aqueous solution as the cold trap
to decrease the effects of heat from illumination and of UV-light
shorter than 400 nm. The absorbance was measured aer illu-
minating for different times. We also measured the thermo-
stability of FP1 (6 mmol Lꢂ1 in DMSO) at 80 ꢁC in oil bath. The
absorption intensities of the compound were measured over
time.
2.2 General procedure
2.2.1 Synthesis (E)-1-(15-carboxypentadecyl)-4-(2-(9-ethyl-
9H-carbazol-3-yl)vinyl)pyridine-1-ium bromide (FP1). 16-
Hydroxyhexadecanoic acid was substituted by HBr to obtain 16-
bromohexadecanoic acid,20 which substituted 4-methylpyridine
to
give
1-(15-carboxypentadecyl)-4-methylpyridin-1-ium
2.2.5 Probe labeling of M. parvicella. Appropriate doses of
FP1, FP2 and FP3 solution (500 mmol Lꢂ1 in EtOH) was added to
1 mL of the sludge samples (1 g SS per L) as the carbon source
(other wastewater units are in normal operation) and stirred at
bromide.21 FP1 was synthesized via a condensation reaction of
4-methylpyridin derivative and 3-formyl carbazole derivative22
(Scheme 1).
1-(15-Carboxypentadecyl)-4-methylpyridin-1-ium
bromide
ꢁ
15–20 C. The sludge samples were then examined with a uo-
(0.477 g, 1.0 mmoL), 9-ethyl-9H-carbazole-3-carbaldehyde
(0.245 g, 1.1 mmoL), ethanol (10.0 mL) and piperidine (0.1
mL) were mixed together and reuxed for 8 h. The mixture was
concentrated and eluted over silica gel to give the product (FP1).
(0.379 g, 60%). Purity: 98.8% (HPLC). Mp 92–93 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d: 1.23–1.28 (m, 18H, CH2), 1.33–1.39 (m,
7H, J ¼ 6.80 Hz, CH2), 1.42–1.47 (m, 2H, CH2), 1.86–1.92 (m, 2H,
CH2), 2.16–1.18 (m, 2H, CH2), 4.46–4.51 (m, 4H, CH2), 7.28–7.31
(t, J ¼ 7.4 Hz, 1H, Ar), 7.54–7.57 (m, 2H), 7.69 (d, 1H, J ¼ 8.4 Hz,
Ar), 7.75 (d, 1H, J ¼ 8.8 Hz, Ar), 7.91 (d, J ¼ 8.8 Hz, 1H, Ar), 8.20–
rescence inverted microscope (OLYMPUS-IX71) to investigate
the labeling and metabolism in M. parvicella. To avoid biasing,
15–20 images were taken randomly rather than according to the
photo quality or labeling effect during the imaging.25 The image
analysis used Image Pro Plus 6 (IPP 6) soware.
3. Results and discussion
3.1 The spectral properties of FP1 in different solvents
8.26 (m, 4H, Ar), 8.60 (s, 1H, Ar), 8.91 (d, 2H, J ¼ 6.80 Hz, Ar), Fluorescent compounds oen show different spectra in
11.97 (br, 1H, COOH). HRMS (ESI-TOF) m/z: calcd for 553.3789 different solvents. Fig. 2 shows the spectral results of FP1, and
[M ꢂ Br]+; found: 553.3799 [M ꢂ Br]+.
35856 | RSC Adv., 2018, 8, 35855–35862
Table 1 shows the spectral data.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Fig. 2 The (a) UV-vis spectra and (b) fluorescent spectra of FP1 in
different solvents (6 mmol Lꢂ1) (lex ¼ 450 nm).
Fig. 3 The (a) UV-vis spectra and (b) fluorescence spectra of FP1 at
different pH values.
Fig. 2a shows that the absorption wavelength (labs) of FP1 in
all solvents were located at about 450 nm and shied blue with
increasing dipole moment and solvent polarity. This showed
a negative solvent effect. The dipole moment of the ground state
is larger than that of the excited state. When intramolecular
charge transfer (ITC) occurs, the ground state changed to an
excited state with a lower dipole moment. Solvents with a larger
polarity can decrease the energy of the ground state and
increase that of the excited state. This increases the energy
difference between them and results in an absorption blue shi.
