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: chryan@ust.hk; tangbenz@ust.hk

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: wx.wang@cityu.edu.hk

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: chryan@ust.hk; tangbenz@ust.hk

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: wx.wang@cityu.edu.hk

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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