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doi.org/10.26434/chemrxiv.12739241.v1
Boosting Algal Bloom by Five-Fold with AIEgens: Towards theDevelopment of BiofactoryHaixiang Liu, Haotian Bai, Neng Yan, Tin-Yan Wong, Dongfeng Dang, Jen-Shyang Ni, Ryan Tsz Kin Kwok,Wen-Xiong Wang, Ben Zhong Tang
Submitted date: 30/07/2020 • Posted date: 31/07/2020Licence: CC BY-NC-ND 4.0Citation information: Liu, Haixiang; Bai, Haotian; Yan, Neng; Wong, Tin-Yan; Dang, Dongfeng; Ni,Jen-Shyang; et al. (2020): Boosting Algal Bloom by Five-Fold with AIEgens: Towards the Development ofBiofactory. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12739241.v1
Human population is now faced with grand challenges such as global warming, food shortage and energysustainability, which could be partially solved by massively increasing the growth and yield of photosyntheticorganisms which capture the light energy to convert carbon dioxide and water into usable chemical energy.Cyanobacteria and eukaryotic microalgae are considered as attractive targets to be exploited by the algalfactory because of their fast growth, low cost cultivation, less arable land and the diversity of high-valuechemical substances produced. Many optical approaches have been introduced to increase the efficiency inartificial culturing systems, such as adding a luminescent layer that absorbs ultraviolet light and emitsphotosynthetic active radiation for cyanobacteria. In this work, we introduced luminogens withaggregation-induced emission characteristics (AIEgens) into the growth medium of a marine cyanobacteria.These hydrophobic AIEgens formed highly emissive luminogenic aggregates in the aqueous medium anddispersed around the cyanobacteria. Remarkedly, the number of cyanobacteria incubated in the medium withAIE aggregates was 5-fold more than the control group after 14-day culturing. The increased photosyntheticactive radiation and the change of cyanobacteria protein expression in photosynthesis and metabolism mightbe the reason. Our study is the first using organic luminogenic aggregates as optical engineering inside thegrowth medium to dramatically increase the growth of cyanobacteria and demonstrated that AIEgens ispromising technologies in the development of algal factories.
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Boosting Algal Bloom by Five-Fold with AIEgens: towards
the Development of Biofactory
Haixiang Liu, Haotian Bai, Neng Yan, Tin Yan Wong, Dongfeng Dang, Jen-Shyang
Ni, Ryan T. K. Kwok*, Wen-Xiong Wang* and Ben Zhong Tang*
Dr. H. Liu, Dr. H. Bai, T. Y. Wong, Dr. D. Dang, Dr. J.-S. Ni, Dr. R. T. K. Kwok and
Prof. B. Z. Tang
Department of Chemical and Biological Engineering, Department of Chemistry, The
Hong Kong Branch of Chinese National Engineering Research Center for Tissue
Restoration and Reconstruction and Institute for Advanced Study, The Hong Kong
University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong
Kong, China
E-mail: [email protected]; [email protected]
Dr. N. Yan and Prof. W-X. Wang
School of Energy and Environment, State Key Laboratory of Marine Pollution, City
University of Hong Kong, Kowloon, Hong Kong, China
E-mail: [email protected]
Dr. R. T. K. Kwok and Prof. B. Z. Tang
HKUST-Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park,
Nanshan, Shenzhen 518057, China
Prof. B. Z. Tang
NSFC Center for Luminescence from Molecular Aggregate, Guangdong Innovative
Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of
Luminescent Materials and Devices, South China University of Technology,
Guangzhou 510640, China
Prof. B. Z. Tang
AIE Institute, Guangzhou Development District, Huangpu, Guangzhou 510530, China
H. Liu, H. Bai and N. Yan equally contributed to this work.
Keywords: Optical engineering; cyanobacteria; aggregation-induced emission; algae
factory
Abstract:
Human population is now faced with grand challenges such as global warming,
food shortage and energy sustainability, which could be partially solved by massively
increasing the growth and yield of photosynthetic organisms which capture the light
energy to convert carbon dioxide and water into usable chemical energy. Cyanobacteria
and eukaryotic microalgae are considered as attractive targets to be exploited by the
algal factory because of their fast growth, low cost cultivation, less arable land and the
diversity of high-value chemical substances produced. Many optical approaches have
been introduced to increase the efficiency in artificial culturing systems, such as adding
a luminescent layer that absorbs ultraviolet light and emits photosynthetic active
radiation for cyanobacteria. In this work, we introduced luminogens with aggregation-
induced emission characteristics (AIEgens) into the growth medium of a marine
cyanobacteria. These hydrophobic AIEgens formed highly emissive luminogenic
aggregates in the aqueous medium and dispersed around the cyanobacteria. Remarkedly,
the number of cyanobacteria incubated in the medium with AIE aggregates was 5-fold
more than the control group after 14-day culturing. The increased photosynthetic active
radiation and the change of cyanobacteria protein expression in photosynthesis and
metabolism might be the reason. Our study is the first using organic luminogenic
aggregates as optical engineering inside the growth medium to dramatically increase
the growth of cyanobacteria and demonstrated that AIEgens is promising technologies
in the development of algal factories.