The uorescence spectra were recorded with a lex of 450 nm
and a lem of 570 nm in all solvents (Fig. 2b). The Stokes shi
and F increased with increasing solvent polarity except water.
This might be because of FP1 aggregation in water increased the
nonradiative decay process and caused quenching.
Fig. 4 The photostability curve of FP1 (light source intensity: 7000 lm,
wavelength: 400–2500 nm).
3.3 Photostability
The photostability of uorescent probes plays a big role in their
quality and applications as uorescent labels.26 Excellent pho-
tostability is required, but organic probes can be easily
decomposed under illumination resulting in instability or error
when labeling. The absorption intensities of FP1 were measured
aer exposure to illumination with different times. The curve
between the intensity and time was shown in Fig. 4. There is no
apparent change aer exposure, even aer irradiation for 8 h,
there was only 3.3% signal loss indicating excellent stability—
this makes it a promising probe for applications in medicine,
biology, chemistry, environmental studies and other elds.
To measure the photostability, the rate of photooxidation
was described and referred to as the photooxidation
mechanism:27
3.2 The spectral properties of FP1 at different pHs
The pH is an important factor that affects the spectra of uo-
rescent compounds. Here, FP1(6 mmol Lꢂ1) in 10 mmol Lꢂ1 of
Na2HPO4–citric acid buffer with pH of 2.2 to 8.0 were prepared,
and the spectra were measured with a UV-vis and uorescence
spectrophotometer, respectively (Fig. 3).
It shows slight changes in lmax, lem and the spectra shapes
with pH. The absorption and uorescent intensities changed
little as pH increased from 2.2 to 6.0. The chemical structure
dissolved badly and was stable across this regime, which is
typical in medical, bioorganic and environment elds. However,
when the pH increased to 7–8, the uorescent intensities
increased, which may be because the carboxy group dissociate
into the anion form (carboxylate) in alkaline conditions, and
then the dissolvability of dye improved at this condition.
d[Dye]/dt ¼ K1[Dye][O2].
(2)
Table 1 The solvent properties and UV-vis characteristics of the probe
Solvent
H2O
DMF
DMSO
CH3CN
MeOH
EtOH
DCM
nD20a
1.333
1.85
80.0
405
572
167
1.431
3.82
36.7
432
566
134
1.478
3.96
48.9
433
566
133
1.344
3.92
37.5
434
567
133
1.328
1.7
33.0
437
565
128
1.361
1.69
24.3
442
564
122
6.28
1.424
0.51
8.9
469
570
101
mb
3c
lmax/nm
lem/nm
Stokes shi
Fd/10ꢂ3
0.834
8.70
13.85
6.44
6.59
74.45
a
Refractive index. b Dipole moment. c Dielectric constant. d Fluorescence quantum yield.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 35855–35862 | 35857

View Article Online
RSC Advances
Paper
Fig. 5 The lightfastness of FP1.
Fig. 6 The thermostability of FP1.
Here, [O2] is a constant of 3 ꢀ 10ꢂ4 mol Lꢂ1, so the above
equation can be changed to
ꢂd[Dye]0/dt ¼ K[Dye]
(3)
and
ln[Dye]0/[Dye]t ¼ Kt.
(4)
[Dye]0 and [Dye]t are the original concentration of dye and
that being illuminated for t hours, respectively. The Lambert–
Beer law states that there is a linear relationship between the
concentration of the dye and the maximum absorbance at low
concentrations. Thus, eqn (4) can be written as
Fig. 7 Images of sludge samples with Gram staining ((a) ꢀ400) and
FISH staining ((b and c) 10 ꢀ 100).
bacteria were detected by recommended uorescent probe
EUBmix probe mixed by 1 : 1:1 of EUB338 (Cy3-
GCTGCCTCCCGTAGGAGT, targeting for most bacteria),
EUB338-II (Cy3-GCAGCCACCCGTAGGTGT, targeting for Planc-
tomycetales), and EUB338-III (cy3-GCTGCCACCCGTAGGTGT,
targeting for Verruco microbiales). M. parvicella were targeted by
uorescent probes MPAmix mixed by 1 : 1 : 1 of MPA223 (Cy3-
GCCGCGAGACCCTCCTAG, targeting or most M. parvicella),
MPA60 (Cy3-GGATGGCCGCGTTCGACT, targeting for most M.
parvicella), and MPA645 (Cy3-CCGGACTCTAGTCAGAGC, tar-
geting for most M. parvicella).7,34,35
ln(A0/At) ¼ kt.
