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Enhancing lipid production in attached culture of a thermotolerant microalga Desmodesmus sp. F51 using light-related strategies
Accepted Manuscript Title: Enhancing lipid production in attached culture of a thermotolerant microalga Desmodesmus sp. F51 using light-related strategies Authors: Ying Shen, Sha Wang, Shih-Hsin Ho, Youping Xie, Jianfeng Chen PII: DOI: Reference:
S1369-703X(17)30248-6 http://dx.doi.org/10.1016/j.bej.2017.09.017 BEJ 6790
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
4-7-2017 7-9-2017 22-9-2017
Please cite this article as: Ying Shen, Sha Wang, Shih-Hsin Ho, Youping Xie, Jianfeng Chen, Enhancing lipid production in attached culture of a thermotolerant microalga Desmodesmus sp.F51 using light-related strategies, Biochemical Engineering Journalhttp://dx.doi.org/10.1016/j.bej.2017.09.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A revised manuscript (BEJ-D-17-00665R1) submitted to Biochemical Engineering Journal
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Enhancing lipid production in attached culture of a thermotolerant microalga Desmodesmus sp. F51 using light-related strategies
Ying Shen1,2, Sha Wang1, Shih-Hsin Ho3, Youping Xie3*, Jianfeng Chen3**
1
College of Mechanical Engineering and Automation, Fuzhou University,
Fuzhou350116, China 2
Collaborative Innovation Center of High-End Equipment Manufacturing in Fujian,
Fuzhou 350116, China 3
College of Biological Science and Engineering, Fuzhou University, Fuzhou 350116,
China
*Corresponding author: Youping Xie Tel: +86-591-22866373 Fax: +86-591-22866373 Email: [email protected] **
Co-corresponding author: Jianfeng Chen
Tel: +86-591-22866373 Fax: +86-591-22866373 Email: [email protected]
Highlights
Efficiency of light related strategies on attached microalgal culture was assessed.
Light-switching strategy enhanced both biofilm and lipid production. A maximum biofilm production of 241.67 g m-2 was achieved. A maximum lipid production of 66.65 g m-2 was achieved. 1
ABSTRACT In attached culture, concentrated cells are grown on the substratum, which makes the light adsorption mechanism quite different from those of suspended cultures. An energy conservation biofilm reactor based on capillary-effect was firstly introduced for attached culture of the thermotolerant microalga Desmodesmus sp. F51. The effects of light-related strategies (including light intensity, photoperiod and light-switching strategies) on lipid production were investigated. It was found that the light requirements for biomass or lipid accumulation varied greatly at different growth stages, probably due to increasing biofilm thickness. By switching the light intensity from 700 to 1134 μmol m-2 s-1 at day 3 in Strategy I, a maximum biofilm/lipid production of 241.67/53.62 g m-2 was achieved at day 8. A similar operation was conducted in Strategy II except a further switch to light intensity of 938 μmol m-2 s-1 at day 5, a maximum biofilm/lipid production of 223.58/66.65 g m-2 was achieved at day 8. This performance is better than most of the previously reported values. Light-switching operation was proved to be an effective strategy to supply the appropriate light for each growth stage of the immobilized cells, and was beneficial for biofilm/lipid production.
Key-words: Microalgae; attached culture; light intensity; photoperiod; lipid production
1. INTRODUCTION 2
With the development of economy and society, a large amount of fossil fuels are unduly consumed and climatic changes are becoming more and more severe, which requires us to seek alternative energy sources. Microalgae, unicellular photosynthetic organisms, are regarded as a renewable feedstock for biodiesel production compared to most oil rich crops [1, 2]. Moreover, cultivation of microalgae can utilize non-arable land and municipal wastewater as a nutrient source [3], reducing the severe problems of various environmental pollutants. Large volumes of water and energy are required for suspended cultures, which significantly hinder the commercial feasibility [4]. Compared to suspended cultures, the biofilm cultivation of microalgae, in which microalgal cells are grown on the surface of supporting materials with high density and fed with nutrient solutions, is a novel cultivation mode. Attached microalgal cultures grow as immobilized cells on the substratum. There are many advantages to this cultivation method including: 1) the capital cost and energy consumption for biomass harvesting are significantly reduced [5, 6, 7]; 2) since the immobilized cells were highly concentrated on the substratum, the cell growth area can be better controlled when the system is set up in the ocean or in lagoons; 3) immobilized algae have been reported to have excellent efficiency in wastewater treatment [8, 9]. Although the attached microalgal culture seems to provide an innovative solution for the microalgal industry, there are still many issues to be solved. Firstly, the immobilized cells are exposed to air instead of the medium and, as a result, the temperature on the cells is higher than those grown in suspended culture in summer. 3
Fortunately, it has been reported that Desmodesmus sp. F51cells can be grown at 45C for 24 h and 50C for 8 h with satisfactory mortality rates [10], which suggests that strain F51 may be suitable for attached cultivation as they can handle the high temperatures in the summer in southern China. Secondly, the light absorption mechanism of attached cultures is quite different from suspended culture. In suspended culture, cells are supposedly exposed to a similar light intensity when proper mixing is provided. Since immobilized cells are attached to the substratum, the inner-layer of the immobilized cells (old cells) was believed to receive less light than cells on the outer layer (new cells). Wang et al. [11] stated that the specific growth rate of the inner layer decreased as the biomass of the upper layer increased, which was mainly because the light traveling through the upper layer became weaker and weaker. In addition, light intensity and photoperiods are critical factors that influence the growth rate of microalgal cultures [12]. Thus, to enhance the economic potential of attached microalgal cultures, selecting a proper light exposure strategy is of great importance. To the best of our knowledge, information on the effects of light-related strategies on biomass production and lipid accumulation in attached culture is still lacking. It has been recognized that many microalgal species can use light as their energy source for synthesizing cell protoplasm. The light requirements of microalgae are not only species dependent, but may differ for each growth phase of the microalgal strain [13]. For example, most microalgae require less intense light at the initial lag phase to avoid photoinhibition, however, on the other hand, higher light intensity is essential at exponential phase to prevent cell shading (photo limitation) [14]. Therefore, selecting 4
an appropriate light intensity for each growth phase is vital to improve algal growth with reduced power consumption [15]. Numerous studies have reported that the lipid content in microalgae can accumulate under higher intensity light [12, 16]. However, the effect of light intensity on lipid accumulation in attached culture of Desmodesmus sp. is still unknown. Taken together, due to the advantage of low energy consumption, a capillary-effect-based biofilm reactor (CBR) was used in this study to assess its potential for generating microalgal biomass and lipid production. As light significantly influences photosynthetic efficiency in microalgae, it is important to understand the effect of light-related strategies on microalgal lipid production in this attached culture system. Thus, in this study, light intensity (700 to 1134 μmol m-2 s-1) and photoperiod cycles (24:0 to 8:16 light: dark) were first investigated to improve cell growth and lipid production from attached culture of Desmodesmus sp. F51. Moreover, in order to produce a large amount of biomass with high lipid content, different light-switching strategies were also assessed.
