Biomass and Astaxanthin Productivities of Haematococcus Pluvialis in an Angled Twin-Layer Porous Substrate Photobioreactor: Eﬀect of Inoculum Density and Storage Time

biology

Article Biomass and Astaxanthin Productivities of Haematococcus pluvialis in an Angled Twin-Layer Porous Substrate Photobioreactor: Eﬀect of Inoculum Density and Storage Time

Thanh-Tri Do 1, Binh-Nguyen Ong 2, Minh-Ly Nguyen Tran 3, Doan Nguyen 4, Michael Melkonian 5 and Hoang-Dung Tran 2,*

1 Faculty of Biology, Ho Chi Minh City University of Education, 280 An Duong Vuong Street, District 5, Ho Chi Minh City 72711, Vietnam 2 Faculty of Biotechnology, Nguyen-Tat-Thanh University, 298A-300A Nguyen-Tat-Thanh Street, District 04, Hochiminh City 72820, Vietnam 3 Vietnam-United States-Australia Biotechnology Company, Group 4, Cay Da Ward, Nguyen Thi Lang Street, Cu Chi District, Hochiminh City 71609, Vietnam 4 Faculty of Business and Law, Swinburne University of Technology, Mail H25 PO Box 218, Hawthorn, Victoria 3122, Australia 5 Biozentrum Köln, Universität zu Köln, Zülpicher Str. 47 b, 50674 Köln, Germany * Correspondence: [email protected]; Tel.: +84-772-999-537

Received: 17 May 2019; Accepted: 22 August 2019; Published: 18 September 2019

Abstract: The microalga Haematococcus pluvialis is mainly cultivated in suspended systems for astaxanthin production. Immobilized cultivation on a Twin-Layer porous substrate photobioreactor (TL-PSBR) has recently shown promise as an alternative approach. In Vietnam, a TL-PSBR was constructed as a low-angle (15 ◦) horizontal system to study the cultivation of H. pluvialis for astaxanthin production. In this study, the biomass and astaxanthin productivities and astaxanthin content in the dry biomass were determined using different initial biomass (inoculum) densities (from 2 2.5 to 10 g dry weight m− ), different storage times of the initial biomass at 4 ◦C (24, 72, 120 and 168 h) 2 1 and different light intensities (300–1000 µmol photons m− s− ). The optimal initial biomass density 2 1 2 at light intensities between 400–600 µmol photons− s− was 5–7.5 g m− . Algae stored for 24 h after harvest from suspension for immobilization on the TL-PSBR yielded the highest biomass and 2 1 2 1 astaxanthin productivities, 8.7 g m− d− and 170 mg m− d− , respectively; longer storage periods decreased productivity. Biomass and astaxanthin productivities were largely independent of light 2 1 intensity between 300–1000 µmol photons m− s− but the efficiency of light use per mole photons 2 1 was highest between 300–500 µmol photons m− s− . The astaxanthin content in the dry biomass varied between 2–3% (w/w). Efficient supply of CO2 to the culture medium remains a task for future improvements of angled TL-PSBRs.

Keywords: Haematococcus pluvialis; astaxanthin; porous substrate photobioreactor; a horizontal (low-angled) twin-layer photobioreactor

1. Introduction Recent studies have shown strong antioxidant activity of astaxanthin [1] with beneﬁts to the immune system, cardiac muscles, and to treatments of cancer and skin aging [1–4]. The green alga Haematococcus pluvialis is the most widely used source for natural astaxanthin at a commercial scale and has been reported to accumulate astaxanthin up to 5.9% (w/w) of the dry biomass [5–7].

Biology 2019, 8, 68; doi:10.3390/biology8030068 www.mdpi.com/journal/biology Biology 2019, 8, 68 2 of 14