(5)
In the equation, A0 and At are the maximum absorbances of
the dye and that aer illumination, respectively. While k is the
photooxidation rate constant obtained from the slope (Fig. 5).
In this paper, the photooxidation rate constant of FP1 is 7.062 ꢀ
10ꢂ5 mol minꢂ1, which is the same order of magnitude with
some other carbazole dyes26 and much smaller than some
cyanines reported.19,28,29 The lower photoreduction rate constant
might be attributed to a stable trans structure.30
The FISH results showed that the fragmented images with
green uorescence (EUBmix, Fig. 7b) were the entire bacteria and
red uorescence (MPAmix, Fig. 7c) were M. parvicella. Both
results of distinct morphotype and oligonucleotide probes
conrmed M. parvicella in the activated sludge samples.
3.4 Thermostability
Thermostability is another important property of dyes. The
absorbance of FP1 (6 mmol Lꢂ1 in DMSO) was measured aer
ꢁ
storing it at 80 C in a water bath for different times (Fig. 6).
The results showed that the absorption intensity reduced
only 8.2% even aer heating for 8 h at 80 ꢁC. This suggests that
the dye has excellent thermostability.
3.6 Labeling of M. parvicella by novel probes
Here, 10 mmol Lꢂ1 of FP1, FP2, and FP3 were used to label M.
parvicella at different times respectively, and the images were
quantitated with Image Pro Plus 6.0 aer different treatment
3.5 Analysis of activated sludge samples
The distinct morphotype of lamentous bacteria species and M. times (Fig. 8). The mean uorescent intensities of M. parvicella
parvicella in activated sludge were studied with Gram staining as a function of time were shown in Fig. 9.
and FISH probes (Fig. 7). Fig. 7a shows lamentous bacteria
FP1 and FP2 can both distinguish between M. parvicella and
identied by Gram-positive staining. They have unbranched zooglea to label M. parvicella, but FP3 could not. M. parvicella
long and curly laments with diameters that are approximately had bright green uorescence with a distinct morphotype when
of 0.7–0.9 mm and a length of 200–400 mm. This agrees with the FP1 and FP2 were added to the activated sludge samples. At
distinct morphotype of M. parvicella.31,32
longer times the mean uorescence intensities of both M. par-
FISH analysis could identify and classify lamentous vicella and zooglea labeled by FP1 were gradually diminished.
bacteria and provide more detailed information.33 The total Aer 8 h, M. parvicella could not be clearly distinguished from
35858 | RSC Adv., 2018, 8, 35855–35862
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
3.7 The relationship between mean uorescent intensity
and SVI
The sludge volume index (SVI) is a metric of active sludge
settling in wastewater treatment. A SVI of 50–120 suggests that
the active sludge is suitable for processing. Lower values imply
low sludge activity, and higher values imply sludge bulking.
When the sludge of lamentous bacteria bulks, loose sludge
structures have increased to increase volumes, leading to
oating, and the difficult separation of solids and liquids leads
to sludge ocs in the effluent water, which affects effluent
quality. Early warning and control measures could assure that
the wastewater treatment process would operate normally. We
established an abundance model targeting M. parvicella in
activated sludge system.
Fig. 8 Images of activated sludge samples labeled by FP1, FP2, and
FP3 (10 mmol Lꢂ1) at different duration times (10 ꢀ 40).