1. Introduction
Global warming, shortage of foods and lack of sustainable energy are the global
grand challenges in the 21st century.[1] The massive usage of fossil fuels has generated
large amount of carbon dioxide, one of the primary greenhouse gases. On the other
hand, fossil fuels are classified as non-renewable resources which are being depleted
much faster than the new ones are generated. Therefore, efficient utilization of clean
and renewable energy is critically important to maintain sustainable development. Solar
energy is the most abundant energy resource on earth. There are a variety of
technologies that have been developed to take advantage of solar energy. For examples,
semiconductor-based solar panels that convert the sun’s light into usable electricity
have been developed, while their conversion efficiency is still not high enough with
high cost.[2] On the other hand, photosynthesis is a highly efficient light energy
utilization method evolved naturally.[3] Oxygenic photosynthetic organisms such as
plants and algae harvest light energy to convert atmospheric carbon dioxide and water
into oxygen and usable biomass such as carbohydrate, lipids and other bioactive
metabolites.[4] Although mass cultivation of the oxygenic photosynthetic organisms is
a promise in solving these global challenges, the amount and productivity of oxygenic
organisms are far below the demand for energy.[5]
Some startup algae and plant factories have been established in order to
massively increase the amount and yield of photosynthetic species.[6] Cyanobacteria
(CB) can dominate marine environments and have been reef builders on Earth for more
than three billion years. As most ancient oxygenic photosynthetic species, CB are a
great promise in algae factory because they grow very fast and can be cultured without
fresh-water and arable lands.[7] CB has some strong and broad absorption bands in the
light spectrum due to the different pigments inside.[8] Chlorophyll a and b are present
in photosynthetic organisms (cyanobacteria, algae and plant) which have absorbance
peaks in the blue and red regions (440 and 680 nm). In addition to chlorophyll, there is
principal light harvesting complex in the CB that is phycobilisome.[9] The
phycobilisome absorbs the green and yellow lights (500−650 nm range), which are
inaccessible to chlorophyll. Therefore, their presence allows absorption and transfer of
light energy to chlorophyll of the photosystem for photosynthesis.[10] Although the
future of algae factory is fascinating, the yield of cyanobacteria is not satisfactory,
which is limited by various conditions such as light intensity and quality.[11]
Some efforts have been made to increase the growth rate and productivity of
CB by optical engineering approaches, which basically increase the photosynthetic
active radiation (PAR) and energy and thus the growth speed.[12] About 30% increase
in algae cell number was achieved by a luminescent material doped shifting layer,
which shifted the ultra-violet (UV) light of sunlight into PAR.[13] However, the overall
setup of the shifting layer is labor intensive and not applicable in large scale
operation.[13, 14] Furthermore, the fluorescent materials such as photoluminescent
phosphor (Ca0.59Sr0.40Eu0.01S) and Uvitex OB used in these earlier studies were either
potentially toxic or inefficient light conversion. The simplest and efficient way to shift
the UV light toward longer wavelength is to directly add the fluorescent materials into
the algae growth medium.[15] Organic fluorescent materials are more suitable because
their biocompatibility is overall higher than inorganic materials.[16] However, seawater
system is very complicated due to their saline environment (normally 35%) and the
presence of various metallic and organic matters, which could induce the aggregation
of organic fluorophores and lower the fluorescence efficiency due to the aggregation-
caused quenching (ACQ). Their fluorescent intensity generally decreased or even
quenched when they formed aggregates in aqueous medium.[17] These ACQ
fluorophores can absorb the short wavelength of sunlight but cannot efficiently emit the
usable light for CB photosynthesis or growth. Therefore, traditional organic fluorescent
materials cannot effectively work in the complicated seawater matrices, showing
limited effects on promoting the growth and photosynthesis of marine cyanobacteria.
Aggregation-induced emission (AIE), on the contrary, is an opposite
phenomenon, where the AIE fluorophores emit extensively in the aggregated state.[18]
Due to their good biocompatibility, high photostability and intense emission, many
biological applications of AIE luminogens (AIEgens) have been explored, including
biosensing, bioimaging and image-guided therapy.[19] In the present study, two
previously reported AIEgens (TPBA and APO) were used as the models to promote
photosynthetic CB growth (Scheme 1). These hydrophobic AIEgens formed highly
emissive luminogenic aggregates in the aqueous medium as well as around CB. The
aggregates absorbed the UV and blue light and emitted efficient green fluorescence,
serving as numerous and multidirectional nano light sources. With the addition of the
AIE aggregates, we demonstrated that the growth rate of CB increased by 5-fold after
14-day culture as compared to the normal algal cultivation. We further conducted
proteomic analysis and the result also suggested increased protein expression in
pathways of photosynthesis and metabolism, which in turn supported the dramatically
increased growth rate.
2. Experiment Section
Materials: All chemicals and reagents were commercially available and used as
received without further purification. 2,6-Dichlorophenolindophenol (DCPIP), methyl-
tert-butyl ether (MTBE), F/2 media, dithiothreitol (DTT), iodoacetamide (IAA) were
purchased from Sigma. Cyanobacteria, Synechococcus bacillaris
WH5701(CCMP1333) was obtained from Institute of Hydrobiology, Chinese Academy
of Sciences. The AIEgens 3-diphenylamino-6-(2-pyridinyl)phenyldiphenylboron
(TPBA) and 4-((2,2-difluoro-5-phenyl-2,3-dihydro-1,3,4,2-oxadiazaborol-3-
ylidene)methyl)-N,N-dimethylaniline (APO) were synthesized according to the
previous procedure.[20]
Instrumentation: Absorption spectra were measured on a PerkinElmer UV/VIS
Lambda 365 spectrophotometer. Steady-state photoluminescence (PL) spectra were
measured on a Horiba FL-3000 fluorescence spectrophotometer. Particle size analysis
was determined at room temperature using a Zetaplus Potential Analyzer (Brookhaven
Instruments Corporation, USA). Laser confocal scanning microscope images were
collected and analyzed on Zeiss laser scanning confocal microscope (LSM810).