Due to its life history, H. pluvialis is usually grown in two-phase cultivation, in which the cells are grown by cell multiplication in the “green phase” and are stimulated to accumulate astaxanthin in the successive “red phase” (e.g., [6,8]). The two-phase cultivation method is technically demanding and is characterized by high energy consumption and relatively long cultivation times [9,10]. Two-phase suspended cultivation of H. pluvialis is the predominant cultivation system at both exploratory and commercial scales. In suspended cultivation, a maximum light intensity of 150 µmol 2 1 photons m− s− should not be exceeded to maintain cell growth and divisions and environmental parameters such as temperature, CO2, and pH need to be closely monitored [6,8]. As the needed biomass is reached, the second phase, known as the stressed or red phase is switched on to stimulate astaxanthin accumulation [6,8]. Suspended cultivation of Haematococcus is performed in open ponds/raceways or in closed tubular or flat-plate photobioreactors (PBR). Open pond cultivation is usually used only for the stressed phase with short cultivation time (4–6 days) to minimize contamination and apply stress conditions [8]. The closed PBR can minimize contamination and control growth parameters better, but has the drawbacks of high assembly and maintenance costs (on average, a tenfold increase in costs) [11–13]. Suspended cultivation usually yields low biomass density (0.05–0.06% of the total cultivation liquid) due to the ineffective light and carbon dioxide distribution caused by the low surface-to-volume ratio of the cultivation systems and demands additional effort for the harvest of the algae [14]. Flat or tubular closed systems increase productivity by increasing the efficiency of light usage and limiting contamination, but there are other limitations, especially in the adhesion of cells to the inner PBR surfaces of many microalgae including H. pluvialis [15]. Porous substrate photobioreactors (PSBR) such as the vertical Twin-Layer system can be used to cultivate various algal species including H. pluvialis (for a review, see Podola et al. [16]). PSBRs are a recently developed technique for microalgal cultivation in immobilized systems that has also shown considerable promise for application in Haematococcus [7,17–21]. The Twin-Layer system is a modular PSBR. The prototype (large scale) TL-PSBR is capable to operate up to eight Twin-Layer modules, the area for growth of microalgae on both sides is 2 0.67 m2 for each module. A tube-type TL-PSBR × for small-scale holding of 12 Twin-Layer modules of 10 90 cm was used to monitor growth kinetics × of H. pluvialis [15,22,23]. Each TL module consists of a vertically orientated source layer (e.g., glass fiber nonwoven), through which a flow of medium is established to feed nutrient. Onto both sides of the source layer, the substrate layers (e.g., printing paper or filter paper that has small porosity) are applied by self-adhesion of hydrogen bonds. Microalgae will be immobilized on the substrate layers and will develop a biofilm [15,22,23]. The concurrent production of biomass and astaxanthin of H. pluvialis in one-phase cultivation at high light intensity with CO2 supplementation using a vertical Twin-Layer photobioreactor yielded 2 1 2 biomass productivities of up to 19.4 g m− d− and final biomass of 213 g dry weight m− growth area after 16 days of cultivation [23]. In comparison with two-phase cultivation using the same photobioreactor system, one-phase cultivation yielded a similar astaxanthin output in just half of the cultivation time [23]. In one-phase cultivation on TL-PSBR, growth and astaxanthin accumulation of H. pluvialis happens simultaneously but in different cell layers of the biofilm. Cells on the surface are exposed to direct light and change into red akinetes, whereas green cells in the lower layers divide by spore formation [23]. Hence, the thickness of the biofilm and high light intensity play an important role in one-phase cultivation. However, in modular vertical TL-PSBRs high intensity and uniformity of incident light is difficult to achieve due to mutual shading by adjacent modules, although the vertical arrangement of the TL modules saves space and enhances areal productivity. A horizontal (low-angled) TL-PSBR was developed for research purposes in Vietnam starting in 2016, as it is easier to construct and operate than a vertical TL system and for high light applications such as the production of astaxanthin, only slightly less productive (aerial productivity) than a vertical system. Furthermore, harvesting the biomass is technically less complicated in low-angled TL-systems compared to vertical systems [16]. In Vietnam, the angled Twin-Layer photobioreactor system for immobilized cultivation of microalgae provides significant advantages over traditional suspended Biology 2019, 8, 68 3 of 14 cultivation such as saving of water, energy and cultivation time [24]. This study investigates several factors affecting biomass and astaxanthin accumulation of H. pluvialis cultivated in a small-scale angled TL-PSBR: initial biomass (inoculum) density, algae storage time before immobilization and influence of different light intensities. Furthermore, a larger-scale angled TL-PSBR system for H. pluvialis was designed and operated to probe upscaling astaxanthin production to pilot scale.

2. Materials and Methods

2.1. Algal Strain and Culture Maintenance The H. pluvialis strain CCAC 0125 was supplied from the Culture Collection of Algae at the University of Cologne, Germany (http://www.ccac.uni-koeln.de/). The strain was maintained in 100 mL Erlenmeyer flasks, with 50 mL of modified BG-11 medium [23] and transferred every 3–4 weeks. 2 1 Cultures were grown at 20 ◦C, with a light intensity of 30 µmol photons m− s− provided by fluorescent lamps (Philips Essential energy saver 18W E27 Cool day light lamp, Philips Lighting GmbH, Hamburg, Germany), using a 14/10 h light/dark cycle.

2.2. Experimental Designs The experiments were carried out on small scale (0.1 0.5 = 0.05 m2 growth area) and large scale × (0.5 m2 4 growth area) angled TL-PSBRs [24]. The non-sterile culture medium was stored in 40 L × plastic chambers.

2.2.1. Experiments with the Small-Scale TL-PSBR The small-scale angled TL-PSBR for H. pluvialis immobilized cultivation includes small chambers with source layers, substrate layer (paper) and immobilized algae inside, nutrient circulation system, air circulation system, light supply system and steel frame (Figure1). Each chamber is 0.37 0.52 m in size divided into three compartments. Each compartment × is about 0.12 0.52 m in size and contains three layers: the source layer (nonwoven fiberglass, × 0.11 0.51 m), substrate layers (Kraft paper 70 g m 2, 0.11 0.51 m) and biofilm (immobilized algae × − × on the substrate layer, the growth area, 0.1 0.5 m) (Figure1C). A small-scale TL-PSBR has six small × chambers (Figure1B) that share the same nutrient, air and light supply systems. Therefore, a small system with 18 growth areas (0.05 m2 for each) for microalgae was divided into different treatments for each experiment. Three experiments with the small-scale TL-PSBR were carried out. The most effective outcome from each preceding experiment would be applied for the next experiment. Influence of algal storage time before immobilization: the concentrated algae were stored at 4 ◦C in the dark over 24, 72, 120 and 168 h after centrifugation, respectively. Initial algae biomass for 2 inoculation of the TL-PSBR was 5 g dry weight m− , the light intensity was adjusted to reach the range 2 1 of 400–600 µmol photons m− s− over the growth area. The optimal initial algal density: the initial biomass densities of 2.5, 5, 7.5 and 10 g dry weight 2 m− were tested. Algae were immobilized immediately after centrifugation or were stored for 24 h at 2 –1 4 ◦C. The light intensity was set at 400–600 µmol photons m− s . Influence of light intensity on algal growth and astaxanthin accumulation: The experiment used sodium high-pressure lamps (PHILIPS) as a light source with the intensity of 300–1000 µmol photon 2 1 m− s− . Each point on the cultivation chambers received different light intensity and was considered to be one experiment for one value point of this parameter. The initial biomass density was 7.5 g dry 2 weight m− . Biology 2019, 8, 68 4 of 14 Biology 2019, 8, x FOR PEER REVIEW 4 of 14