Sludge samples with different SVI values were labeled with 10
mmol Lꢂ1 of FP1 (Fig. 10). The mean uorescence intensities (IOD/
Area) of each image were calculated and more than 10 images were
averaged. An early-warning model of targeting M. parvicella in
activated sludge system was established based on the relationship
between the mean uorescence intensity and SVI (Fig. 11).
As SVI increases, the abundance of M. parvicella labeled by FP1
increased, and the uorescence intensities and the mean uores-
cence intensities also enhanced with linear positive correlation (y
¼ 0.904x+7.2474, R2 ¼ 0.9827). Thus, the mean uorescence
intensities reect the abundance of M. parvicella when labeled.
Fig. 9 The mean fluorescent intensities of M. parvicella as a function
of time.
the zooglea. The signal became especially dim aer 12 h in both
M. parvicella and zooglea. Metabolism in FP1-labeled M. parvi-
cella (hydrolases) might underlie this signal change.
When FP2 was used to label, M. parvicella quickly had
a bright green uorescence and a distinct morphotype. The
mean uorescence intensities of both M. parvicella and zooglea
labeled by FP2 were still strong even at 36 h, and M. parvicella
could still be clearly distinguished from zooglea. The FP2 on M.
parvicella remained stable without any digestion of the dye. The
FP3 samples show that the zooglea had bright uorescence, but
M. parvicella could not be seen clearly. This suggests that M.
parvicella could not be labeled or distinguished from zooglea.
FP1, FP2, and FP3 have the same parent structure (carbazole-
stilbazolium salts), while they had different labeling properties
on M. parvicella because of their different side chain polarities.
When labeled by FP1, the extracellular lipase on the cell surface
of M. parvicella might be absorbed by the hydrophobic LCFA.
This would then be slowly ingested by endocytosis resulting in
a reduced concentration of FP1 and a decrease or even disap-
pearance of uorescence. When labeled by FP2, the lipase on
the cell surface of M. parvicella adsorbed the hydrophobic long-
chain alkanes but there was no endocytosis or metabolism—
this stabilized the probe, uorescence, and labeling. FP3 had no
hydrophobic long-chain groups and this resulted in weak
affinity to M. parvicella and thus poor labeling.
Fig. 10 Image of M. parvicella labeled by FP1 under different SVI values
(10 ꢀ 40).
These results agree with the affinity of hydrophobic long-
chain fatty acids to M. parvicella. These offer the theoretical
basis for further studies on M. parvicella and bulking or foam-
ing in activated sludge.
Fig. 11 Relationship between the fluorescence intensity of M. parvi-
cella labeled by FP1 and SVI.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 35855–35862 | 35859

View Article Online
RSC Advances
Paper
The SVI value of normal activated sludge is 50–150, and it is
considered to be bulked when over 200. Traditional SVI methods
or Gram staining can only judge sludge bulking when the SVI is
over 150 with M. parvicella proliferation and sticking out of
zooglea. When the SVI is lower than 60, Gram staining could not
label M. parvicella but the uorescent probe could. The correla-
tion between SVI and the mean uorescence intensity shows that
the probe can label M. parvicella even when there is only a trace of
M. parvicella. This makes the early diagnosis of M. parvicella easy
and offers a shorter warning period.
3.8 Interaction mechanism calculation
To further understand the different labeling results between the
three probes, we calculated the interactions between these three
probes and lipase via computational simulation. The structures
of the three probes were rst optimized by the density func-
tional tight-binding (DFTB) method,36–41 and the frontier
orbitals including the highest occupied molecular orbital
(HOMO) and the lowest unoccupied molecular orbital (LUMO)
were further calculated. A molecular docking study between the
probes and lipase used Autodock program.42 The crystal struc-
ture of the extracellular lipase is unknown, so the crystal
structure of triacylglycerol lipase (PDB ID: 1OIL)43 of Pseudo-
monas cepacia was docked in this study. In the molecular
docking simulation, a rapid grid-based energy evaluation and
torsional degree of freedom search method was used. The grid
Fig. 12 The HOMO (a) and LUMO (b) of FP1. The most stable docking
mode of FP1 and lipase (c) and (d) the binding energy distribution (e),
and binding modes (f) of FP1 and lipase.