2. MATERIAL AND METHODS 2.1 Algal strain and pre-culture Desmodesmus sp. F51, isolated from southern Taiwan, was obtained from Prof. Ching-Nen Nathan Chen’s Laboratory at National Sun Yat-sen University, Taiwan. The medium used for pre-culture and culture of Desmodesmus sp. F51 was BG11 medium which contains: 1500 mg L-1 NaNO3, 40 mg L-1 K2HPO4, 75 mg L-1 MgSO47H2O, 36 5
mg L-1 CaCl22H2O, 6 mg L-1 citric acid, 6 mg L-1 ferric ammonium citrate, 1 mg L-1 EDTANa2, 20 mg L-1 Na2CO3 and 1 mL L-1 A5 trace metal solution. The recipe of A5 trace metal solution was: 2.86 g L-1 H3BO3, 1.86 g L-1 MnCl24H2O, 0.22 g L-1 ZnSO47H2O, 0.39 g L-1 Na2MoO42H2O, 0.08 g L-1 CuSO45H2O, and 0.05 g L-1 Co(NO3)26H2O. The initial pH value of the medium was titrated to 7.5 with 1 mol L-1 HCl. The photobioreactor (PBR) used for pre-culture of microalgal cells was 1 L glass vessel (15.5 cm in length and 9.5 cm in diameter) equipped with external light sources (14 W TL5 tungsten filament lamps, Philips Co., China) mounted on both sides of the PBR. The microalgae were grown at 30 C for 4-5 days under a continuous supply of 2.5% CO2 (0.2 vvm), and an agitation rate of 300 rpm. The microalgal culture was continually illuminated with a light intensity of approximately 150 μmol m-2 s-1. The light intensity was measured by a Li-250 Light Meter with a Li-190SA pyranometer sensor (Li-COR Inc., Lincoln, Nebraska, USA). 2.2 Capillary-effect-based biofilm reactor (CBR) Fig. 1 depicts a schematic of the capillary-effect-based biofilm reactor (CBR). The system was composed of 10 units. In each unit, two pieces of porous substratum (80 cm×10 cm folded to 40 cm × 10 cm) were coated on a rigid polypropylene base (40 cm×10 cm), which were vertically aligned to the top of the nutrient medium reservoir (11 cm (length) × 11 cm (width) × 7 cm (height)). The porous canvas substratum selected from the previous experiments served to provide a growth surface for algal biofilms and diffuse the nutrient medium under capillary force. Light was provided by an array (2×5) of white light emitting diodes (LEDs) that were mounted 6
on the top of the reactor. Since the LED lights were put on the top of the reactor, the light intensities varied vertically. The light meter was set at the center of the microalgal growth area, which was about 15 cm from the top of the medium (shown in Fig.1). 2.3 Experimental design 2.3.1 Effect of light intensity and photoperiod regime The experiments were carried out in three steps. In the first step, four different light intensities (including 700, 812, 938 and 1134 μmol m-2 s-1) were compared under a 12:12 h light: dark photoperiod. The different light intensities were achieved by adjusting the distance between the LEDs and the CBR. The inoculum density was 15 g m-2. For each cultivation unit, an aeration pipe was fixed between the two substratum (Shown in Fig. 1), and the air enriched with 2.5% CO2 was aerated into the atmosphere environment with a speed of 10 mL min-1. The temperature was approximately 30 C. In order to provide sufficient nutrient medium for algal growth, 400 mL standard BG11 medium was kept in the nutrient medium reservoir. To compensate for water evaporation, 60-100 mL BG11 supplemental medium without nitrate was added to the reservoir to reach a total volume of 400 mL once every 24h. In the second step of the experiment, four photoperiods, viz. 24:0, 16:8, 12:12, and 8:16 h light: dark were investigated with a light intensity of 938 μmol m-2 s-1. The other culture conditions were the same as in the previous experiment. 2.3.2 light-switching strategies 7
Based on the light intensity preferences for biomass or lipid accumulation at different culture stages, two light-switching strategies were compared in the third set of experiments. In Strategy I, a light intensity of 700 μmol m-2 s-1 was provided during the first 2 days, and then switched to a light intensity of 1134 μmol m-2 s-1 at day 3 to further enhance the biomass productivity as the biofilm thickness increased. Compared to Strategy I, the only difference in Strategy II was that the light intensity was switched to 938 μmol m-2 s-1 at day 5 to stimulate lipid accumulation. The experiments were conducted at an optimal light: dark cycle of 16:8 h. All other culture conditions were the same as the previous experiments. The experiments were duplicated with the same culture conditions, and an average of the data is presented. 2.4 Analytical methods 2.4.1 Determination of the microalgal growth The biomass dry weight was measured by the gravimetric method [17]. The attached cells were completely washed off from the porous substrate and resuspended with distilled water. The biofilm productivity was identified as the total biomass dry weight obtained from the substratum (4 pieces) per footprint of the single cultivation unit (11 cm×11 cm) per day. The inoculated part was deducted. 2.4.2 Determination of the nitrate concentration The nitrate concentration was determined according to an optical method [18]. Samples were filtered through a 0.22 m pore size filter to remove cells and particles. The filtered medium was diluted with deionized water and its absorption at 220 nm wavelength was measured using a UV/VIS spectrophotometer (model UVmini-2001, 8
Hitachi, Tokyo, Japan). The nitrate level was determined by interpolating the OD220reading into a standard curve. 2.4.3 Determination of the lipid content and fatty acid compositions Lipid content was measured by using a modified Bligh and Dyer’s method [19]. The lipid extraction was carried out using a BioSpec Model 3110 BX bead-beater (Bartlesville, Oklahoma, USA) for cell disruption (3 min) followed by solvent extraction with n-hexane. The oils collected after evaporation was dried at 95C for 1.5 h before weighing. Lipid content was the percentage of lipid weight to disrupted biomass. Lipid productivity was calculated by multiplying biofilm productivity by lipid content. The fatty acids composition was analyzed by gas chromatographic-mass spectrometric analysis (Agilent 6890-5975, USA) introduced in our previous research [20].