Figure 1.1. TheThe small-scalesmall-scale angledangled TL-PSBR: TL-PSBR: nutrient nutrient and and air air supply supply system system for for cultivation cultivation chamber chamber (A), positioning(A), positioning of chambers of chambers and lightsand lights (B), components(B), components inside inside a chamber a chamber (C). (C). 2.2.2. Experiment with the Large Scale TL-PSBR Three experiments with the small-scale TL-PSBR were carried out. The most effective outcome fromThe each large-scale preceding angledexperiment TL-PSBR would uses be applied the same for components the next experiment. of the small system but has four largerInfluence chambers of algal assembled storage in time the before same immobilizati nutrient, airon: and the lightconcentrated supply algae systems, were each stored chamber at 4 °C providesin the dark1 over0.5 = 24,0.5 72, m 2120area and for 168 algae h after growth centrifugation, (Figure2). Thus,respectively. each large-scale Initial algae TL-PSBR biomass will for × haveinoculation0.5 4 of= the2 m TL-PSBR2 growth was area. 5 Twog dry large weight TL-PSBRs m−2, the with light two intensity diﬀerent was lighting adjusted systems to reach were the setrange up × andofBiology 400–600 operated 2019, µmol8, x atFOR thephotons PEER same REVIEW timem−2 s − to1 over perform the growth the experiment. area. 5 of 14 The optimal initial algal density: the initial biomass densities of 2.5, 5, 7.5 and 10 g dry weight m−2 were tested. Algae were immobilized immediately after centrifugation or were stored for 24 h at 4 °C. The light intensity was set at 400–600 µmol photons m−2 s–1. Influence of light intensity on algal growth and astaxanthin accumulation: The experiment used sodium high-pressure lamps (PHILIPS) as a light source with the intensity of 300–1000 µmol photon m−2 s−1. Each point on the cultivation chambers received different light intensity and was considered to be one experiment for one value point of this parameter. The initial biomass density was 7.5 g dry weight m−2.

2.2.2. Experiment with the Large Scale TL-PSBR The large-scale angled TL-PSBR uses the same components of the small system but has four larger chambersFigureFigure 2.assembled2. Design of the thein thenutrient nutrient same andand nutrient, air air supply supply air system and system light for for the supply the large-scale large-scale systems, angled angled each TL-PSBR. TL-PSBR.chamber provides 1 × 0.5 = 0.5 m2 area for algae growth (Figure 2). Thus, each large-scale TL-PSBR will have 0.5 × 4 = 2 In large-scale TL-PSBRs, the experiment was set up to compare the efficiency for lighting of two m2 growthIn large-scale area. Two TL-PSBRs, large TL-PSBRs the experiment with two was differe set up ntto comparelighting systemsthe efficiency were for set lighting up and of operated two different luminaires. The two luminaires used to compare energy efficiency in this experiment are: atdifferent the same luminaires. time to perform The two the luminaires experiment. used to compare energy efficiency in this experiment are: luminair 250W (light intensity of 300–1150 µM photons m−2 s−1, provided by ten PHILIPS 250W sodium high-pressure lamps) and luminair 400W (light intensity of 300–1300 µmol photon m−2 s−1, provided by eight PHILIPS 400W sodium high-pressure lamps). Some experimental conditions were established based on the results obtained with the small TL- PSBR, in which the initial dry biomass density was 7.5 g m−2, and algae were immobilized after centrifugation within 24 h.

2.3. Preparation of H. pluvialis Biomass for Experiments on Angled TL-PSBRs Suspended cultivation before immobilization: The culture medium used was modified BG-11 [23]. Suspended cultivation of microalgae was done in 500 mL (culture time is about 10 days) and 2000 mL (culture time is about 14 days) Erlenmeyer flasks at 23 ± 2 °C, illuminated by a fluorescent lamp at 40–60 µmol photons m−2 s−1, using a 14/10 h light/dark cycle. However, the large-scale angled TL-PSBRs required a larger amount of initial algal biomass, so the volume of suspended algae was increased to 10 L in PE bags (with 6 L of modified BG-11 medium) (Figure 3). Algae from 2000 mL flasks will be transplanted through PE bags with the initial algal density of about 1.5 x 104 cells mL-1, and a culture period of about 16 days. The medium was aerated with ambient air during the cultivation period, illuminated by fluorescent lamps at 40–60 µmol photons m−2 s−1 with 14/10 h light/dark cycle, at 23 ± 2 °C.

Figure 3. Suspended cultivation of H. pluvialis in 10 L PE bags.

Biology 2019, 8, x FOR PEER REVIEW 5 of 14

Figure 2. Design of the nutrient and air supply system for the large-scale angled TL-PSBR. Biology 2019, 8, 68 5 of 14 In large-scale TL-PSBRs, the experiment was set up to compare the efficiency for lighting of two different luminaires. The two luminaires used to compare energy efficiency in this experiment are: 2 1 luminair 250250W W (light (light intensity of 300–1150300–1150 µµMM photonsphotons mm−−2 ss−−1,, provided by tenten PHILIPSPHILIPS 250250W W 2 1 sodium high-pressure lamps) andand luminairluminair 400W400W (light(light intensityintensity ofof 300–1300300–1300µ µmolmol photon photon m m−−2 ss−−1,, provided by eight PHILIPSPHILIPS 400W400W sodiumsodium high-pressurehigh-pressure lamps).lamps). Some experimental experimental conditions conditions we werere established established based based on on the the resu resultslts obtained obtained with with the thesmall small TL- 2 TL-PSBR,PSBR, in which in which the the initial initial dr dryy biomass biomass density density was was 7.5 7.5 g g m m−−2, ,and and algae algae were were immobilized after centrifugation within 24 h.