ꢀ
size was 126 ꢀ 126 ꢀ 126, and the grid spacing was 0.375 A. The
docking was done by the Lamarckian genetic algorithm with
1000 conformations.
As shown in Fig. 12a and b, the active group was localized at
the ring of carbazole and pyridine. This plays an important role
when it interacts with other molecules. When docked with
lipase, the carbazole ring was outside the lipase pocket. Mean-
while, the long-chain was folded and the tail carboxyl group was
inset into the pocket because of the large size of the pocket. The
binding energy between FP1 and the lipase was
ꢂ8.35 kcal molꢂ1. Fig. 12e and f show that the largest binding
energy distribution was about 25 modes with a binding energy
of about ꢂ8.3 kcal molꢂ1 among the 1000 conformations.
The HOMO and LUMO of FP2 were similar to those of FP1,
and the active groups were localized at the ring of carbazole and
pyridine. The most stable binding energy was ꢂ8.8 kcal molꢂ1
when docked with the lipase (Fig. 13). The binding mode was
different from that of FP1. The structure of FP2 was inset into
the pocket, and all functional groups of FP2 interacted with the
lipase residues. This might explain the largest bonding
strength. Of the 1000 docked conformations, there were about 7
conformations that had the strongest binding energy of about
ꢂ8.8 kcal molꢂ1, and there were about 70 conformations with
a binding energy of about ꢂ8.6 kcal molꢂ1. This means that the
docked structural distribution is very dense.
Fig. 13 The HOMO (a) and LUMO (b) of FP2. The most stable
configuration of FP2 and lipase (c) and (d), and the binding energy
distribution (e), and docked binding modes (f) of FP2 and lipase.
and FP2, and the carbazole and long chain were localized at two
small cavities. Of the 1000 docked conformations, there were
about 10 conformations that had the strongest binding energy
of about ꢂ8.0 kcal molꢂ1. There were about 140 conformations
with a binding energy of about ꢂ7.6 kcal molꢂ1. This indicated
that the docking structural distribution is very dense as well.
The docked binding mode of FP3 was quite different from
those of FP1 and FP2. FP1 and PF2 were located at the large
concave cavity of lipase while FP3 was located at the side cavity
of lipase (Fig. 15).
The HOMO and LUMO orbitals were localized throughout
the molecule including the side chain of FP3 (Fig. 14)—these
were different from those of FP1 and FP2.
When docked with lipase, the binding energy was about
ꢂ8.07 kcal molꢂ1. The docked mode was very different from FP1
35860 | RSC Adv., 2018, 8, 35855–35862
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
4. Conclusions
The novel uorescent probe FP1 was designed and synthesized.
The spectra in different solvents and pH values were studied.
The stability results showed that it has excellent thermostability
and photostability. Labeling of M. parvicella was best with FP2.
The theoretical calculation results showed that the binding
energy of FP2 with lipases was the strongest, and this results in
stable uorescence, even for 36 h. The FP1 signal decreased via
lipase metabolism on the surface of M. parvicella. This corre-
sponds to the affinity of LCFAs for M. parvicella.
Conflicts of interest
The authors declare that there are no conicts to declare.
Acknowledgements
Fig. 14 The HOMO (a) and LUMO (b) of FP3. The most stable
configuration of FP3 and lipase (c) and (d), the binding energy distri-
bution (e), and binding modes (f) of FP3 and lipase.
The present study was nancially supported by the National
Natural Science Foundation of China (grant no. 21502137,
51578360, 51778398, 11604238), Key Laboratory of Targeting
Therapy and Diagnosis for Critical Diseases, Henan Province
(grant no. NMZL2018101) and Beijing National Laboratory for
Molecular Sciences (BNLMS201831)
Notes and references
¨
1 T. Dunkel, E. L. D. L. Gallegos, C. D. Schonsee, T. Hesse,
J. Jochmann, J. Wingender and M. Denecke, Water Res.,
2016, 88, 510–523.