3. RESULTS AND DISCUSSION 3.1 Effect of light intensity on biofilm and lipid productivity of Desmodesmus sp.F51 As a primary energy source for photoautotroph, light plays an important role in microalgal growth and lipid accumulation. Because the immobilized algae are exposed directly to light, it is possible that the immobilized cells are more sensitive to fluctuations in light intensity when compared with suspended cells. Fig. 2 shows the influence of light intensity on the biomass (including the suspended and immobilized biomass) and lipid productivity of attached cultures of Desmodesmus sp.F51cells with a light: dark cycle of 12: 12 h at different culture stages. 9
In the first 2 days, the biofilm biomass was inversely proportional to light intensity. The maximum biofilm productivity of 37±0.83 g m-2 d-1 was obtained with the lowest light intensity of 700 μmol m-2 s-1 (as shown in Fig. 2A). However, this tendency varied as the culture period was prolonged, with maximum biofilm productivity of 34.88±0.84 g m-2 d-1 obtained with the highest light intensity of 1134 μmol m-2 s-1 at day 4 (as shown in Fig. 2C). In general, the biofilm productivity decreased as the culture period continued, especially with lower light intensity. The lipid content data shows that a light intensity of 938 μmol m-2 s-1 was preferable for Desmodesmus sp.F51 lipid accumulation over the entire culture period (as shown in Fig. 2B, D and F). Under an optimal light intensity of 938 μmol m-2 s-1, a maximum lipid productivity of 5.18±0.30 g m-2 d-1 and a maximum lipid content of 21.77±0.70% were achieved at day 4 and day 6, respectively. In the attached cultures, concentrated algae were inoculated on the substratum instead of into the medium. The high cell inoculation of microalgae was beneficial for adaptation to the environment [21]. Compared to the suspended cultures, there was barely a lag period in the biomass growth in the attached Desmodesmus sp. F51 cultures. Similar results were also found in other studies, including attached culture of Scenedesmus obliquus, where the biomass productivity reached 45 g m-2 d-1 in 2 days culture [6]. In an attached culture of Chlorella vulgaris, the biofilm growth rates at day 1 were even higher than at day 3 under light intensity that varied from 40 to 280 μmol m-2 s-1 [22]. The results were consistent with those seen in this study. 10
As a thermo-tolerant algal species, the suspended culture of Desmodesmus sp. F51 had the highest specific growth rate of 2.53 d-1 and biomass productivity of 796 mg L-1 d-1 at a light intensity of 750 μmol m-2 s-1 [23]. For attached culture, a light intensity of 700 μmol m-2 s-1 was found to be optimal for biomass growth in the first 2 days. Wang et al. [11] compared the efficiency of light penetration in a suspended open pond system and an attached culture system, and concluded that under nitrogen-replete conditions, the immobilized cells of Scenedesmus dimorphus were 100% illuminated with light intensity of 100 μmol m-2 s-1, and the biofilm production increased from 8.8 to 107.6 g m-2 in 10 days. The efficiency of light penetration can be affected by many factors, such as algal species, light intensity, nitrogen concentration, pH and biofilm thickness [24]. Murphy et al. [25] mentioned that biofilm productivity can be enhanced from 8.2 to 11.9 g m-2 d-1 with increasing biofilm thickness from 20 to 100 µm. However, a further increase in biofilm thickness to 200 µm led to a significant drop in biofilm productivity, indicating the efficiency of light penetration was attenuated exponentially with increasing biofilm thickness. In the current study, under nitrogen-deficient conditions, the biofilm productivities were decreased for all different light intensities used (as shown in Fig. 2A, C and E), which demonstrates that nitrogen deficiency has a negative effect on biofilm productivity. The influence of light intensity on lipid content of microalgae is affected by many factors, such as microalgae species, nitrogen and carbon supplementation, temperature and culture ages [26, 27]. An increase in light intensity is supposed to promote the biosynthesis of fatty acid through enhancing the activities of the enzymes 11
like, acetyl CoA carboxylase, desaturase, and ATP/citrate lyase, as a result, the excessive light might be trapped by the cells and transformed into fatty acid [28, 29]. However, photo-oxidative cell damage could also be happened as the light intensity is higher than the tolerance of the specific microalgae species. In the current study, a light intensity of 938 μmol m-2 s-1 was found optimal for lipid production. 3.2. Effect of light intensity on pH and nitrate consumption of Desmodesmus sp. F51 The pH and nitrate consumption are valuable factors that reflect microalgal growth and biochemical composition [30]. Fig. 3 shows the variation of pH and nitrate concentration obtained under the four different light intensities. Corresponding to a rapid increase in biomass during the initial period, nitrate was rapidly consumed. Under all conditions, the nitrate concentration was almost 0 at day 4. Accumulation of microbial lipids can be induced by environmental stresses [31, 32] in which microalgal cells may shift from actively dividing to storing energy under stressful conditions (e.g., high light intensity and nitrogen starvation). In the present study, the lipid content of the immobilized cells was enhanced along with a decrease in the biofilm productivity under nitrogen-deficient conditions. The results were consistent with several researches [33, 34]. In nitrogen-deficient situation, the cell division can barely be processed. To reserve energy to survive, the proteins are tent to be converted into the lipid, leading to an enhanced lipid content. As the nitrate was consumed, the pH increased. Although a continuous air flow enriched with 2.5% CO2 (v/v) at 10 mL min-1 was provided to the biofilm on the 12
substratum, the CO2 dissolved in the medium was not enough to neutralize the OHgenerated by the nitrate consumption. As a result, the pH increased at first and then dropped from day 3 onwards, probably due to the low nitrate concentration. In addition, increasing the light intensity enhanced the consumption of nitrate, leading to a higher pH at day 2 (as shown in Fig. 3B). Cuellar-Bermudez et al. [32] investigated the effects of light intensity and CO2 supply on pH and total lipid production by Synechocystis sp. and concluded that when the input CO2 was 3% v/v, pH values increased proportionally with light intensity so that the highest pH values were obtained with the highest light intensity (1920 μmol m-2 s-1). In addition, the most C16: 1 and C18: 1 (main components of biodiesel) were obtained with light intensity up to 600 μmol m-2 s-1 at pH 9. The results obtained in this study (a maximum lipid content was achieved at light intensity of 938 μmol m-2 s-1, and a pH range of 8.6 to 9.1) were consistent with previous studies. 3.3. Effect of photoperiod on biofilm and lipid productivity of Desmodesmus sp.F51 The photoperiod is a critical factor in determining the growth rate of microalgal cultures [16]. The biological pathways of lipid/glucan biosynthesis and carbon fixation occur during the light period, while the processes of mitosis, the TCA cycle and lipid β-oxidation occur during the dark period. Fig. 4 shows the influence of photoperiod on biomass (including the suspended and immobilized biomass) and lipid productivity of attached culture of Desmodesmus sp. F51 with a light intensity of 938 μmol m-2 s-1 at different culture stages. Generally, both the biofilm productivity and lipid content were enhanced by increasing the 13
illumination time from 8h to 16h, but decreased with continuous illumination. The maximum biofilm productivity of 35.67±0.75 g m-2 d-1 was obtained with a light: dark cycle of 16:8 h at day 4. The maximum lipid productivity of 7.83±0.12 g m-2 d-1 (lipid content of 23.08±0.74%) was achieved with a light: dark cycle of 16:8 h at day 6. The photoperiod has been reported to influence photosynthetic activity, metabolite production, lipid accumulation and profiles in many microalgae [12, 35]. The results obtained in this study have confirmed that a 16:8 h light: dark cycle as the optimum photoperiod regime for the growth of Desmodesmus sp. F51, which was interpreted as meaning that continuous illumination leads to damage to photosystem II, whereas introducing a short dark period might reduce the light induced damage to photosystem II and enhance the growth in the next light period. Cultures exposed to continuous illumination (24:0 light: dark cycle) reached stationary phase earlier, indicating photo-inhibition caused by excess illumination. Sriram and Seenivasan [36] concluded that a 16:8 h light: dark cycle is the optimal photoperiod regime for Desmodesmus sp. VIT to attain maximum lipid content and lipid productivity. Wahidin et al. [12] also found that the growth and lipid productivity of Nannochloropsis sp. were enhanced by increasing the illumination time up to 16 h. The study of the diatom Phaeodactylum tricornutum showed a clear temporal separation in the carbon capture (light period) and mitosis (dark period) processes after acclimatization to a 16:8 h light: dark cycle. Moreover, an increase in cellular neutral lipids was observed throughout the light period, but a near-linear decline occurred 14