Treatment effects on the photophysiology of S. bacillaris were assessed with a fast
repetition rate fluorometer (FRRf) in combination with a FastAct Laboratory system
(FastOcean PTX), both from Chelsea Technologies Group Ltd. (West Molesey, UK).
Cyanobacteria imaging: S. bacillaris were concentrated to 107cells/mL by
centrifugation at 2200 g, followed by incubation with TPBA or APO (10 μM) for 1 h.
The cyanobacteria were imaged under a confocal microscope (Zeiss LSM 810 Laser
Scanning Confocal Microscope), using proper excitation and emission filters: APO and
TPBA, excitation = 405 nm and emission filter = 420−550 nm; AIEgen sensitized
cyanobacteria fluorescence, excitation = 405 nm and emission filter = 600−740 nm;
intrinsic cyanobacteria autofluorescence, excitation = 488 nm and emission filter =
600−740 nm.
Cyanobacterial culture: Cyanobacteria was grown in f/2 media,[21] under continuous
illumination at a photon flux density of 30 μmol of photons m-2 s-1, on a 16/8 light/dark
cycle. Cell growth was monitored by using a traditional hemocytometer-based method
under the microscope (Olympus cx31rtsf).
Impact of AIEgens on the growth of cyanobacteria: To investigate the capability of
AIEgens in promoting the growth of cyanobacteria, S. bacillaris (1.0 × 104 cells/mL)
was exposed to TPBA or APO (10 μM). At different time intervals, the cell number
was counted by using the method mentioned above. The influence of UV light was
evaluated by exposing the algae to UV light (wavelength: 315−400 nm) for additional
1.5 h after the normal culture condition, which was similar to previous study.[22] The
cells were grown at two different CO2 levels with or without the bubbling aeration
system of atmospheric CO2 level. Again, the cell number was counted at different time
intervals.
Biomass analysis: S. bacillaris cells were first collected by centrifugation (2200 g, 5
min), then washed with a 1% (w/v, g/100 mL) aqueous NaCl solution, centrifuged again
and freeze-dried. The dry biomass was analyzed immediately or stored at −22 °C for
up to 10-days prior to analysis.
Determination of lipid content: Lipid content was determined based on the previous
study.[23] In brief, for 200 mL of S. bacillaris, 1.5 mL of methanol was added and mixed
rigorously (vertexing), followed by addition of 5 mL MTBE, and the mixture was
incubated for 1 h at room temperature. Water (1.25 mL) was added to the mixture and
allowed to stand at room temperature for 10 min to develop phase separation. The upper
organic phase was collected after centrifugation at 1000 × g for 10 min. The lower
phase was re-extracted with a new addition of 2 mL MTBE/methanol/water (10/3/2.5,
v/v/v) to achieve complete lipid recovery. The organic phase containing the lipid extract
was vacuum dried, and the dried vials were weighed, and lipid content was measured
gravimetrically. Lipid production (g L-1) was determined only at the end of the culture
period.
Fv/Fm detection: Variable fluorescence, Fv, was calculated by subtracting maximal,
Fm, with initial fluorescence, F0. The ratio Fv/Fm was a measure of the quantum
efficiency if all PSII centres were open, i.e., maximum photosynthetic efficiency, and
was highly correlated with the quantum yield of net photosynthesis.[24] Treatment
effects on the photophysiology of S. bacillaris were assessed with a fast repetition rate
fluorometer (FRRf) in combination with a FastAct Laboratory system (FastOcean PTX),
both from Chelsea Technologies Group Ltd. (West Molesey, UK). Excitation
wavelength of the fluorometer’s LED was 450 nm with an automated adjustment of the
light intensity to 0.66–1.2 x 1022 μmol photons m-2 s-1. A single turnover mode with
100 flashlets saturation phases on a 2 μs pitch and 40 flashlets relaxation phases on a
40 μs pitch was used to increasingly saturate the PSII. Iterative algorithms for the
induction[25] and relaxation phases[26] were applied to estimate the minimum Chl a
fluorescence (F0) and maximum Chl a fluorescence (Fm). The apparent maximum
quantum yield of photosynthesis of PSII (Fv/Fm) could then be calculated.
Proteomics Analysis: The control and AIEgens treated cyanobacteria cultured after 14
days were collected. Then, the proteins were extracted from cells and tryptic peptides
were prepared for standard shotgun proteomics using LC-MS/MS analysis. The raw
data acquired by the mass spectrometer was processed and searched against the
Synechococcus sp. WH 5701 protein sequence database. Label-free quantification was
applied for determining the relative amount of proteins in both control and AIEgens
treated groups based on spectral counting. To better understand the significantly
regulated proteins, protein networks were studied. Details regarding LC-MS/MS
analysis method, proteomics data analysis and bioinformatics analysis were included in
the supplementary information.