2.3. Preparation of H. pluvialis Bioma Biomassss for Experiments on Angled TL-PSBRs Suspended cultivationcultivation before before immobilization: immobilization: The The culture culture medium medium used used was was modified modified BG-11 BG-11 [23]. Suspended[23]. Suspended cultivation cultivation of microalgae of microalgae was done was in done 500 mLin 500 (culture mL (culture time is abouttime is 10 about days) 10 and days) 2000 and mL (culture2000 mL time(culture is about time is 14 about days) 14 Erlenmeyer days) Erlenmeyer flasks at flasks 23 at2 ◦23C, ± illuminated 2 °C, illuminated by a fluorescent by a fluorescent lamp 2 1 ± atlamp 40–60 at 40–60µmol µmol photons photons m− ms−−2, s using−1, using a 14 a /14/1010 h lighth light/dark/dark cycle. cycle. However, However,the the large-scalelarge-scale angled TL-PSBRs required a larger amount of initial algalalgal biomass, so the volume of suspended algae was increased to 10 L inin PEPE bagsbags (with(with 66 LL ofof modifiedmodified BG-11BG-11 medium)medium) (Figure(Figure3 3).). AlgaeAlgae fromfrom 20002000 mLmL 4 1 flasksflasks will be transplanted throughthrough PEPE bagsbags withwith thethe initialinitial algalalgaldensity densityof of about about 1.5 1.5 x x 10 104cells cellsmL mL−-1,, and aa culture culture period period of aboutof about 16 days. 16 days. The mediumThe me wasdium aerated was aerated with ambient with airambient during air the during cultivation the 2 1 period,cultivation illuminated period, byilluminated fluorescent by lamps fluorescent at 40–60 lampsµmol at photons 40–60 mµmol− s− photonswith 14 /m10−2 h s light−1 with/dark 14/10 cycle, h at 23 2 C. light/dark± ◦ cycle, at 23 ± 2 °C.

Figure 3. Suspended cultivation of H. pluvialis in 10 L PE bags.

Centrifugation to concentrate algae in suspension: Algae in 10 L PE bags after 20 days of culture consisted to 85% of flagellate cells and were harvested from suspension by low-speed centrifugation (800 g for 5 min) with a large bench centrifuge (ROTANTA 460 RC, Hettich GmbHo. KG, Tuttlingen, × Germany). Under these conditions, no damage was observed to the flagellate cells. After centrifugation, the concentrated algal suspension was used to determine the amount of dry biomass (mL). The dry biomass of the concentrated algal suspension was determined by filtering 1 mL of the suspension onto a filter paper (0.4 µm pore size, Whatman GmbH, Dassel, Germany) with a predetermined dry weight of W1 and dried the whole set in 2 h at 105 ◦C. The dried filter paper with algae was weighed, and the drying process repeated until an unchanged total weight of W2 was achieved. The dry biomass in 1 mL concentrated algae liquid was calculated as D = W2 –W1. Biology 2019, 8, 68 6 of 14

2.4. Algae Immobilization on Angled TL-PSBRs The inoculation of algae on the substrate layer was tested with several methods. However, using a brush to apply the concentrated algal suspension x showed many advantages. The required volume V (mL) of algal suspension immobilized onto a surface area S was calculated as V (mL) = (M S)/D 2 × with M as the initial immobilized dry biomass density (g m− ). The concentrated algal suspension was immobilized by a soft brush onto substrate layer on 0.05 and 0.5 m2 TL-PSBRs, respectively.

2.5. Culture Conditions after Algal Immobilization Immobilized cultivation time was 10 days. A nutrient-replete BG-11 medium was used during days 1–7 by replacing 20 L (on small-scale TL-PSBR) or 40 L (on large scale TL-PSBR) of medium every 2 or 3 days. Twenty or forty litres of BG-11 medium without N and P (excludes NaNO and K HPO 3H O 3 2 4 × 2 in modified BG-11 medium) [18] were used on days 8, 9 and 10 for astaxanthin induction. The culture medium was supplemented with CO2 1% (v/v) as a carbon source and also as a means for pH adjustment. The temperature was kept in 23–26 ◦C, pH = 6.5–8. The electrical conductivity of the 1 medium was kept in 1800–2000 µS cm− by adding filtered water after every 24 h. 2 1 The following variables were monitored: Dry algae biomass productivity (g m− d− ) was calculated based on equation Mp = (Ma-M)/10 (Ma: dry biomass after 10 days, M: initial 2 1 immobilized dry biomass); astaxanthin productivity (mg m− d− ) was calculated based on equation Ap = (Ma 1000 %A)/10, %A is the astaxathin content in the dry biomass. × × Measurement of immobilized dry biomass: after 10 days of cultivation, the thin algal layer and 2 substrate layer (Kraft paper 70 g/m , Vietnam) were harvested and dried for 2 hours at 105 ◦C, and cooled in a desiccator in 30 min. The dried product was weighed and the drying process repeated until an unchanged total weight of m was obtained. The dry biomass was calculated as Ma = m m (g), 2 2 − 1 m1 is the weight of dry paper without algae. Measurement of astaxanthin content in the dry biomass (%A): Astaxanthin was determined spectrophotometrically according to Li et al. [25]. All processes were carried out in the dark. A sample of 1 mg of freeze-dried biomass was placed with 0.5 mL acetone 90% in a 2 mL tube with screw cap to limit the vaporization, incubated in water bath at 70 ◦C for 5 min with intervals for vortexing, and then ground using a glass pestle. The mixture was centrifuged at 4000 g for 5 min and the supernatant × retrieved. Extraction with acetone was repeated until the pellet turned white. The extract was supplied with acetone (90%) to reach a volume of 3 mL in a 5 mL sealed centrifuge tube with screw cap until spectrophotometric analysis. The OD value of the extracted pigments was measured at a wavelength of 530 nm with Ultrospec 2100 Pro Spectrophotometer (Amersham Biosciences). Astaxanthin content was calculated based on the calibration curve equation of y = 0.0577x + 0.0131 (established with astaxanthin 98 % (HPLC), ≥ Sigma-Aldrich, St. Louis, MO, USA) (Figure4), in which y was the OD value measured and x was the 1 astaxanthin content (µg mL− ). Astaxanthin content in biomass was calculated in percentage (A%), 2 1 and subsequent estimation of astaxanthin production per biofilm surface (in mg m− d− ).