during the dark period [35]. Carbon metabolism pathways (e.g., photosynthesis and carbon assimilation) appeared to be tightly regulated by photoperiod during the daytime and catabolic utilization of energy reserves occurred mainly during the night period [37]. Therefore, it is reasonable that a light regime of 16:8 h light: dark could trigger the lipid accumulation. It was also found that under the nitrogen-deficient conditions, increasing the light intensity to 938 μmol m-2 s-1 as well as prolonging the light period to 16h could enhance the photosynthetically active region inside the biofilm, and achieved a relatively high biofilm productivity of 33.92±0.52 g m-2 d-1 at day 6. A similar result was reported by Schnurr and Allen [24], who found that light penetration inside a biofilm was related to the light regime. Increasing the light intensity could enhance the photosynthetically active region, leading to a higher biomass productivity. 3.4. Effect of photoperiod on pH and nitrate consumption of Desmodesmus sp. F51 Fig. 5 shows the pH and nitrate concentration obtained during the four different photoperiods. Prolonging the light period, greatly enhanced the consumption of nitrate, and a correspondingly high pH resulted. With a photoperiod of 24:0 h light: dark, the nitrate concentration decreased to 0.07 g L-1 at day 3, leading to a maximum pH of 10.89. After that, the pH decreased again, which was probably due to the dissolved CO2 provided to the biofilm. As previously explained, the process of carbon fixation and lipid biosynthesis mainly occur during the light period [35]. With sufficient nitrogen and carbon 15
supplementation, increasing the light period would enhance the biomass growth and nitrate consumption. The source of nitrogen used in this study was NaNO3, and when the NO3- was consumed, the pH of medium became basic due to the excessive OH- and Na+. In addition, since the medium recipe contains 20 mg L-1 Na2CO3, and 2.5% CO2 gas was contentiously aerated into the cultivation unit, there is dissolved inorganic carbon (DIC) in the medium. Several researches have pointed out that when using NaHCO3 or Na2CO3 as carbon source for microalgae growth, the increase in culture pH could be mainly attributed to the accumulation of OH- after the microalgal uptake of CO2 [38, 39, 40]. As shown in Fig. 4A, rising the illumination time generally enhanced the biomass productivity, which may represent higher assimilation rates of DIC and nitrate and lead to a higher pH value. Therefore, the pH varied differently as shown in Fig. 5. It has to be pointed out that since the pH in the culture medium with light: dark cycle of 24:0 h was over 10, the microalgae growth might be suffered from a higher inhibitory effect than that of the light: dark cycle of 16:8 h resulting in a lower biomass productivity [32]. The maximum adhesion ratio of 98.37% occurred with a light: dark cycle of 24:0 h at day 4. 3.5. Effect of light-switching strategies on biofilm and lipid productivity Generally, higher light levels can simultaneously increase both biomass and lipid accumulation to eventually achieve large amounts of lipid products [41]. The efficiency of light penetration affects optimal photosynthesis of the immobilized microalgae. The results described in the previous sections show that a light intensity of 700 μmol m-2 s-1 is adequate for cell growth during initial growth stages. However, the enhancement of 16
light intensity from 700 to 1134 μmol m-2 s-1 is required to maintain the high cell growth rate, due primarily to the self-shading effect arising from the increase in biofilm thickness. Moreover, a light intensity of 938 μmol m-2 s-1 was preferable for lipid accumulation of Desmodesmus sp. F51 in this attached culture system. Hence, these results suggest that light-switching strategy can be adopted as an effective strategy for enhancing both biofilm production and lipid accumulation. In this study, two light-switching strategies with a photoperiod of 16: 8 h (light:dark) were compared (as shown in Fig. 6). In the first 2 days, a low light intensity of 700 μmol m-2 s-1 was provided, and resulted in a high biofilm productivity of 35.83±0.33 g m-2 d-1. When the light intensity was switched to 1134 μmol m-2 s-1 at day 3, the biomass productivity slightly declined at first (probably due to strong light inhibition), and then increased (as shown in Fig. 6A). A maximum biofilm production of 241.67±3.50 g m-2 was obtained after 8 days of culture. Although the nitrate concentration was almost 0 at day 4, the biomass (instead of lipids) accumulated under strong light intensity of 1134 μmol m-2 s-1. As shown in Fig. 6C and D, the maximum lipid content and lipid production of 22.18±0.72% and 53.62±0.98 g m-2 (lipid productivity of 7.43±0.12 g m-2 d-1), respectively, were obtained with Strategy I. In Strategy II, the light intensity was switched again to 938 μmol m-2 s-1 at day 5. Compared to Strategy I, the biofilm productivity was slightly lower (as shown in Fig. 6A), but the lipid content was greatly enhanced to 29.81±0.59% at day 8 (as shown in Fig. 6C). A maximum biofilm production of 223.58±0.58 g m-2 and a maximum lipid production of 66.65±1.48 g m-2 (lipid productivity of 8.33±0.18 g m-2 d-1) were 17
achieved with Strategy II. Table 1 compares the algae biofilm productivities from different studies with different light regimes. This indicates that the energy conservation capillary-effect-based biofilm reactor is beneficial for increased biofilm/lipid production. In addition, to evaluate the suitability of the lipid produced by Desmodesmus sp. F51 for biodiesel production, the fatty acid compositions of the lipid produced by Desmodesmus sp. F51 was analysed. The fatty acid compositions obtained from day 5 to 8 in strategy II (nitrogen replete conditions) were mainly consisted of C16:0 (28-30%), C18:1 (30-35%), C18:2 (12-16%) and C18:3 (8-12%), which means that the lipid produced from Desmodesmus sp. F51 is suitable for biodiesel production. Compared with the constant light intensity, the light-switching strategies achieved higher biofilm and lipid production. As the biofilm was thin at the initial growth stage, low light intensity could provide proper illumination for the photosynthetically active area with the avoidance of light saturation. As the culture continued, the biofilm became thick and the nitrate concentration decreased, which decreased the efficiency of light penetration inside the biofilm. Thus, incrementally increasing the light intensity with a photoperiod of 16: 8 h (light:dark) greatly enhanced the photosynthetic efficiency inside the biofilm, along with an improvement in biomass production and lipid accumulation. Therefore, incremental light intensity was beneficial during this phase to avoid the effect of insufficient lighting, as higher light intensity penetrated deeper into the biofilm, 18
resulting in higher photosynthetic activity in the microalgal culture [42]. Furthermore, incremental light intensity resulted in increased accumulation of lipids in the cells [43]. Therefore, the optimal light-switching strategy was able to avoid insufficient light intensity in the latter culture phase.
4. CONCLUTIONS Optimization of light-related strategies is important for biofilm and lipid production in attached microalgal cultures. A maximum lipid production of 66.65 g m-2 was obtained by switching light intensity from 700 to 1134 μmol m-2 s-1 at day 3 and further switching to 938 μmol m-2 s-1 at day 5 with a light: dark cycle of 16: 8h (Strategy II). The light-switching strategy could supply the appropriate light for each growth stage of the immobilized cells, and was beneficial for lipid production. Therefore, a light-switching strategy is a suitable light supply method for indoor microalgae cultivation in attached culture.
ACKNOWLEDGMENTS The authors thank the financial support from the Natural Science Foundation of Fujian Province, China (No. 2017N0013 & 2015H0018) and Fuzhou Administration of Science and Technology, China (No. 2017-G-58).