3.Results and Discussion:
Absorbance and fluorescence coupling of AIEgen and Cyanobacteria:
Cyanobacteria was mainly cultured under two types of light sources: natural
sunlight and artificial white LED light. Therefore, the absorption spectrum of
cyanobacteria (CB) and the emission profiles of the light sources were first measured
to evaluate the energy matching degree between CB and the light sources. As shown in
Figure 1A, CB exhibited broad absorption bands in the blue region and red region
(peaks around 450nm, 640nm and 680 nm) correlated to the chlorophyll a and b, and
green region (peaks around 500nm) correlated to the phycoerythrin. However, there
were obvious mismatches between the cyanobacteria absorption and the two light
sources. The strong UV light (300−400 nm) in sunlight was not absorbed by the CB for
photosynthesis. Similarly, the LED light in the region of 450−550 nm did not overlap
with the CB absorption. These data indicated that the CB could not efficiently capture
and covert the sunlight or artificial light for photosynthesis and growth. These
measurements strongly suggested that new material is required to convert the non-
utilized light energy to usable light to be captured by CB.
The AIEgens with high quantum yield and matched absorption and emission
spectrum were the promising candidates. The TPBA and APO were employed in the
following investigations, which owned greenish-blue fluorescence emission with high
quantum yield (88.4% and 25% respectively).[20] The particle size and photophysical
property of the AIEgens were measured in seawater. Dynamic light scattering (DLS)
results showed that the average particle sizes of TPBA were around 260 nm and APO
were around 150 nm (Figure S1). The UV-vis absorption spectra of TPBA and APO
showed that they both exhibited strong absorptivity in the UV and blue regions with the
peaks around 420 nm and 380 nm, respectively (Figure 1B). The photoluminescence
(PL) spectra showed that the TPBA emitted intense fluorescence in 450−550 nm
(Figure 1C). After adding the algae in the seawater, the green fluorescence of TPBA
decreased, while a new red fluorescent peak around 680 nm increased. The excitation
of chlorophyll could cause charge separation and photochemical reaction, and the
separated charges could recombine with bright fluorescence emission at around 680 nm
with high intensity of light.[27] Thus, the enhanced red fluorescence was from the
chlorophyll in the CB. Similarly, the emission of APO covered 500 nm to 650 nm and
exhibited a similar trend with the TPBA after adding CB (Figure 1D). Moreover, they
both exhibited the energy transfer function under the excitation of 350 nm and 450 nm
(Figure S2), suggesting that the reabsorption of cyanobacteria from luminescence of
AIEgens was possible under a broad spectrum of UV and blue lights. Subsequently, the
roles of TPBA and APO in the CB photosynthesis were tested with the common probe
DCPIP. During the light-dependent reaction, DCPIP could capture and react with the
produced electron from pigment chlorophyll, leading to the decreased absorbance
around 600 nm, which could reflect the electron generation and transportation in
photosynthesis. As shown in Figure S3, the absorbance of DCPIP in CB mixed AIEgen
groups decreased much faster than the control group under light irradiation, which
demonstrated that the AIEgens could transfer the light energy to CB and promoted the
light-dependent reaction.
Confocal imaging of cyanobacteria with AIEgens:
CLSM was used to investigate the spatial distribution and interaction between
the AIE aggregates and the CB. No fluorescent signal was detected for the CB incubated
in the growth medium under excitation of 405 nm (Figure 2C1 and 2C2). On the other
hand, when the AIEgen was added to the CB growth medium, the fluorescence of
AIEgens (green) and chlorophyll (red) were detected under excitation of 405 nm
(Figure 2A1, A2, B1 and B2), while the red fluorescence of chlorophyll in all groups
was observed under laser excitation of 488 nm (Figure 2A4, B4 and C4). By closer
examining the merged images, we found that the two FL signals were spatially
separated, suggesting that the AIE aggregates surrounded the CB (Figure 2A3 and B3).
To further investigate the long-lasting stability during the algal growth, the CB mixed
with TPBA and APO were imaged after 14 days of culture. The results showed that the
formed AIE aggregates dispersed around the CB in the growth medium (Figure S4).
Thus, the emission of AIE aggregates could luminate the CB under the light excitation.
The light radiation by AIEgens could be effectively reabsorbed by CB and the
converted energy could be used for CB photosynthesis.
Growth of cyanobacteria
We first studied the cytotoxicity of AIE materials to the cyanobacteria. There
was negligible cytotoxicity of TPBA and APO toward cyanobacteria up to 20 μM
(Figure S5). Then, the growth of cyanobacteria was monitored under the condition of
16 h white light LED illumination and 8 h dark cycles. It was obvious that the cell
density dramatically increased under TPBA and APO treatment (Figure 3A). The
differences occurred on Day 3, and the maximum fold change was about five in the two
AIEs treated groups compared to the control after 14-day culture. Apart from the cell
density, the biomass and lipid production of cyanobacteria were also measured. Clearly,
both the biomass and lipid production increased in both TPBA treated and APO treated
group, consistent with the cell density measurement (Figure 3B and 3C). Thus, with
AIE aggregates the light could be much more efficiently utilized by the cyanobacteria,
resulting in higher photosynthetic efficiency and growth rate. Our results demonstrated
the great potential of AIE aggregates as a boosting agent, promoting the growth and
photosynthesis activity of cyanobacteria (or even other photosynthetic species),
eventually increasing the production of biofuel. Besides the light source, the availability
of CO2 could be another parameter that limited the growth and photosynthetic activity
of cyanobacteria. Accordingly, cyanobacteria were cultured with continuous supply of
the CO2 (0.035%) and compared with that cultured under normal condition. The result
showed that the cell concentration of TPBA or APO treated cyanobacteria with constant
supply of CO2 increased about 50% as compared to that without supply of CO2 (Figure
3D). Under this condition, the cell concentration reached about 9 times more than the
control group cultured without adding AIEgens and CO2 supply, which is promising in
culturing cyanobacteria and other photosynthetic organisms.