BiologyBiology 20192019, ,88, ,x 68 FOR PEER REVIEW 77 of of 14 14

Figure 4. Astaxanthin standard calibration curve for spectrophotometry.

2.6. Data Analysis The data were analyzed using Microsoft Excel 2016. Statistical analysis and resulting charts were made using R program version 3.4.2. The presented values were averaged value of more than three replicates with corresponding standard deviation. Figure 4. Astaxanthin standard calibration curve for spectrophotometry. Figure 4. Astaxanthin standard calibration curve for spectrophotometry. 3. Results 2.6. Data Analysis 3.1. Dry Biomass Growth andand AstaxanthinAstaxanthin AccumulationAccumulation ofof ImmobilizedImmobilized H. H. pluvialis pluvialis Cultivated Cultivated in in an an Angled The data were analyzed using Microsoft Excel 2016. Statistical analysis and resulting charts were TL-PhotobioreactorAngled TL-Photobioreactor at Different at Different Initial Biomass Initial Biomass Density Density made using R program version 3.4.2. The presented values were averaged value of more than three 2 replicatesAt an with initialinitial corresponding biomassbiomass densitydensity standard ofof 2.5,2.5, deviation. 55 andand 7.57.5 gg mm−−2, the biomass productivities were 5.6, 6 andand 2 1 6.2 gg mm−−2 dd−−1, ,respectivelyrespectively (Figure (Figure 5B).5B). These These values values were were not significantly significantly different di fferent from from each each other. other. 2 1 3.The Results highesthighest averageaverage drydry biomassbiomass productivityproductivity reachedreached 7.23 7.23 g g m m−−2 dd−−1 atat an initial biomass density of 2 1010 gg mm−−2 andand was was significantly significantly different different from the other three values (p < 0.05).0.05). 3.1. Dry Biomass Growth and Astaxanthin Accumulation of Immobilized H. pluvialis Cultivated in an Angled TL-Photobioreactor at Different Initial Biomass Density

At an initial biomass density of 2.5, 5 and 7.5 g m−2, the biomass productivities were 5.6, 6 and 6.2 g m−2 d−1, respectively (Figure 5B). These values were not significantly different from each other. The highest average dry biomass productivity reached 7.23 g m−2 d−1 at an initial biomass density of 10 g m−2 and was significantly different from the other three values (p < 0.05).

Figure 5. Final biomass and astaxanthin content in dry weight (%) in H. pluvialis at different initial biomassFigure 5. densities Final biomass after 10and days astaxanthin of growth content in a TL-BSBR. in dry weight (A) Final (%) biomass in H. pluvialis and astaxanthin at different content; initial (biomassB) Biomass densities growth after rate 10 (average days of of growth biomass in increasea TL-BSBR. in 10 (A days)) Final and biomass astaxanthin and astaxanthin productivity. content; (B) Biomass growth rate (average of biomass increase in 10 days) and astaxanthin productivity. Final astaxanthin content in the dry biomass reached the highest value of 2.17 % at initial biomass 2 2 densitiesFinal of astaxanthin 5 and 7.5 g content m− , respectively. in the dry However,biomass reached the astaxanthin the highest content value at lowerof 2.17 (2.5 % gat m initial− ) or 2 higherbiomass (10 densities g m− ) of initial 5 and biomass 7.5 g m densities−2, respectively. was lower However, (1.87 %)the andastaxanthin the difference content was at lower statistically (2.5 g significant (p < 0.05). Figure 5. Final biomass and astaxanthin content in dry2 weight (%) in H. pluvialis at different initial Except for low initial biomass densities (2.5 g m− ), astaxanthin productivities at initial biomass biomass densities after 10 days2 of growth in a TL-BSBR. (A) Final2 biomass and astaxanthin2 - content;1 densities of 5, 7.5 and 10 g m− were similar: 141 (5 g m− ), 151 and 154 mg.m− d − (at 7.5 and (B) Biomass2 growth rate (average of biomass increase in 10 days) and astaxanthin productivity. 10 g m− ). Final astaxanthin content in the dry biomass reached the highest value of 2.17 % at initial biomass densities of 5 and 7.5 g m−2, respectively. However, the astaxanthin content at lower (2.5 g

Biology 2019, 8, x FOR PEER REVIEW 8 of 14

m−2) or higher (10 g m−2) initial biomass densities was lower (1.87 %) and the difference was statistically significant (p < 0.05). Except for low initial biomass densities (2.5 g m−2), astaxanthin productivities at initial biomass Biology 2019, 8, 68 8 of 14 densities of 5, 7.5 and 10 g m−2 were similar: 141 (5 g m−2), 151 and 154 mg.m−2 d-−1 (at 7.5 and 10 g m−2).

