Light-Emitting Diode Power Conversion Capability and CO 2 Fixation Rate of Microalgae Bioﬁlm Cultured Under Di ﬀ erent Light Spectra

: Microalgae bioﬁlm-based culture has attracted much interest due to its high harvest e ﬃ ciency and low energy requirements. Using light-emitting diodes (LEDs) as light source for microalgae culture has been considered as a promising choice to enhance the economic feasibility of microalgae-based commodities. In this work, the LED power conversion capability and CO 2 ﬁxation rate of microalgae bioﬁlms ( Chlorella ellipsoidea and Chlorella pyrenoidosa ) cultured under di ﬀ erent light spectra (white, blue, green and red) were studied. The results indicated that the power-to-biomass conversion capabilities of these two microalgae bioﬁlms cultured under blue and white LEDs were much higher than those under green and red LEDs ( C. ellipsoidea : 32%–33% higher, C. pyrenoidosa : 34%–46% higher), and their power-to-lipid conversion capabilities cultured under blue LEDs were 61%–66% higher than those under green LEDs. The CO 2 ﬁxation rates of these two bioﬁlms cultured under blue LEDs were 13% and 31% higher, respectively, than those under green LEDs. The results of this study have important implications for selecting the optimal energy-e ﬃ cient LEDs using in microalgae bioﬁlm-based culture systems.


Introduction
Microalgae are photosynthetic microorganisms that can convert light, CO 2 and nutrients into biomass [1][2][3]. Due to the high growth rates, high lipid contents and ability to mitigate CO 2 emissions, microalgae have potential to be promising biological sources to produce high-value biomolecules and biofuels [4][5][6]. However, the success of microalgae-based commodities is dependent on the biomass productivity and production cost [7][8][9]. Recently, some researchers reported that cultivating microalgae as biofilm (i.e., cells are attached to solid surface) can enhance the economic feasibility of microalgae-based commodities, due to its lower water consumption, higher volumetric productivity, higher harvest efficiency and reduced energy requirements compared with suspended systems [10][11][12]. Therefore, developing more efficient biofilm-based microalgae culture system has attracted much attention recently [13][14][15].

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To evaluate microalgae growth, microalgae biofilm were harvested after 6 days culture. The 105 collected biofilm were resuspended with deionized water to remove the soluble nutrients, followed 106 by centrifuging and drying to a constant weight at 105 °C. After cooling in a desiccator, the microalgae

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The chlorophyll contents of microalgae cells cultured under white, blue, green and red LEDs 109 were determined according to the methods described by Wellburn [36]. In detail, the chlorophyll was  During cultivation, four kinds of LEDs (J&K Photoelectric Technology, Shanghai, China), including white (JK-W300200, 400-750 nm), blue (JK-B300200, 440-500 nm), green (JK-G300200, 500-550 nm), and red (JK-R300200, 610-650 nm) LEDs were fixed above the bioreactors as the light sources for microalgae cultivation. All the LEDs in the biofilm cultivation were continuous lighting. The photon flux densities of these LEDs at the biofilm surfaces were set to be approximately 100 µmol photons m −2 s −1 , which were measured with a 4π quantum scalar sensor (QSL 2100, Biospherical Instruments Inc., San Diego, CA, USA). The light spectra of these four LEDs were characterized with a fiber spectrometer (USB4000, Ocean Optics Inc., Dunedin, FL, USA) between 350 and 750 nm, as shown in Figure 2a. Additionally, the absorption spectra of the C. ellipsoidea and C. pyrenoidosa were characterized by a microplate spectrophotometer (Epoch, BioTek, Winooski, VT, USA), as shown in Figure 2b.

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The photon flux densities of these LEDs at the biofilm surfaces were set to be approximately 100 μmol

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The chlorophyll contents of microalgae cells cultured under white, blue, green and red LEDs

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were determined according to the methods described by Wellburn [36]. In detail, the chlorophyll was

Determining the Biomass and Chlorophyll Contents
To evaluate microalgae growth, microalgae biofilm were harvested after 6 days culture. The collected biofilm were resuspended with deionized water to remove the soluble nutrients, followed by centrifuging and drying to a constant weight at 105 • C. After cooling in a desiccator, the microalgae biomass was weighed by an analytical balance (XS105, METTLER TOLEDO, Switzerland).
The chlorophyll contents of microalgae cells cultured under white, blue, green and red LEDs were determined according to the methods described by Wellburn [36]. In detail, the chlorophyll was extracted with 80 vol% acetone. The absorbance of chlorophyll solvent was measured at 646 and 663 nm with a visible spectrophotometer (721, INESA, Shanghai, China). The chlorophyll a (chl-a), chlorophyll b (chl-b) concentrations (mg L −1 ) were calculated by: The measurements for biomass and chlorophyll contents were repeated three times. The results were shown as mean ± standard deviation.