As natural sunlight is the most available light source for photosynthesis, the
growth of cyanobacteria under natural sunlight was also studied. The cell concentration
increased after the incubation with TPBA or APO under 12 h sunlight and dark (Figure
3E). Considering that AIE aggregates absorbs UV light, which is harmful to
cyanobacteria, the protection effect of AIE aggregates was studied. Under UV light
irradiation (1.5 h) after the white light incubation (16 h), cyanobacteria were unable to
grow due to UV light, but were able to grow with incubation of TPBA or APO (Figure
3F). Furthermore, we found that the protection effect was associated with the
concentration of AIE aggregates.
Adaptation of cyanobacteria in the AIE aggregates environment
The spatial and spectrum change of light distribution caused by AIE aggregates
created unique environment for cyanobacteria to grow. Earlier study indicated that the
protein expression changed in response to light quality.[28] We then examined the
photosynthesis and protein responses to AIE aggregates. We measured the maximum
photosynthetic quantum yield (Fv/Fm) and efficiency of protein machinery of light
reaction in cyanobacteria. The results showed that the quantum yield in TPBA and APO
treatments was much higher than the control during the cell culture (Figure 4A),
reaching earlier to around 0.8 than the control group. This value was maintained
afterwards as the optimum state of light reaction. Thus, the protein machinery changed
to its optimal state under this specific environment. Detailed proteomic changes were
studied after 14-day of incubation with AIE aggregates. There were 188 common
proteins found in APO treatment and control, 187 common proteins in TPBA and
control, and 166 common proteins in APO treatment, TPBA treatment and control
(Figure S6). Next, differences in protein expression were identified with fold change
higher or lower than ±1.5 and p-values from a t-test < 0.05 (Figure S7).
To get a further insight of the influence of AIE aggregates on protein expression
of cyanobacteria, proteins that are significantly up-regulated or down-regulated in both
APO and TPBA treatments were plotted in a heat map with color indicating the fold
change (Figure 4B). There were 21 proteins up-regulated and 18 proteins down-
regulated characterized respectively with identical name and function. Among the up-
regulated proteins, those related to photosynthesis and metabolism were marked with a
red star. These proteins were then classified in different groups including
phycobilisome, photosystem Ⅱ, photosystem Ι, ATP synthesis, carbon fixation and
metabolism (Figure 4C). In these groups, proteins were identified including
phycocyanin, phycobilisome rod-core linker, Photosystem II CP47 reaction center
protein, Photosystem II D2 protein, Photosystem I reaction center, NAD(P)H-quinone
oxidoreductase, thioredoxin, phosphoribulokinase, ATP-synthase, pyruvate kinase and
so on. Many proteins are in the core of the protein machinery such as light reaction,
ATP synthesis and carbon fixation.[29] Detailed differentially expressed proteins
between APO-treated group and normal as well as proteins between TPBA-treated
group and normal were identified and studied respectively (Figure S8). These results
indicated that the unique environment created by AIE aggregates induced the change
of protein expressions of cyanobacteria, which supported the dramatically enhanced
photosynthesis and growth rate.
4. Conclusion:
Algae may provide sustainable foods and biofuels, as well as reduce the
atmospheric carbon dioxide. Cyanobacteria is a promising candidate due to their
widespread distribution and importance in the ocean environment. In this study, we
introduced highly emissive organic luminogenic AIE aggregates (TPBA and APO)
inside the growth medium and demonstrated the dramatic increase of photosynthesis
and growth of cyanobacteria. The APO and TPBA absorbed the UV and blue light from
the light source, which could cause harmful effect to cyanobacteria. Further, the highly
efficient fluorescence of AIE aggregates in the range of 450−600 nm was reabsorbed
by cyanobacteria for photosynthesis. The increased photosynthetic active radiation by
both spectra shifting and spatial surroundings of APO and TPBA aggregates
dramatically enhanced the growth rate of cyanobacteria, leading to as much as five
times faster than control. At the meantime, cyanobacteria adapted to the unique
environment created by APO and TPBA aggregates, with up-regulated protein
expression in light harvesting, light reaction, ATP synthesis, carbon fixation, which
correlates to the enhanced photosynthesis and growth. Further studies are required to
explore the effects of size, absorbance and emission spectrum of AIE aggregates. This
optical engineering by utilizing organic luminogenic aggregates provides an exciting
and promising way to develop algae and plant factory and solving the global challenges.