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oceanica IMET1 under high light intensity and nitrogen replete conditions, Algal Res. 11 (2015) 55-62. [42] C. U. Ugwu, H. Aoyagi, H. Uchiyama, Influence of irradiance, dissolved oxygen concentration, and temperature on the growth of Chlorella sorokiniana, Photosynthetica 45 (2007) 309-311. [43]
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photoperiods (6:18 h, 12:12 h, 18:6 h, 24:0 h) with light intensity of 938 μmol m-2 s-1 at different culture stages. A, C and E represent the biomass productivity and adhesion ratio obtained at days 2, 4 and 6; B, D, and F represent lipid content and lipid productivity obtained at days 2, 4 and 6 Figure5 The effect of light photoperiods (6:18 h, 12:12 h, 18:6 h, 24:0 h) on the pH (A) and nitrate concentration (B) in the culture medium Figure6 Comparison of two light incremental strategies (Strategy I: illumination with light intensity of 700 μmol m-2 s-1 during the first 2 days, and then shifted to 1134 μmol m-2 s-1 from day 3 to day 8; Strategy II: illumination with light intensity of 700 μmol m-2 s-1 during the first 2 days, shifted to 1134 μmol m-2 s-1 from day 3 to day 4, and shifted again to 938 μmol m-2 s-1 from day 5 to day 8, the photoperiod of 16:8 h (light: dark) was applied for the two strategies)
27
Fig. 1
28
Fig. 2
29
Fig. 3
30
Fig. 4
31
Fig. 5
32
Fig. 6
33
Table 1. Comparing biofilm productivity of Desmodesmus sp. F51 with that obtained in the related reports using attached culture system. Microalgae strains
Reactor type
Light intensity (μmol m-2 s-1)
Photoperiod (light: dark h)
Temperature (C)
Biofilm productivity (g m-2 d-1)
Lipid content (%)
References
Mixed-Filamen tous dominated
Algal turf scrubber
390
23:1
19-24
17.2
N/A
[44]
Chlorella vulgaris
Rotating algal biofilm growth system
642
15:9
~25.5
14
7.72
[5]
Chlorella vulgaris
A pilot-scale revolving algal bofilm cultivation system
~457
Yearly average
11-35
18.9
7.2-10.2
[7]
Chlorella sorokiniana
Rotating biological contactor based PBR
422
24:0
38
17.5-20.1
N/A
[45]
Scenedesmus dimorphus
Single layer attached biofilm cultivation system
100
24:0
25
10.8
Desmodesmus sp.F51
Capillary-effect-based biofilm reactor
Strategy I
30.21 16:8
Strategy II
[11]
22.19
30
This study 27.95
34
N/A
29.81
35
S1369-703X(17)30248-6 http://dx.doi.org/10.1016/j.bej.2017.09.017 BEJ 6790
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
4-7-2017 7-9-2017 22-9-2017
Please cite this article as: Ying Shen, Sha Wang, Shih-Hsin Ho, Youping Xie, Jianfeng Chen, Enhancing lipid production in attached culture of a thermotolerant microalga Desmodesmus sp.F51 using light-related strategies, Biochemical Engineering Journalhttp://dx.doi.org/10.1016/j.bej.2017.09.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A revised manuscript (BEJ-D-17-00665R1) submitted to Biochemical Engineering Journal
(All the changes are marked with yellow highlight)
Enhancing lipid production in attached culture of a thermotolerant microalga Desmodesmus sp. F51 using light-related strategies
Ying Shen1,2, Sha Wang1, Shih-Hsin Ho3, Youping Xie3*, Jianfeng Chen3**
1
College of Mechanical Engineering and Automation, Fuzhou University,
Fuzhou350116, China 2
Collaborative Innovation Center of High-End Equipment Manufacturing in Fujian,
Fuzhou 350116, China 3
College of Biological Science and Engineering, Fuzhou University, Fuzhou 350116,
China
*Corresponding author: Youping Xie Tel: +86-591-22866373 Fax: +86-591-22866373 Email: [email protected] **
Co-corresponding author: Jianfeng Chen
Tel: +86-591-22866373 Fax: +86-591-22866373 Email: [email protected]
Highlights
Efficiency of light related strategies on attached microalgal culture was assessed.
Light-switching strategy enhanced both biofilm and lipid production. A maximum biofilm production of 241.67 g m-2 was achieved. A maximum lipid production of 66.65 g m-2 was achieved. 1
ABSTRACT In attached culture, concentrated cells are grown on the substratum, which makes the light adsorption mechanism quite different from those of suspended cultures. An energy conservation biofilm reactor based on capillary-effect was firstly introduced for attached culture of the thermotolerant microalga Desmodesmus sp. F51. The effects of light-related strategies (including light intensity, photoperiod and light-switching strategies) on lipid production were investigated. It was found that the light requirements for biomass or lipid accumulation varied greatly at different growth stages, probably due to increasing biofilm thickness. By switching the light intensity from 700 to 1134 μmol m-2 s-1 at day 3 in Strategy I, a maximum biofilm/lipid production of 241.67/53.62 g m-2 was achieved at day 8. A similar operation was conducted in Strategy II except a further switch to light intensity of 938 μmol m-2 s-1 at day 5, a maximum biofilm/lipid production of 223.58/66.65 g m-2 was achieved at day 8. This performance is better than most of the previously reported values. Light-switching operation was proved to be an effective strategy to supply the appropriate light for each growth stage of the immobilized cells, and was beneficial for biofilm/lipid production.
Key-words: Microalgae; attached culture; light intensity; photoperiod; lipid production
1. INTRODUCTION 2
With the development of economy and society, a large amount of fossil fuels are unduly consumed and climatic changes are becoming more and more severe, which requires us to seek alternative energy sources. Microalgae, unicellular photosynthetic organisms, are regarded as a renewable feedstock for biodiesel production compared to most oil rich crops [1, 2]. Moreover, cultivation of microalgae can utilize non-arable land and municipal wastewater as a nutrient source [3], reducing the severe problems of various environmental pollutants. Large volumes of water and energy are required for suspended cultures, which significantly hinder the commercial feasibility [4]. Compared to suspended cultures, the biofilm cultivation of microalgae, in which microalgal cells are grown on the surface of supporting materials with high density and fed with nutrient solutions, is a novel cultivation mode. Attached microalgal cultures grow as immobilized cells on the substratum. There are many advantages to this cultivation method including: 1) the capital cost and energy consumption for biomass harvesting are significantly reduced [5, 6, 7]; 2) since the immobilized cells were highly concentrated on the substratum, the cell growth area can be better controlled when the system is set up in the ocean or in lagoons; 3) immobilized algae have been reported to have excellent efficiency in wastewater treatment [8, 9]. Although the attached microalgal culture seems to provide an innovative solution for the microalgal industry, there are still many issues to be solved. Firstly, the immobilized cells are exposed to air instead of the medium and, as a result, the temperature on the cells is higher than those grown in suspended culture in summer. 3
Fortunately, it has been reported that Desmodesmus sp. F51cells can be grown at 45C for 24 h and 50C for 8 h with satisfactory mortality rates [10], which suggests that strain F51 may be suitable for attached cultivation as they can handle the high temperatures in the summer in southern China. Secondly, the light absorption mechanism of attached cultures is quite different from suspended culture. In suspended culture, cells are supposedly exposed to a similar light intensity when proper mixing is provided. Since immobilized cells are attached to the substratum, the inner-layer of the immobilized cells (old cells) was believed to receive less light than cells on the outer layer (new cells). Wang et al. [11] stated that the specific growth rate of the inner layer decreased as the biomass of the upper layer increased, which was mainly because the light traveling through the upper layer became weaker and weaker. In addition, light intensity and photoperiods are critical factors that influence the growth rate of microalgal cultures [12]. Thus, to enhance the economic potential of attached microalgal cultures, selecting a proper light exposure strategy is of great importance. To the best of our knowledge, information on the effects of light-related strategies on biomass production and lipid accumulation in attached culture is still lacking. It has been recognized that many microalgal species can use light as their energy source for synthesizing cell protoplasm. The light requirements of microalgae are not only species dependent, but may differ for each growth phase of the microalgal strain [13]. For example, most microalgae require less intense light at the initial lag phase to avoid photoinhibition, however, on the other hand, higher light intensity is essential at exponential phase to prevent cell shading (photo limitation) [14]. Therefore, selecting 4
an appropriate light intensity for each growth phase is vital to improve algal growth with reduced power consumption [15]. Numerous studies have reported that the lipid content in microalgae can accumulate under higher intensity light [12, 16]. However, the effect of light intensity on lipid accumulation in attached culture of Desmodesmus sp. is still unknown. Taken together, due to the advantage of low energy consumption, a capillary-effect-based biofilm reactor (CBR) was used in this study to assess its potential for generating microalgal biomass and lipid production. As light significantly influences photosynthetic efficiency in microalgae, it is important to understand the effect of light-related strategies on microalgal lipid production in this attached culture system. Thus, in this study, light intensity (700 to 1134 μmol m-2 s-1) and photoperiod cycles (24:0 to 8:16 light: dark) were first investigated to improve cell growth and lipid production from attached culture of Desmodesmus sp. F51. Moreover, in order to produce a large amount of biomass with high lipid content, different light-switching strategies were also assessed.