2 L. Vanysacker, C. Denis, J. Roels, K. Verhaeghe and
I. F. Vankelecom, J. Microbiol. Methods, 2014, 97, 6–14.
3 D. Mamais, G. Nikitopoulos, E. Andronikou, E. Gavalakis,
A. Andreadakis, C. Giotakis and G. Tsimarakis, Global
NEST J., 2006, 8, 82–88.
Fig. 15 Docked binding modes of FP1, FP2 and FP3 with fatty
hydrolase (the green model represents FP1, purple is FP2, and pink is
FP3).
4 J. Wang, R. Qi, M. Liu, Q. Li, H. Bao, Y. Li, S. Wang, V. Tandoi
and M. Yang, Water Sci. Technol., 2014, 70, 367–375.
The binding strength order for the three probes and lipase
was FP2 > FP1 > FP3, while the dipole moment order (Table 2)
of the probes were FP2 < FP1 < FP3. This indicated that the
binding strength increased as the dipole moment decreased.
The theoretical results were consistent with the experimental
results: FP2 has the strongest interaction strength with
lipase, which was adsorbed on the surface of M. parvicella.
The uorescence intensity remained constant over time. FP1
has a weaker interaction with lipase than with FP2, but the
long chain fatty acid can metabolize the lipase of M.
parvicella.
´
5 D. S. L. Araujo, V. Ferreira, M. M. Neto, M. A. Pereira,
M. Mota and A. Nicolau, Appl. Microbiol. Biotechnol., 2015,
99, 1–10.
6 L. Speirs, T. Nittami, S. Mcilroy, S. Schroeder and
R. J. Seviour, Appl. Environ. Microbiol., 2009, 75, 2446–2452.
7 D. H. Eikelboom and H. J. J. Van Buijsen, Microscopic sludge
investigation manual, TNO Research Institute of
Environmental Hygiene, Del. 1983.
´
8 D. S. Wagner, E. Ramin, P. Szabo, A. Dechesne and
´
B. G. Plosz, Water Res., 2015, 78, 121–132.
9 F. D. L. Reyes, D. Rothauszky and L. Raskin, Water Environ.
Res., 2002, 74, 437–449.
10 J. C. Costa, D. P. Mesquita, A. L. Amaral, M. M. Alves and
E. C. Ferreira, Environ. Sci. Pollut. Control Ser., 2013, 20,
5887–5912.
Table 2 The dipole moments of FP1, FP2 and FP3 (unit: debye)
X
Y
Z
Total
11 X. Fei, W. Sun, L. Cao, X. Jiao, D. Lin and G. Jia, Appl.
Microbiol. Biotechnol., 2016, 100, 961–968.
12 S. Li, X. Fei, X. Jiao, D. Lin, B. Zhang and L. Cao, Appl.
Microbiol. Biotechnol., 2015, 100, 2883–2894.
FP1
FP2
FP3
ꢂ13.46
ꢂ10.33
ꢂ11.72
ꢂ4.15
5.76
2.80
1.06
15.22
12.36
16.63
ꢂ6.18
ꢂ11.75
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 35855–35862 | 35861

View Article Online
RSC Advances
Paper
13 X. Jiao, X. Fei, S. Li, D. Lin, H. Ma and B. Zhang, Materials,
2017, 10, 804.
T. V. Nasedkina, A. S. Zasedatelev and A. V. Chudinov, Dyes
Pigm., 2013, 97, 353–360.
14 P. H. Nielsen, P. Roslev, T. E. Dueholm and J. L. Nielsen, 30 Q. Kang, F. Fan, W. Bi, Y. Huang, Y. Fu and L. Wang, Chin. J.
Water Sci. Technol., 2002, 46, 73–80. Org. Chem., 2012, 32, 567–573.
15 M. Hesselsoe, J. L. Nielsen, P. Roslev and P. H. Nielsen, Appl. 31 S. Rossetti, C. Christensson, L. L. Blackall and V. Tandoi, J.
Environ. Microbiol., 2005, 71, 646–655. Appl. Microbiol., 1997, 82, 405–410.
16 A. Casey, R. S. Ashraf, Z. Fei and M. Heeney, Macromolecules, 32 F. Yan, R. Zhang, Y. Huang, D. Kong, Q. Ye, J. Xu and
2014, 47, 2279–2288. L. Chen, RSC Adv., 2016, 6, 50732–50760.