Conflicts of interest
There are no conflicts to declare
Acknowledgements
This work is supported by Research Grants Council of Hong Kong (N_HKUST604/14
and C6009-17G), the Innovation and Technology Commission (ITC-CNERC14SC01),
and the National Key Research and Development program of China
(2018YFE0190200), the Science and Technology Plan of Shenzhen
(JCYJ20160229205601482 and JCYJ20180507183832744).
Scheme 1. Schematic illustration of luminogenic AIE aggregates for dramatically
enhanced photosynthesis and growth of cyanobacteria. UV: ultraviolet; Vis: visible
light; Abs: absorption; Em: emission; CB: cyanobacteria
Figure 1. Absorbance and photoluminescence (PL) of APO, TPBA, cyanobacteria (CB)
and their combination. (A) The absorption of cyanobacteria in seawater and the light
spectrum of sunlight on sea level and commercially used cool light LED light for plant
growth; (B) the absorbance of TPBA and APO in seawater containing 0.1% DMSO.
(C) the PL spectrum of TPBA, cyanobacteria and cyanobacteria incubated with TPBA
under the excitation of 400 nm; (D) the PL spectrum of APO and cyanobacteria
incubated with APO under the excitation of 400 nm. APO and TPBA were used as 10-
5 M, the concentration of cyanobacteria is 106 cells/mL.
Figure 2. Confocal images of Cyanobacteria (C) and cyanobacteria stained with TPBA
(A) and APO (B). Green signal (A1, B1, C1) and red signal (A2, B2, C2) was taken
under excitation of 405 nm, which represents the fluorescence of APO or TPBA and the
cyanobacteria. Merged images (A3, B3, C3) were merged by green and red signal.
Another red signal (A4, B4, C4) was taken under excitation of 488 nm, which represents
autofluorescence signal excited directly by laser. APO and TPBA were used as 10-5M,
the concentration of cyanobacteria is 107 cells/mL.
Figure 3. Growth of cyanobacteria with or without AIE aggregates. (A) the cell
concentration, (B) the biomass of dry weight and (C) the lipid production of
cyanobacteria incubated with APO or TPBA under 16h white light, 8 h dark cycle for
a growth period of 14 days;(D) the growth curve of cyanobacteria and cyanobacteria
with APO or TPBA under 16 h white light irradiation and 8h dark under supply of
carbon dioxide; (E) the growth of cyanobacteria and cyanobacteria incubated with
TPBA or APO under 12 h sunlight near the sea for 12 days; (F)the growth curve of
cyanobacteria incubated with different concentrations of TPBA (solid) or APO (hallow)
under 16 h white light, 1.5 h UV and 6.5 dark cycle for 14 days. Concentration of APO
or TPBA is 10-5 M. Cyanobacteria were seeded as 104 cells/mL. Insert are photographs
of cyanobacteria grown after 14 days.
Figure 4. The change of cyanobacteria protein machinery after incubation with TPBA
or APO. (A) the maximum quantum yield Fv/Fm of cyanobacteria under normal
condition and that incubated with 10-5 M TPBA or APO for 14 days; (B) Heat map of
the differentially expressed proteins in TPBA-treated WH5701 and APO-treated
WH5701 compared to the normal WH5701. Proteins are defined if all the fold changes
are above or below 1, and p-values are < 0.05. (C) Schematic diagram of possible
increases in groups of photosynthesis pathway in WH5701 after the treatment of TPBA
or APO.
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download fileview on ChemRxivmanuscript_Boosting Algal Bloom by Five-Fold with AIE... (757.48 KiB)
Boosting Algal Bloom by Five-Fold with AIEgens: towards
the Development of Biofactory
Haixiang Liu, Haotian Bai, Neng Yan, Tin Yan Wong, Dongfeng Dang, Jen-Shyang
Ni, Ryan T. K. Kwok*, Wen-Xiong Wang* and Ben Zhong Tang*
Dr. H. Liu, Dr. H. Bai, T. Y. Wong, Dr. D. Dang, Dr. J.-S. Ni, Dr. R. T. K. Kwok and
Prof. B. Z. Tang
Department of Chemical and Biological Engineering, Department of Chemistry, The
Hong Kong Branch of Chinese National Engineering Research Center for Tissue
Restoration and Reconstruction and Institute for Advanced Study, The Hong Kong
University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong
Kong, China
E-mail: [email protected]; [email protected]
Dr. N. Yan and Prof. W-X. Wang
School of Energy and Environment, State Key Laboratory of Marine Pollution, City
University of Hong Kong, Kowloon, Hong Kong, China
E-mail: [email protected]
Dr. R. T. K. Kwok and Prof. B. Z. Tang
HKUST-Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park,
Nanshan, Shenzhen 518057, China
Prof. B. Z. Tang
NSFC Center for Luminescence from Molecular Aggregate, Guangdong Innovative
Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of
Luminescent Materials and Devices, South China University of Technology,
Guangzhou 510640, China
Prof. B. Z. Tang
AIE Institute, Guangzhou Development District, Huangpu, Guangzhou 510530, China
H. Liu, H. Bai and N. Yan equally contributed to this work.
Keywords: Optical engineering; cyanobacteria; aggregation-induced emission; algae
factory
Synthesis of TPBA:
Briefly, To a solution of 4-(diphenylamino) phenylboronic acid and 2-
bromopyridine in a mixture of toluene and ethanol were added tetrakis-
(triphenylphosphine)palladium [Pd(PPh3)4] and potassium carbonate under a nitrogen
atmosphere. After reflux for 20 h, the mixture was purified to afford TPAP as a white
solid. Compound TPAP and N,N-diisopropyl-ethylamine were dissolved in DCM and
then boron tribromide solution in DCM was added dropwise on ice. Afterward, the
mixture was stirred overnight at room temperature. The mixture was purified to afford
compound TPAPBr as an orange solid. To a solution of compound TPARBr in toluene
was added diphenylzinc under a nitrogen atmosphere. Then the mixture was stirred at
70 °C for 12 h. The mixture was purified to get the pure TPBA as a yellow solid.