2. MATERIAL AND METHODS 2.1 Algal strain and pre-culture Desmodesmus sp. F51, isolated from southern Taiwan, was obtained from Prof. Ching-Nen Nathan Chen’s Laboratory at National Sun Yat-sen University, Taiwan. The medium used for pre-culture and culture of Desmodesmus sp. F51 was BG11 medium which contains: 1500 mg L-1 NaNO3, 40 mg L-1 K2HPO4, 75 mg L-1 MgSO47H2O, 36 5
mg L-1 CaCl22H2O, 6 mg L-1 citric acid, 6 mg L-1 ferric ammonium citrate, 1 mg L-1 EDTANa2, 20 mg L-1 Na2CO3 and 1 mL L-1 A5 trace metal solution. The recipe of A5 trace metal solution was: 2.86 g L-1 H3BO3, 1.86 g L-1 MnCl24H2O, 0.22 g L-1 ZnSO47H2O, 0.39 g L-1 Na2MoO42H2O, 0.08 g L-1 CuSO45H2O, and 0.05 g L-1 Co(NO3)26H2O. The initial pH value of the medium was titrated to 7.5 with 1 mol L-1 HCl. The photobioreactor (PBR) used for pre-culture of microalgal cells was 1 L glass vessel (15.5 cm in length and 9.5 cm in diameter) equipped with external light sources (14 W TL5 tungsten filament lamps, Philips Co., China) mounted on both sides of the PBR. The microalgae were grown at 30 C for 4-5 days under a continuous supply of 2.5% CO2 (0.2 vvm), and an agitation rate of 300 rpm. The microalgal culture was continually illuminated with a light intensity of approximately 150 μmol m-2 s-1. The light intensity was measured by a Li-250 Light Meter with a Li-190SA pyranometer sensor (Li-COR Inc., Lincoln, Nebraska, USA). 2.2 Capillary-effect-based biofilm reactor (CBR) Fig. 1 depicts a schematic of the capillary-effect-based biofilm reactor (CBR). The system was composed of 10 units. In each unit, two pieces of porous substratum (80 cm×10 cm folded to 40 cm × 10 cm) were coated on a rigid polypropylene base (40 cm×10 cm), which were vertically aligned to the top of the nutrient medium reservoir (11 cm (length) × 11 cm (width) × 7 cm (height)). The porous canvas substratum selected from the previous experiments served to provide a growth surface for algal biofilms and diffuse the nutrient medium under capillary force. Light was provided by an array (2×5) of white light emitting diodes (LEDs) that were mounted 6
on the top of the reactor. Since the LED lights were put on the top of the reactor, the light intensities varied vertically. The light meter was set at the center of the microalgal growth area, which was about 15 cm from the top of the medium (shown in Fig.1).
Based on the light intensity preferences for biomass or lipid accumulation at different culture stages, two light-switching strategies were compared in the third set of experiments. In Strategy I, a light intensity of 700 μmol m-2 s-1 was provided during the first 2 days, and then switched to a light intensity of 1134 μmol m-2 s-1 at day 3 to further enhance the biomass productivity as the biofilm thickness increased. Compared to Strategy I, the only difference in Strategy II was that the light intensity was switched to 938 μmol m-2 s-1 at day 5 to stimulate lipid accumulation. The experiments were conducted at an optimal light: dark cycle of 16:8 h. All other culture conditions were the same as the previous experiments. The experiments were duplicated with the same culture conditions, and an average of the data is presented. 2.4 Analytical methods 2.4.1 Determination of the microalgal growth The biomass dry weight was measured by the gravimetric method [17]. The attached cells were completely washed off from the porous substrate and resuspended with distilled water. The biofilm productivity was identified as the total biomass dry weight obtained from the substratum (4 pieces) per footprint of the single cultivation unit (11 cm×11 cm) per day. The inoculated part was deducted. 2.4.2 Determination of the nitrate concentration The nitrate concentration was determined according to an optical method [18]. Samples were filtered through a 0.22 m pore size filter to remove cells and particles. The filtered medium was diluted with deionized water and its absorption at 220 nm wavelength was measured using a UV/VIS spectrophotometer (model UVmini-2001, 8
Hitachi, Tokyo, Japan). The nitrate level was determined by interpolating the OD220reading into a standard curve. 2.4.3 Determination of the lipid content and fatty acid compositions Lipid content was measured by using a modified Bligh and Dyer’s method [19]. The lipid extraction was carried out using a BioSpec Model 3110 BX bead-beater (Bartlesville, Oklahoma, USA) for cell disruption (3 min) followed by solvent extraction with n-hexane. The oils collected after evaporation was dried at 95C for 1.5 h before weighing. Lipid content was the percentage of lipid weight to disrupted biomass. Lipid productivity was calculated by multiplying biofilm productivity by lipid content. The fatty acids composition was analyzed by gas chromatographic-mass spectrometric analysis (Agilent 6890-5975, USA) introduced in our previous research [20].