3.2. Influence Inﬂuence of Algal Storage Time afterafter CentrifugationCentrifugation ofof aa SuspensionSuspension CultureCulture onon BiomassBiomass GrowthGrowth andand AstaxanthinAstaxanthin AccumulationAccumulation inin aa TL-PSBRTL-PSBR

2 After aa storagestorage time time of of 24 24 hours hours before before immobilization immobilization and and at an at initialan initial biomass biomass density density of 5 gof m 5− g, them−2,highest the highest value value of average of average dry dry biomass biomass productivity productivity after after 10 10 days days of of cultivation cultivation waswas achievedachieved −2 −11 −22 (8.75(8.75 gg mm− d−).). AtAt an an initial initial biomass biomass density density of 5 g m− , ,storage storage times times of of 72, 72, 120 120 and 168 hh yieldedyielded −22 −11 average productivityproductivity ofof 6.3,6.3, 1.71.7 andand 2.72.7 gg mm− d− , ,respectively. respectively. Biomass Biomass productivities productivities at storage time of 24,24, 7272 andand 120120 hh werewere significantlysignificantly didifferentfferent ((pp << 0.05)0.05) whilewhile productivitiesproductivities at storage times of 120 and 168 hh werewere notnot ((pp >> 0.05). Average astaxanthinastaxanthin content reached 2.15 % drydry biomassbiomass at storage time of 24 h andand anan initialinitial 2 density of 5 5 g.m g.m−−2. It. decreased It decreased with with increased increased time time storage storage until until 168 h 168 when h when it was it higher was higher again again(1.92, (1.92,1.68 and 1.68 2.30 and 2.30% at %72, at 72,120, 120, 168 168 h storage h storage time, time, respectively). respectively). However, However, there there was was no significantsignificant didifferencefference betweenbetween these these values. values. Storage Storage time time did did not not influence influence the the final final astaxanthin astaxanthin content content (% in(% dry in weight)dry weight) but onlybut only the final the biomassfinal biomass content content (Figure (Fig6A).ure Consequently, 6A). Consequently, the storage the timestorage of 24time h yielded of 24 h 2 1 theyielded highest the astaxanthinhighest astaxanthi productivityn productivity (205 mg m (205− d −mg) measured, m−2 d−1) measured, astaxanthin astaxant productivityhin productivity decreased significantlydecreased significantly at longer storage at longer times storage (Figure times6B). (Figure 6B).

Figure 6. Biomass and astaxanthin in H. pluvialis at different storage times of initial biomass after Figure 6. Biomass and astaxanthin in H. pluvialis at different storage times of initial biomass after centrifugation from suspension culture. (A) Final biomass and astaxanthin content; (B) Growth rate centrifugation from suspension culture. (A) Final biomass and astaxanthin content; (B) Growth rate (average of biomass increase in 10 days) and astaxanthin productivity. (average of biomass increase in 10 days) and astaxanthin productivity. 3.3. Influence of Light Intensity on Biomass Growth and Astaxanthin Accumulation of H. pluvialis in an Angled3.3. Influence TL-PSBR of Light Intensity on Biomass Growth and Astaxanthin Accumulation of H. pluvialis in an Angled TL-PSBR 2 1 The effect of different light intensities was studied in the range of 300–1000 µmol photons m− s− in 8 experiments.The effect of different light intensities was studied in the range of 300–1000 µmol photons m−2 2 1 s−1 inA 8 lightexperiments. intensity of 400 µmol photons m− s− yielded the highest average dry biomass productivity 2 1 2 1 2 1 (6.6 gA m −lightd− )intensity and 300 µ molof 400 photons µmol m −photonss− yielded m−2 thes−1 lowestyielded productivity the highest (5.56 average g m− d −dry). However,biomass 2 1 theproductivity productivity (6.6 valuesg m−2 d in−1) the and whole 300 µmol range photons of 300–1000 m−2 sµ−1mol yielded photons the lowest m− s− productivitywere not significantly (5.56 g m−2 did−ff1).erent However, (p > 0.05). the productivity values in the whole range of 300–1000 µmol photons m−2 s−1 were not 2 1 significantlyIntensities different of 500 and(p > 6000.05).µmol photons m− s− yielded highest astaxanthin content (2.56 %) and 2 1 700 µIntensitiesM photons of m 500− s and− yielded 600 µmol lowest photons value m (1.96−2 s−1 yielded %), the dihighestfference, astaxant however,hin content was not (2.56 statistically %) and significant700 µM photons (p > 0.05) m−2 (Figures−1 yielded7). lowest value (1.96 %), the difference, however, was not statistically significant (p > 0.05) (Figure 7). The highest average astaxanthin productivity reached 183 mg m-2 d-1 at 500 µmol photons m−2 s−1 but it was not significantly different from the value at other light intensities.