1H NMR (400 MHz, CDCl3), δH = 8.44 (d, J = 5.3 Hz, 1H), 7.98−7.95 (t, J =
7.5 Hz, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.49 (s, 1H), 7.30−7.17
(m, 19H), 7.09−7.06 (t, J = 7.0 Hz, 2H), 6.96 (d, J = 7.6 Hz, 1H). 13C NMR (100 MHz,
CDCl3), δC = 158.14, 150.60, 147.47, 143.98, 140.21, 133.12, 129.71, 129.24, 127.32,
125.59, 125.30, 123.73, 123.47, 122.59, 120.35, 120.22, 117.44. MALDI-MS
calculated for C35H27BN2 [M]+ 486.23; found 486.2288.
Synthesis of APO:
Benzhydrazide and 4-(Dimethylamino)benzaldehyde were dissolved in
methanol, and the resulting mixture was then refluxed overnight. The above hydrazone
intermediate was suspended in 1,2-dichloroethane and allyltrimethylsilane and boron
trifluoride etherate were successively added. The reaction flask was immersed in a
boiling oil bath of 120.1C and stirred overnight. The crude product was purified to get
APO as yellow solid.
1H NMR (400 MHz, THF-d8), d (ppm): 8.45–8.43 (m, 2H), 8.16 (d, 2H, J = 7.2
Hz), 7.86 (s, 1H), 7.59–7.55 (m, 1H), 7.51–7.49 (m, 2H), 6.89–6.87 (m, 2H), 3.16 (s,
6H). 13C NMR (100 MHz, THF-d8), d (ppm): 170.65, 155.63, 151.25, 138.14, 133.58,
129.66, 129.41, 129.27, 128.47, 117.98, 112.88, 112.53, 40.54, 40.22. HRMS
(MALDI-TOF):calcd for C16H16BF2N3O [M]+: 316.1354, found, 316.1354.
DCPIP measurement: DCPIP was dissolved in seawater at final concentration of 100
μg/mL. The treatment group had 10 μM TPBA or APO. The cyanobacteria were used
as 106cells/mL. The absorbance was measured in every minute under UV or white light
irradiation.
Protein extraction and sample preparation:
Cells were harvested at 5000 × g by centrifugation and washed with cold PBS
buffer for three times. The collected cell pellet was then suspended in 300 μL of lysis
buffer (8M urea) and flash-frozen in liquid nitrogen for 1 min, followed by sonication
at 95 °C for 5 min. The cell lysate was centrifuged at 16000 × g for 15 min at 4 °C.
Then, supernatant obtained was mixed with four-volume of cold acetone and put in -
20 °C overnight. Precipitated proteins were collected by centrifuging at 16000 × g for
10 min at 4 °C and washed with 80% acetone. The remaining solvents were allowed to
evaporate and then the precipitated proteins were reconstituted in a buffer of 4M urea
in 25mM ammonium bicarbonate. The concentration of protein in the samples were
determined by Bradford assay using the Bio-Rad Protein Assay Kit. The protein
solution was incubated with 10mM dithiothreitol (DTT) at 37°C for 1h to reduce the
disulfide bonds. Iodoacetamide (IAA) was added to a final concentration of 20mM and
the treated samples were incubated at room temperature in the dark for 30 min. DTT
solution with a final concentration of 10mM was then added to quench the alkylation
reaction and the samples were diluted with 25mM ammonium bicarbonate solution to
decrease the concentration of urea to 1M. The reduced and alkylated proteins were
digested with sequencing grade-modified trypsin (1:50 w/w, Promega, Madison, WI)
at 37 °C for 10h.The digestion was acidified by 10% formic acid to a final concentration
of 0.1% (v/v) and desalted and purified by a ZipTipC18 (C18-resin packed pipette tip).
Finally, the purified samples were dried with a SpeedVac (Eppendorf, Hamburg,
Germany) and stored at -20 °C prior to LC-MS/MS analysis.
LC-MS/MS analysis:
Standard shotgun proteomics was performed to identify the tryptic peptides in the
samples on Accela HPLC system coupled to a dual-cell linear ion trap MS, LTQ Velos
(Thermo Fisher Scientific, Bremen, Germany), which is interfaced to a
nanoelectrospray ion source. Approximately 10 μg of the tryptic peptides was injected
into the HPLC system and separated on a C18 column (Thermo Bio-Basic-18, 150 ×
0.1 mm, 300 Å pore size, 5 μm particle size) at a flow rate of 220 μL/min. The mobile
phase composition was 0.1% formic acid in water for solvent A and 0.1% formic acid
in acetonitrile for solvent B. The gradient began at 2% solvent B, increased to 10% in
4 min, 10% to 32% in 60 min, 32% to 60% in 6 min, 60% to 80% in 2 min, followed
by 25 min 100% solvent B. Finally, the column was re-equilibrated with 2% solvent B
prior to the next injection. The m/z range acquired in the MS full scan was 400 to 2000
Da. The top 10 most intense precursor ions were selected for the MS/MS by using
collision-induced dissociation (CID) with collision energy set at 30V and MS//MS
isolation width was set at 2.0 m/z. Dynamic exclusion (60s) was also used to minimize
repeated fragmentation of the same peptides.