3. RESULTS AND DISCUSSION 3.1 Effect of light intensity on biofilm and lipid productivity of Desmodesmus sp.F51 As a primary energy source for photoautotroph, light plays an important role in microalgal growth and lipid accumulation. Because the immobilized algae are exposed directly to light, it is possible that the immobilized cells are more sensitive to fluctuations in light intensity when compared with suspended cells. Fig. 2 shows the influence of light intensity on the biomass (including the suspended and immobilized biomass) and lipid productivity of attached cultures of Desmodesmus sp.F51cells with a light: dark cycle of 12: 12 h at different culture stages. 9
In the first 2 days, the biofilm biomass was inversely proportional to light intensity. The maximum biofilm productivity of 37±0.83 g m-2 d-1 was obtained with the lowest light intensity of 700 μmol m-2 s-1 (as shown in Fig. 2A). However, this tendency varied as the culture period was prolonged, with maximum biofilm productivity of 34.88±0.84 g m-2 d-1 obtained with the highest light intensity of 1134 μmol m-2 s-1 at day 4 (as shown in Fig. 2C). In general, the biofilm productivity decreased as the culture period continued, especially with lower light intensity. The lipid content data shows that a light intensity of 938 μmol m-2 s-1 was preferable for Desmodesmus sp.F51 lipid accumulation over the entire culture period (as shown in Fig. 2B, D and F). Under an optimal light intensity of 938 μmol m-2 s-1, a maximum lipid productivity of 5.18±0.30 g m-2 d-1 and a maximum lipid content of 21.77±0.70% were achieved at day 4 and day 6, respectively.
As a thermo-tolerant algal species, the suspended culture of Desmodesmus sp. F51 had the highest specific growth rate of 2.53 d-1 and biomass productivity of 796 mg L-1 d-1 at a light intensity of 750 μmol m-2 s-1 [23]. For attached culture, a light intensity of 700 μmol m-2 s-1 was found to be optimal for biomass growth in the first 2 days. Wang et al. [11] compared the efficiency of light penetration in a suspended open pond system and an attached culture system, and concluded that under nitrogen-replete conditions, the immobilized cells of Scenedesmus dimorphus were 100% illuminated with light intensity of 100 μmol m-2 s-1, and the biofilm production increased from 8.8 to 107.6 g m-2 in 10 days. The efficiency of light penetration can be affected by many factors, such as algal species, light intensity, nitrogen concentration, pH and biofilm thickness [24]. Murphy et al. [25] mentioned that biofilm productivity can be enhanced from 8.2 to 11.9 g m-2 d-1 with increasing biofilm thickness from 20 to 100 µm. However, a further increase in biofilm thickness to 200 µm led to a significant drop in biofilm productivity, indicating the efficiency of light penetration was attenuated exponentially with increasing biofilm thickness. In the current study, under nitrogen-deficient conditions, the biofilm productivities were decreased for all different light intensities used (as shown in Fig. 2A, C and E), which demonstrates that nitrogen deficiency has a negative effect on biofilm productivity. The influence of light intensity on lipid content of microalgae is affected by many factors, such as microalgae species, nitrogen and carbon supplementation, temperature and culture ages [26, 27]. An increase in light intensity is supposed to promote the biosynthesis of fatty acid through enhancing the activities of the enzymes 11
like, acetyl CoA carboxylase, desaturase, and ATP/citrate lyase, as a result, the excessive light might be trapped by the cells and transformed into fatty acid [28, 29]. However, photo-oxidative cell damage could also be happened as the light intensity is higher than the tolerance of the specific microalgae species. In the current study, a light intensity of 938 μmol m-2 s-1 was found optimal for lipid production. 3.2. Effect of light intensity on pH and nitrate consumption of Desmodesmus sp. F51 The pH and nitrate consumption are valuable factors that reflect microalgal growth and biochemical composition [30]. Fig. 3 shows the variation of pH and nitrate concentration obtained under the four different light intensities. Corresponding to a rapid increase in biomass during the initial period, nitrate was rapidly consumed. Under all conditions, the nitrate concentration was almost 0 at day 4. Accumulation of microbial lipids can be induced by environmental stresses [31, 32] in which microalgal cells may shift from actively dividing to storing energy under stressful conditions (e.g., high light intensity and nitrogen starvation). In the present study, the lipid content of the immobilized cells was enhanced along with a decrease in the biofilm productivity under nitrogen-deficient conditions. The results were consistent with several researches [33, 34]. In nitrogen-deficient situation, the cell division can barely be processed. To reserve energy to survive, the proteins are tent to be converted into the lipid, leading to an enhanced lipid content.
substratum, the CO2 dissolved in the medium was not enough to neutralize the OHgenerated by the nitrate consumption. As a result, the pH increased at first and then dropped from day 3 onwards, probably due to the low nitrate concentration. In addition, increasing the light intensity enhanced the consumption of nitrate, leading to a higher pH at day 2 (as shown in Fig. 3B). Cuellar-Bermudez et al. [32] investigated the effects of light intensity and CO2 supply on pH and total lipid production by Synechocystis sp. and concluded that when the input CO2 was 3% v/v, pH values increased proportionally with light intensity so that the highest pH values were obtained with the highest light intensity (1920 μmol m-2 s-1). In addition, the most C16: 1 and C18: 1 (main components of biodiesel) were obtained with light intensity up to 600 μmol m-2 s-1 at pH 9. The results obtained in this study (a maximum lipid content was achieved at light intensity of 938 μmol m-2 s-1, and a pH range of 8.6 to 9.1) were consistent with previous studies. 3.3. Effect of photoperiod on biofilm and lipid productivity of Desmodesmus sp.F51 The photoperiod is a critical factor in determining the growth rate of microalgal cultures [16]. The biological pathways of lipid/glucan biosynthesis and carbon fixation occur during the light period, while the processes of mitosis, the TCA cycle and lipid β-oxidation occur during the dark period. Fig. 4 shows the influence of photoperiod on biomass (including the suspended and immobilized biomass) and lipid productivity of attached culture of Desmodesmus sp. F51 with a light intensity of 938 μmol m-2 s-1 at different culture stages. Generally, both the biofilm productivity and lipid content were enhanced by increasing the 13
illumination time from 8h to 16h, but decreased with continuous illumination. The maximum biofilm productivity of 35.67±0.75 g m-2 d-1 was obtained with a light: dark cycle of 16:8 h at day 4. The maximum lipid productivity of 7.83±0.12 g m-2 d-1 (lipid content of 23.08±0.74%) was achieved with a light: dark cycle of 16:8 h at day 6.
during the dark period [35]. Carbon metabolism pathways (e.g., photosynthesis and carbon assimilation) appeared to be tightly regulated by photoperiod during the daytime and catabolic utilization of energy reserves occurred mainly during the night period [37]. Therefore, it is reasonable that a light regime of 16:8 h light: dark could trigger the lipid accumulation. It was also found that under the nitrogen-deficient conditions, increasing the light intensity to 938 μmol m-2 s-1 as well as prolonging the light period to 16h could enhance the photosynthetically active region inside the biofilm, and achieved a relatively high biofilm productivity of 33.92±0.52 g m-2 d-1 at day 6. A similar result was reported by Schnurr and Allen [24], who found that light penetration inside a biofilm was related to the light regime. Increasing the light intensity could enhance the photosynthetically active region, leading to a higher biomass productivity. 3.4. Effect of photoperiod on pH and nitrate consumption of Desmodesmus sp. F51 Fig. 5 shows the pH and nitrate concentration obtained during the four different photoperiods. Prolonging the light period, greatly enhanced the consumption of nitrate, and a correspondingly high pH resulted. With a photoperiod of 24:0 h light: dark, the nitrate concentration decreased to 0.07 g L-1 at day 3, leading to a maximum pH of 10.89. After that, the pH decreased again, which was probably due to the dissolved CO2 provided to the biofilm.