Determining Cellular Composition
The cellular compositions of microalgae are critical evaluation parameters in various microalgae-based commodities. Generally, the main organic components of microalgae are lipids, proteins, and carbohydrates, which represent approximately 80% of the microalgal dry biomass. To investigate the cellular compositions of microalgae biofilm cultured under different LEDs, the harvested cells were frozen at −70 • C and lyophilized. The total lipids were measured according to the methods described by Bligh and Dyer [37], in which chloroform was used to extract the lipid and evaporated at 60 • C. The protein content was measured by a colorimetric method [38,39], in which microalgae biomass was pretreated with thermal alkaline and the standard sample was bovine serum albumin (see Figure S1). The carbohydrate content was determined by the phenol-sulfuric method [40,41], the standard sample was glucose (see Figure S2). All the experiments were repeated in triplicate and the results are shown in mean ± standard deviation.

LED Power Conversion Capability
We determined the conversion capability of LED power-to-biomass, power-to-lipid, power-to-protein, and power-to-carbohydrate for these microalgae biofilms cultured under different LEDs, which were calculated by [30]: where C t is the accumulation of biomass, lipid, protein and carbohydrate (g m −2 ) at time t, C 0 is the accumulation of biomass, lipid, protein and carbohydrate (g m −2 ) at time t 0 (at the beginning of inoculation), P is the power consumption of different LED units.

Determining CO 2 Fixation Rate
The CO 2 fixation rate of microalgae biofilms cultured under white, blue, green and red lights were determined by [42]: where X t is microalgae biomass (g m −2 ) at time t, X 0 is microalgae biomass (g m −2 ) at time t 0 (at the beginning of inoculation), C% is the carbon content of the biomass, which was determined by an elemental analysis (vario EL cube, Elementar, Germany). The experiments were repeated at least three times. The results were shown as mean ± standard deviation.

Growth of Microalgae Biofilms Cultured Under Different LEDs
The dry biomass yields and chlorophyll contents (including chl-a and chl-b) of C. ellipsoidea and C. pyrenoidosa biofilms were determined to evaluate their growth. Figure 3 indicates that the microalgae biomasses cultivated under different light spectra were obviously different. In general, for both microalgae, the biomass cultured under the blue LED was much higher than those under the white, green and red LEDs. The total chlorophyll contents of microalgae cultured under the blue and white LEDs were much higher than those under the green and red LEDs. Previously, many studies reported that both the blue and red light can promote the growth of green algae in suspended culture system, because the green algae can absorb the blue light and red irradiation spectra of blue LEDs match with the absorption peaks of C. ellipsoidea and C. pyrenoidosa

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The LED power conversion capability is an important factor in the assessment of microalgae 166 biofilm cultivation. In this work, we determined the conversion capabilities of LED power-to-167 biomass, power-to-lipid, power-to-protein, and power-to-carbohydrate. Figure 4 shows the LED  Previously, many studies reported that both the blue and red light can promote the growth of green algae in suspended culture system, because the green algae can absorb the blue light and red light more efficiently [43]. In this work, Figure 3a indicates that the biomass of C. ellipsoidea was~14% higher under the blue light than that under the green light. Similarly, Figure 3b shows that the biomass of C. pyrenoidosa was~26% higher under the blue light than that under the green light. Evidently, the above results suggested that the blue LED was efficient in enhancing cell growth, whereas, we found that the red LED had little influence on cell growth. We think that it may be related to the characteristics of microalgae pigments. On one hand, as shown in Figure 2b, the irradiation spectra of blue LEDs match with the absorption peaks of C. ellipsoidea and C. pyrenoidosa at the light spectra of 420-480 nm (blue), whereas, the irradiation spectra of red LEDs do not match well with the microalgal absorption peaks at the light spectra of 620-680 nm (red). On the other hand, we found that the chlorophyll contents of these two microalgae cultivated under red LEDs were much lower than those cultured under blue LEDs.