Biology 2019, 8, x FOR PEER REVIEW 9 of 14

Biology 2019, 8, 68 9 of 14 Biology 2019, 8, x FOR PEER REVIEW 9 of 14

Figure 7. Biomass productivity and astaxanthin in H. pluvialis under different values of light intensities in the range of 300–1000 µmol photons m−2 s−1. (A) Final biomass and astaxanthin content; (B) Biomass growth rate and astaxanthin productivity. Figure 7. Biomass productivity and astaxanthin in H. pluvialis under different values of light intensities Figure 7. Biomass productivity and astaxanthin2 1 in H. pluvialis under different values of light inIn thegeneral, range ofwith 300–1000 a totalµ molof 140 photons h of illumination m− s− .(A) Final in 10 biomass days, the and dry astaxanthin biomass content; productivity (B) Biomass per each molegrowthintensities of photons rate in and thedecreased astaxanthinrange of 300–1000with productivity. increased µmol photons light intensity, m−2 s−1. (A )therefore Final biomass the highestand astaxanthin light intensity content; only yielded(B) Biomassthe productivity growth rate of and0.12 astaxanthin g m−2 mol photonsproductivity.−1. The intensities of 300 and 400 µmol photons m−2 -2 -1 µ 2 1 s−1 yieldedThe highest 0.37 and average 0.33 astaxanthing m−2 mol photons productivity−1, respectively reached 183 (p mg> 0.05), m dwhichat 500 wasmol significantly photons m higher− s− In general, with a total of 140 h of illumination in 10 days, the dry biomass productivity per each butthan it at was the not other significantly light intensities different (p from< 0.05). the The value light at otherintensity light of intensities. 500 µmol photons m−2 s−1 yielded mole of photons decreased with increased light intensity, therefore the highest light intensity only 0.262In g general,m−2 mole with photons a total−1, ofhigher 140 h than of illumination the value of in 600–1000 10 days, µmol the dry photons biomass m− productivity2 s−1 (p < 0.05) per (Figure each yielded the productivity of 0.12 g m−2 mol photons−1. The intensities of 300 and 400 µmol photons m−2 mole8). of photons decreased with increased light intensity, therefore the highest light intensity only s−1 yielded 0.37 and 0.33 g m−2 mol photons2 −1, respectively1 (p > 0.05), which was significantlyµ higher yieldedAstaxanthin the productivity productivity of 0.12 per g meach− mol mole photons of photons− . The decreased intensities with of increased 300 and 400 lightmol intensity. photons At than2 at1 the other light intensities (p2 < 0.05). The light1 intensity of 500 µmol photons m−2 s−1 yielded mthe− intensitiess− yielded of 0.37 300, and 400 0.33and g500 m− µmolmol photons −m,−2 respectively s−1 the productivities (p > 0.05), were which 9.2, was 8.5, significantly 7.2 mg m−2 0.262 g m−2 mole photons−1, higher than the value of 600–1000 µmol photons mµ−2 s−1 (p < 0.05) (Figure2 1 highermole photons than at−1 the, respectively other light (p intensities > 0.05) (Figure (p < 0.05).8). These The values light intensity were significantly of 500 mol higher photons than mat− others− 8). 2 1 µ 2 1 yieldedintensities 0.262 (p g< m0.05).− mole The photons lowest −astaxanthin, higher than productivity the value of per 600–1000 mole of molphotons photons was m 3.5− mgs− (mp −<2 mole0.05) Astaxanthin productivity per each mole of photons decreased with increased light intensity. At (Figurephotons8).−1 at 1000 µmol photons m−2 s−1. the intensities of 300, 400 and 500 µmol photons m−2 s−1 the productivities were 9.2, 8.5, 7.2 mg m−2 mole photons−1, respectively (p > 0.05) (Figure 8). These values were significantly higher than at other intensities (p < 0.05). The lowest astaxanthin productivity per mole of photons was 3.5 mg m−2 mole photons−1 at 1000 µmol photons m−2 s−1.

Figure 8. Dry biomass productivity and astaxanthin content in H. pluvialis per mole of photons at Figure 8. Dry biomass productivity and astaxanthin content in H. pluvialis2 per1 mole of photons at different values of light intensity in the range of 300–1000 µmol photons m− s− . different values of light intensity in the range of 300–1000 µmol photons m−2 s−1. Astaxanthin productivity per each mole of photons decreased with increased light intensity. At the 2 1 2 2 intensities3.4. Influence of 300,of the 400 Light and Source 500 µ onmol H. photons pluvialis m Immobilized− s− the productivities Cultivation in werea 0.5 m 9.2,x 8.5,4 (Large 7.2 mg Scale) m− Twin-mole Figure1 8. Dry biomass productivity and astaxanthin content in H. pluvialis per mole of photons at photonsLayer Photobioreactor− , respectively (p > 0.05) (Figure8). These values were significantly higher than at other different values of light intensity in the range of 300–1000 µmol photons m−2 s−1. 2 intensitiesThis study (p < 0.05). investigated The lowest the astaxanthinillumination productivity efficiency of per two mole different of photons luminaires: was 3.5 luminair mg m− 250Wmole photons 1 at 1000 µmol photons m 2 s 1. 3.4.and Influenceluminair− of 400W. the Light The Source luminair on H. −400W pluvialis− yielded Immobilized average Cultivation dry biomass in a productivity 0.5 m2 x 4 (Large of 5.4 Scale) g m Twin-−2 d−1, significantly higher than the productivity under luminair 250W (4.8 g m−2 d−1) (p < 0.05). Astaxanthin 3.4.Layer Influence Photobioreactor of the Light Source on H. pluvialis Immobilized Cultivation in a 0.5 m2 x 4 (Large Scale) contents in biomass under luminair 250W and luminair 400W (3.34 and 3.13 %, respectively) were Twin-LayerThis study Photobioreactor investigated the illumination efficiency of two different luminaires: luminair 250W not significantly different (p > 0.05) (Figure 9). and luminair 400W. The luminair 400W yielded average dry biomass productivity of 5.4 g m−2 d−1, This study investigated the illumination efficiency of two different luminaires: luminair 250 W −2 −1 2 1 andsignificantly luminair higher 400 W. than The luminairthe productivity 400 Wyielded under luminair average dry250W biomass (4.8 g m productivity d ) (p < 0.05). of 5.4 Astaxanthin g m− d− , contents in biomass under luminair 250W and luminair 400W (3.34 and 3.13 %, respectively) were not significantly different (p > 0.05) (Figure 9).