supplementation, increasing the light period would enhance the biomass growth and nitrate consumption. The source of nitrogen used in this study was NaNO3, and when the NO3- was consumed, the pH of medium became basic due to the excessive OH- and Na+. In addition, since the medium recipe contains 20 mg L-1 Na2CO3, and 2.5% CO2 gas was contentiously aerated into the cultivation unit, there is dissolved inorganic carbon (DIC) in the medium. Several researches have pointed out that when using NaHCO3 or Na2CO3 as carbon source for microalgae growth, the increase in culture pH could be mainly attributed to the accumulation of OH- after the microalgal uptake of CO2 [38, 39, 40]. As shown in Fig. 4A, rising the illumination time generally enhanced the biomass productivity, which may represent higher assimilation rates of DIC and nitrate and lead to a higher pH value. Therefore, the pH varied differently as shown in Fig. 5. It has to be pointed out that since the pH in the culture medium with light: dark cycle of 24:0 h was over 10, the microalgae growth might be suffered from a higher inhibitory effect than that of the light: dark cycle of 16:8 h resulting in a lower biomass productivity [32]. The maximum adhesion ratio of 98.37% occurred with a light: dark cycle of 24:0 h at day 4. 3.5. Effect of light-switching strategies on biofilm and lipid productivity Generally, higher light levels can simultaneously increase both biomass and lipid accumulation to eventually achieve large amounts of lipid products [41]. The efficiency of light penetration affects optimal photosynthesis of the immobilized microalgae. The results described in the previous sections show that a light intensity of 700 μmol m-2 s-1 is adequate for cell growth during initial growth stages. However, the enhancement of 16
light intensity from 700 to 1134 μmol m-2 s-1 is required to maintain the high cell growth rate, due primarily to the self-shading effect arising from the increase in biofilm thickness. Moreover, a light intensity of 938 μmol m-2 s-1 was preferable for lipid accumulation of Desmodesmus sp. F51 in this attached culture system. Hence, these results suggest that light-switching strategy can be adopted as an effective strategy for enhancing both biofilm production and lipid accumulation. In this study, two light-switching strategies with a photoperiod of 16: 8 h (light:dark) were compared (as shown in Fig. 6). In the first 2 days, a low light intensity of 700 μmol m-2 s-1 was provided, and resulted in a high biofilm productivity of 35.83±0.33 g m-2 d-1. When the light intensity was switched to 1134 μmol m-2 s-1 at day 3, the biomass productivity slightly declined at first (probably due to strong light inhibition), and then increased (as shown in Fig. 6A). A maximum biofilm production of 241.67±3.50 g m-2 was obtained after 8 days of culture. Although the nitrate concentration was almost 0 at day 4, the biomass (instead of lipids) accumulated under strong light intensity of 1134 μmol m-2 s-1. As shown in Fig. 6C and D, the maximum lipid content and lipid production of 22.18±0.72% and 53.62±0.98 g m-2 (lipid productivity of 7.43±0.12 g m-2 d-1), respectively, were obtained with Strategy I. In Strategy II, the light intensity was switched again to 938 μmol m-2 s-1 at day 5. Compared to Strategy I, the biofilm productivity was slightly lower (as shown in Fig. 6A), but the lipid content was greatly enhanced to 29.81±0.59% at day 8 (as shown in Fig. 6C). A maximum biofilm production of 223.58±0.58 g m-2 and a maximum lipid production of 66.65±1.48 g m-2 (lipid productivity of 8.33±0.18 g m-2 d-1) were 17
achieved with Strategy II. Table 1 compares the algae biofilm productivities from different studies with different light regimes. This indicates that the energy conservation capillary-effect-based biofilm reactor is beneficial for increased biofilm/lipid production. In addition, to evaluate the suitability of the lipid produced by Desmodesmus sp. F51 for biodiesel production, the fatty acid compositions of the lipid produced by Desmodesmus sp. F51 was analysed. The fatty acid compositions obtained from day 5 to 8 in strategy II (nitrogen replete conditions) were mainly consisted of C16:0 (28-30%), C18:1 (30-35%), C18:2 (12-16%) and C18:3 (8-12%), which means that the lipid produced from Desmodesmus sp. F51 is suitable for biodiesel production.
resulting in higher photosynthetic activity in the microalgal culture [42]. Furthermore, incremental light intensity resulted in increased accumulation of lipids in the cells [43]. Therefore, the optimal light-switching strategy was able to avoid insufficient light intensity in the latter culture phase.
4. CONCLUTIONS Optimization of light-related strategies is important for biofilm and lipid production in attached microalgal cultures. A maximum lipid production of 66.65 g m-2 was obtained by switching light intensity from 700 to 1134 μmol m-2 s-1 at day 3 and further switching to 938 μmol m-2 s-1 at day 5 with a light: dark cycle of 16: 8h (Strategy II). The light-switching strategy could supply the appropriate light for each growth stage of the immobilized cells, and was beneficial for lipid production. Therefore, a light-switching strategy is a suitable light supply method for indoor microalgae cultivation in attached culture.
ACKNOWLEDGMENTS The authors thank the financial support from the Natural Science Foundation of Fujian Province, China (No. 2017N0013 & 2015H0018) and Fuzhou Administration of Science and Technology, China (No. 2017-G-58).
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photoperiods (6:18 h, 12:12 h, 18:6 h, 24:0 h) with light intensity of 938 μmol m-2 s-1 at different culture stages. A, C and E represent the biomass productivity and adhesion ratio obtained at days 2, 4 and 6; B, D, and F represent lipid content and lipid productivity obtained at days 2, 4 and 6 Figure5 The effect of light photoperiods (6:18 h, 12:12 h, 18:6 h, 24:0 h) on the pH (A) and nitrate concentration (B) in the culture medium Figure6 Comparison of two light incremental strategies (Strategy I: illumination with light intensity of 700 μmol m-2 s-1 during the first 2 days, and then shifted to 1134 μmol m-2 s-1 from day 3 to day 8; Strategy II: illumination with light intensity of 700 μmol m-2 s-1 during the first 2 days, shifted to 1134 μmol m-2 s-1 from day 3 to day 4, and shifted again to 938 μmol m-2 s-1 from day 5 to day 8, the photoperiod of 16:8 h (light: dark) was applied for the two strategies)
27
Fig. 1
28
Fig. 2
29
Fig. 3
30
Fig. 4
31
Fig. 5
32
Fig. 6
33
Table 1. Comparing biofilm productivity of Desmodesmus sp. F51 with that obtained in the related reports using attached culture system. Microalgae strains
Reactor type
Light intensity (μmol m-2 s-1)
Photoperiod (light: dark h)
Temperature (C)
Biofilm productivity (g m-2 d-1)
Lipid content (%)
References
Mixed-Filamen tous dominated
Algal turf scrubber
390
23:1
19-24
17.2
N/A
[44]
Chlorella vulgaris
Rotating algal biofilm growth system
642
15:9
~25.5
14
7.72
[5]
Chlorella vulgaris
A pilot-scale revolving algal bofilm cultivation system
~457
Yearly average
11-35
18.9
7.2-10.2
[7]
Chlorella sorokiniana
Rotating biological contactor based PBR
422
24:0
38
17.5-20.1
N/A
[45]
Scenedesmus dimorphus
Single layer attached biofilm cultivation system
100
24:0
25
10.8
Desmodesmus sp.F51
Capillary-effect-based biofilm reactor
Strategy I
30.21 16:8
Strategy II
[11]
22.19
30
This study 27.95
34
N/A
29.81
35