The LED Power Conversion Capability of Microalgae Biofilm
The LED power conversion capability is an important factor in the assessment of microalgae biofilm cultivation. In this work, we determined the conversion capabilities of LED power-to-biomass, power-to-lipid, power-to-protein, and power-to-carbohydrate. Figure 4 shows the LED power-to-biomass conversion capability for these two microalgae biofilms cultivated under different light spectra, which were calculated based on the biomass accumulation and power consumption of different LEDs (right axis in Figure 4). The results indicate that the power-to-biomass conversion capability of C. ellipsoidea and C. pyrenoidosa cultured under different LEDs ranged from 862 to 1147 mg/kW·h, and from 618 to 905 mg/kW·h, respectively. The LED power-to-biomass conversion capabilities for these two microalgae cultured under white and blue lights were much higher than those cultured under green and red lights, indicating that these microalgae cells utilized blue and white lights more effectively. Particularly, for C. ellipsoidea, the power-to-biomass conversion capability of cells cultured under blue and white lights increased by 32%-33% compared with those under green light. Similarly, for C. pyrenoidosa, the power-to-biomass conversion capability of cells cultured under blue and white lights increased by 34%-46% compared with those under green light.
Furthermore, cellular compositions of microalgae are generally critical evaluation parameters in various applications. Figure S3 shows the lipids, proteins and carbohydrates contents for the C. ellipsoidea and C. pyrenoidosa cultured under different LEDs. It was found that the main organic components in the microalgae represented approximately 80% of microalgae dry biomass, which were consistent with the literature [35]. Based on the cellular composition and power consumption of different LEDs, we determined the conversion capabilities of LED power-to-lipid, power-to-protein, and power-to-carbohydrate. Figure 5 indicates that the power-to-carbohydrate conversion capabilities for both microalgae under white LEDs were the highest. The power-to-protein conversion capabilities for both microalgae cultured under white and blue LEDs were higher than those under green and red lights. Moreover, we found that both microalgae cultured under blue LED showed the highest power-to-lipid conversion capability. In particular, the power-to-lipid conversion capability for C. ellipsoidea and C. pyrenoidosa were 12.6% and 14.8% higher, respectively, under blue light than under white light. The power-to-lipid conversion capability for C. ellipsoidea and C. pyrenoidosa were 60.7% and 66.3% higher, respectively, under blue light than under green light. Similar results have been reported for the suspended culture system. Ra et al. evaluated the effects of LED wavelength on the lipid production of Picochlorum atomus with two-phase suspended cultivation, and found that the lipid accumulation of P. atomus was 329% higher under blue light than that under green light [44]. Kang et al. determined the effect of using wastewater and wavelength filters on microalgal productivity and lipid accumulation with open raceway ponds, and found that the lipid productivity was highest under blue wavelength, at least 46.8% higher than that under white wavelength [45].This may be because blue light can promote the synthesis of lipids [46,47] and has low energy consumption.

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Furthermore, cellular compositions of microalgae are generally critical evaluation parameters in 182 various applications. Figure S3 shows the lipids, proteins and carbohydrates contents for the C.

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Furthermore, cellular compositions of microalgae are generally critical evaluation parameters in 182 various applications. Figure S3 shows the lipids, proteins and carbohydrates contents for the C.
183 ellipsoidea and C. pyrenoidosa cultured under different LEDs. It was found that the main organic 184 components in the microalgae represented approximately 80% of microalgae dry biomass, which 185 were consistent with the literature [35]. Based on the cellular composition and power consumption 186 of different LEDs, we determined the conversion capabilities of LED power-to-lipid, power-to-187 protein, and power-to-carbohydrate. Figure 5 indicates that the power-to-carbohydrate conversion   Overall, these results suggested that these two microalgae biofilms cultured under blue and white LEDs showed higher conversion capabilities of power-to-biomass and power-to-protein compared with those under green and red LEDs. These two microalgae cultured under blue LEDs possessed the highest power-to-lipid conversion capabilities. The results revealed that it is feasible to induce the synthesis of different chemical components in cells by adjusting the light spectrum in microalgae biofilm-based culture systems.