Sequence database searching:
The raw data acquired by the mass spectrometer were first converted to mzML files by
msconvert of the ProteoWizard (version 3.0.11676 64-bit). Synechococcus sp. WH
5701 protein sequence database downloaded from UniProt (Swissprot) was used for
database searching of the mzML files using Comet (version 2013.01 rev.0), with
detailed parameters as follows: 3 amu peptide mass tolerance (monoisotopic mass),
1.005 amu fragment bin tolerance, 0.4 amu fragment bin offset, both termini tryptic.
Decoy protein sequences While carbamidomethylated cysteine is the fixed
modification and oxidated methionine is the variable modifications. Decoy protein
sequences was created by shuffling and reversing amino acid sequences between tryptic
cleavage sites and subsequently concatenated to the database. The searched results were
then processed in the Trans-Proteomics Pipeline (TPP). False discovery rate (FDR) was
set at 0.01 for protein identification.
Label-free quantification:
The label-free quantification analysis was performed based on spectral counting using
NSAF values on Crux v1.4 (http://cruxtoolkit.sourceforge.net/spectral-counts.html).
The normalized spectral abundance factor (NSAF) were employed for the
normalization of the total number of peptide spectrum matches (PSMs) divided by the
lengths of protein for all identified proteins. NSAF values were calculated by the
number of PSMs for each protein divided by the length of the corresponding protein.
The search results were generated from the combination of the two technical replicates
of each biological replicate and the proteins identified in at least two out of three
biological replicates. Student’s t test was applied on the NSAF values to obtain
differentially expressed proteins between the TPBA/APO group and the control group.
The Benjamini-Hochberg (BH) multiple testing correction was used for the t-test p
values to control at the FDR 0.01. Only proteins with fold changes higher or lower than
±1.5-fold were considered as differentially expressed in our analysis.
Bioinformatics analysis:
To understand the interaction of significantly changed proteins, protein interaction
networks were drawn. STRING v11.0 (https://string-db.org/) was used to predict and
visualize the protein-protein interaction network of the proteins that were significantly
changed with highest confidence (interaction score=0.900).
Figure S1. The hydrodynamic size of APO (A) and TPBA (B) in seawater containing
0.1% DMSO. APO and TPBA were used as 10-5M
Figure S2. The photoluminescence (PL) of AIEgens and chlorophyll fluorescence of
cyanobacteria. The PL spectrum of TPBA under the excitation of (A) 350 nm and (C)
450 nm; the PL spectrum of APO under the excitation of (B) 350 nm and (D) 450 nm.
APO and TPBA were used as 10-5 M, the concentration of cyanobacteria is 106 cells/mL.
Figure S3. The change of absorbance of DCPIP at 600 nm with the incubation of TPBA
or APO under white light (A) and UV (B) for 5 mins. Cyanobacteria were used as 10-5
M, Concentration of DCPIP is 100 μg/mL.
Figure S4. Confocal images of Cyanobacteria cultured with TPBA (A) or APO (B) for
14 days. Green and red signal are under excitation of 405 nm (A1, A2, B1, B2); merge
images are signal from green, red and bright field (A3, B3); another red signal is under
excitation of 488 nm (A4, B4). APO and TPBA were used as 10-5 M, the concentration
of cyanobacteria is 107 cells/mL.
Figure S5. The cytotoxicity of TPBA and APO at different concentrations. The
cyanobacteria (1.0×107 cells/mL) were exposed to AIEgens for 96 h at different
concentrations under the simulated daily cycles. Afterwards, the cell number was
measured. The control group has also conducted with the same experimental setup
except exposing to noting.
Figure S6. Venn diagrams for proteome comparison on (A) APO-treated WH5701 and
normal WH5701 and (B) TPBA-treated WH5701 and normal WH5701 and (C) APO-
treated WH5701, TPBA-treated WH5701 and normal WH5701.
Figure S7. Protein expression difference between (A) APO-treated WH5701 compared
with control and (B)TPBA-treated WH5701 compared with control. Volcano plots for
differentially expressed proteins that are defined to be those with p-values from a t-test
< 0.05, and fold change higher or lower than ±1.5, corresponding to the rectangular
regions. The green rectangular regions are the up-regulated proteins and red rectangular
regions are the down-regulated proteins
Figure S8. Protein-protein interaction of the differentially expressed proteins of APO-
treated WH5701 (A) and TPBA-treated WH5701 (B)compared to normal WH5701 was
analyzed by STRING v11.0. Each node is a protein found to be differentially expressed,
as determined by label-free quantitative proteomics, and the lines represent protein
functional association as predicted by STRING with highest confidence (interaction
score=0.900). Node in different colors represents different protein functions. The
outlines of the nodes represent the upregulation (in green) and downregulation (in red).
Nodes without function enrichment are hidden from the network.
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