17 Y. Gu, D. Lin, Y. Tang, X. Fei, C. Wang, B. Zhang and J. Zhou, 33 J. Guo, Y. Peng, S. Wang, X. Yang and Z. Yuan, Chem. Eng. J.,
Spectrochim. Acta, Part A, 2018, 191, 180–188. 2014, 255, 453–461.
18 D. Lin, X. Fei, Y. Gu, C. Wang, Y. Tang, R. Li and J. Zhou, 34 J. Pernthaler, F. O. Glockner, W. Schonhuber and R. Amann,
Analyst, 2015, 140, 5772–5780. Methods Microbiol., 2001, 30, 207–210.
19 Y. Gu, D. Lin, R. Li, Y. Tang, X. Fei and J. Zhou, Luminescence, 35 D. Jenkins, M. G. Richard and G. T. Daigger, Manual On the
¨
¨
2016, 31, 380–387.
Causes and Control of Activated Sludge Bulking, Foaming And
Other Solids Separation Problems, Crc Press Inc. 2003.
36 G. Seifert, D. Porezag and T. Frauenheim, Int. J. Quantum
Chem., 1996, 58, 185–192.
20 T. Griesser, J. Adams, J. Wappel, W. Kern, G. J. Leggett and
G. Trimmel, Langmuir, 2008, 24, 12420–12425.
21 S. T. Yue, T. H. Steinberg, W. F. Patton, C. Y. Cheung and
R. P. Haugland, Carbazolylvinyl dye protein stains, US Pat. 37 M. Elstner, D. Porezag, G. Jungnickel, J. Elsner, M. Haugk,
US06579718, 2003.
22 Y. Gu, D. Lin, X. Fei, C. Wang, L. Li, Y. Tang and J. Zhou,
Luminescence, 2016, 31, 1229–1236.
23 D. Magde, R. Wong and P. G. Seybold, Photochem. Photobiol.,
2002, 75, 327–334.
24 R. T. Myers, J. Phys. Chem., 1980, 84, 1871.
T. Frauenheim, S. Suhai and G. Seifert, Phys. Rev. B:
Condens. Matter Mater. Phys., 1998, 58, 7260–7268.
38 M. Elstner, P. Hobza, T. Frauenheim, S. Suhai and E. Kaxiras,
J. Chem. Phys., 2001, 114, 5149–5155.
39 M. Elstner, T. Frauenheim and S. Suhai, J. Mol. Struct.:
THEOCHEM, 2003, 632, 29–41.
25 H. Daims, S. Lucker and M. Wagner, Environ. Microbiol., 40 M. Gaus, Q. Cui and M. Elstner, J. Chem. Theory Comput.,
2006, 8, 200–213. 2011, 7, 931–948.
26 X. Fei, R. Li, D. Lin, Y. Gu and L. Yu, J. Fluoresc., 2015, 25, 41 M. Gaus, A. Goez and M. Elstner, J. Chem. Theory Comput.,
1251–1258. 2013, 9, 338–354.
27 X. Chen, X. Peng, A. Cui, B. Wang, L. Wang and R. Zhang, J. 42 G. M. Morris, H. Ruth, W. Lindstrom, M. F. Sanner,
Photochem. Photobiol., A, 2006, 181, 79–85.
28 Y. Gu, X. Fei, Y. Liu, Y. Wang and X. Yang, J. Lumin., 2013,
134, 184–190.
29 V. E. Shershov, M. A. Spitsyn, V. E. Kuznetsova,
E. N. Timofeev, O. A. Ivashkina, I. S. Abramov,
R. K. Belew, D. S. Goodsell and A. J. Olson, J. Comput.
Chem., 2009, 30, 2785–2791.
43 K. K. Kim, H. K. Song, H. S. Dong, K. Y. Hwang and
S. W. Suh, Structure, 1997, 5, 173–185.
35862 | RSC Adv., 2018, 8, 35855–35862
This journal is © The Royal Society of Chemistry 2018