The CO 2 Fixation Rate of Microalgae Biofilm
The CO 2 fixation rates of microalgae biofilms cultivated under different LEDs were determined by evaluating the difference in the carbon content (C%) of microalgae between the inoculation and harvest (see Table S3). Figure 6 indicates that the CO 2 fixation rates for the C. ellipsoidea and C. pyrenoidosa cultured under blue LEDs were~13% and~31% higher, respectively, than those under green light. This may be attributed to the higher photosynthetic performance of microalgae. Additionally, previous study reported that, in suspended culture system, white light was the most effective light for CO 2 fixation compared with the blue, red and yellow lights [48]. Evidently, this study indicated that the influence of the light spectra on the CO 2 fixation rate of microalgae would be different between the biofilm cultured system and the suspended culture system. This may be due to the different characteristics of light transmission and refraction in these two microalgae culture systems. Further studies should be conducted to understand the light transfer phenomena in microalgae biofilm.
Overall, these results suggested that these two microalgae biofilms cultured under blue and white 206 LEDs showed higher conversion capabilities of power-to-biomass and power-to-protein compared   Table S3). Figure 6 indicates that the CO2 fixation rates for the C. ellipsoidea and C.
green light. This may be attributed to the higher photosynthetic performance of microalgae. Additionally, 217 previous study reported that, in suspended culture system, white light was the most effective light for 218 CO2 fixation compared with the blue, red and yellow lights [48]. Evidently, this study indicated that 219 the influence of the light spectra on the CO2 fixation rate of microalgae would be different between 220 the biofilm cultured system and the suspended culture system. This may be due to the different

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Previous studies reported that using LEDs with specific narrow bands in suspended microalgae 227 culture systems may be more economical than the fluorescent lamp and filament lamp [29,49]. The 228 results of this study suggest that the LEDs are also promising in biofilm-based microalgae culture 229 systems. Furthermore, the study also indicates that the light spectra of LEDs can significantly affect

Implications of LED Selection in Biofilm-Based Microalgae Cultivation
Previous studies reported that using LEDs with specific narrow bands in suspended microalgae culture systems may be more economical than the fluorescent lamp and filament lamp [29,49]. The results of this study suggest that the LEDs are also promising in biofilm-based microalgae culture systems. Furthermore, the study also indicates that the light spectra of LEDs can significantly affect the power conversion capability and CO 2 fixation rate of microalgae biofilms. For the C. ellipsoidea and C. pyrenoidosa biofilms, the power-to-biomass conversion capabilities and power-to-protein conversion capabilities were much higher for cells cultured under blue and white LEDs than those under green and red LEDs. Meanwhile, the power-to-lipid conversion capabilities and CO 2 fixation rate were the highest for these two microalgae biofilms cultured under blue LEDs. Moreover, considering the simultaneous improvement of power-to-biomass conversion capabilities, power-to-lipid conversion capabilities and CO 2 fixation rate, blue LEDs may have great potential using in microalgae biofilm cultivation for the biofuel production and CO 2 mitigation. The results of this study will have important implications for selecting the optimal energy-efficient LEDs to use in microalgae biofilm-based culture systems.

Conclusions
The results of this study indicated that the LED power conversion capability and CO 2 fixation rate of C. ellipsoidea and C. pyrenoidosa biofilms cultured under white, blue, green and red LEDs were significantly different. These two microalgae biofilms cultured under white and blue LEDs showed higher power-to-biomass conversion capabilities and power-to-protein conversion capabilities than those under green and red LEDs. The power-to-lipid conversion capabilities and CO 2 fixation rate were the highest for these two microalgae biofilms cultured under blue LEDs. This study would provide guidance in selection of suitable LEDs and reducing energy consumption for developing more efficient microalgae biofilm-based culture systems.

Supplementary Materials:
The following are available online at http://www.mdpi.com/1996-1073/13/7/1536/s1: Tables S1 and S2. The culture medium for microalgae. Figure S1. The relationship between the content of bovine serum albumin and the optical density of solution. Figure S2. The relationship between the content of glucose and the optical density of solution. Figure S3. Biochemical composition for the C. ellipsoidea (a) and C. pyrenoidosa (b) under different LEDs. Table S3. The contents of carbon, nitrogen, hydrogen, and oxygen in microalgae determined with an elemental analyzer.
Author Contributions: H.Y. and X.Z. (Xinru Zhang) carried out the experiments, analyzed the data, and drafted the manuscript. Y.W., Y.X., and X.W. carried out part of experiments. X.Z. (Xinru Zhang), Z.J. and X.Z. (Xinxin Zhang) designed the study, and revised the manuscript. All authors have read and agreed to the published version of the manuscript.