Biology 2019, 8, 68 10 of 14

2 1 significantly higher than the productivity under luminair 250 W (4.8 g m− d− )(p < 0.05). Astaxanthin contents in biomass under luminair 250 W and luminair 400 W (3.34 and 3.13 %, respectively) were not significantlyBiology 2019, 8, x di FORfferent PEER (p REVIEW> 0.05) (Figure9). 10 of 14

Figure 9. Dry biomass productivity and astaxanthin content and productivity in H. pluvialis with twoFigure diff 9.erent Dry lightbiomass sources. productivity (A) Final and biomass astaxa andnthin astaxanthin content and content; productivity (B) Biomass in H. pluvialis growth with rate andtwo astaxanthindifferent light productivity. sources. (A) Final biomass and astaxanthin content; (B) Biomass growth rate and astaxanthin productivity. 2 1 Astaxanthin productivities using luminair 250 W and 400 W (179.4 and 180.8 mg m− d− , respectively)Astaxanthin were notproductivities significantly using different luminair (p > 0.05). 250W The and luminair 400W 400 (179.4 W yielded and higher180.8 mg dry biomassm−2 d−1, productivityrespectively) butwere lower notastaxanthin significantly content different than (p the > other;0.05). thereforeThe luminair astaxanthin 400W productivitiesyielded higher were dry similarbiomass under productivity both luminaires. but lower astaxanthin content than the other; therefore astaxanthin productivities were similar under both luminaires. 4. Discussion 4. DiscussionAt the lab scale, small-scale vertical TL systems were used to monitor growth kinetics and the influenceAt the of lab several scale, factors small-scale such as vert initialical density,TL systems light were intensity, used COto mo2 andnitor nutritent growth concentrationskinetics and the on theinfluence growth of and several accumulation factors such of astaxanthin as initial density, of H. pluvialis light intensity,in biofilms CO [218 and]. nutritent concentrations on theHere, growth we extendedand accumulation these studies of astaxanthin to an angled of H. TL pluvialis system in likely biofilms more [18]. suitable for applications in Vietnam.Here, we extended these studies to an angled TL system likely more suitable for applications in Vietnam. 4.1. Influence of Initial Biomass Density 4.1. InfluenceGrowth ofof HaematococcusInitial Biomass pluvialisDensity in a TL-PSBR under nutrient-replete conditions is characterized by twoGrowth different of processes:Haematococcus (1) growth pluvialis by cellin enlargementa TL-PSBR withoutunder cellnutrient-replete divisions (in akinetes),conditions and is (2)characterized growth by cellby multiplicationtwo different processes: (spore formation (1) growth in green by cell cells). enlargement Both processes without occur cell simultaneously divisions (in (andakinetes), over severaland (2) growth days with by similarcell multiplication biomass growth (spore rates) formation but in in di greenfferent cells). layers Both of theprocesses biofilm occur [23]. Insimultaneously the upper layers (and (that over are several exposed days to high with light similar intensities) biomass red growth akinetes rates) enlarge, but in whereas different in thelayers lower of layersthe biofilm green [23]. cells In divide the upper by spore layers formation (that are (in expose immobilizedd to high systemslight intensities) the spores red are akinetes non-flagellate). enlarge, Inwhereas suspension in the culture, lower layers the organization green cells divide of cell by layers spore similar formation to immobilized (in immobilized culture systems was also the used spores in aare small-scale non-flagellate). “double-layered” In suspension photobioreactor culture, the organization [10]. In that system,of cell cellslayers in similar the outer to jacketimmobilized region 2 1 wereculture exposed was also to excessiveused in airradiation small-scale (770 “double-layereµmol photonsd” mphotobioreactor− s− ) and accumulated [10]. In that astaxanthin system, cells in the in biomassthe outer to reachjacket 5.79region % of were the dry exposed weight. to Meanwhile, excessive withirradiation attenuated (770 light µmol energy photons (40 µ molm−2 photonss−1) and 2 1 maccumulated− s− ) and suastaxanthinfficient nutrients in the cellsbiomass in the to inner reac coreh 5.79 region % of continued the dry toweight. grow byMeanwhile, cell division with to provideattenuated the light inoculum energy for (40 the µmol next photons batch run m− [210 s−].1) and sufficient nutrients cells in the inner core region continuedAn inoculum to grow from by cell a growing division suspensionto provide the culture inoculum usually for consists the next of abatch majority run of[10]. flagellate cells. AfterAn inoculation inoculum on from a TL-PSBR a growing these suspension transform readilyculture intousually the palmellaconsists stage.of a majority Almost of 100% flagellate of the cells. cells shedAfter their inoculation flagella on during a TL-PSBR cell immobilization these transform and readily enhanced into acclimation the palmella to stage. high lightAlmost was 100% associated of the withcells thisshed stage their [26 flagella]. Cells during on the topcell ofimmobilization the biofilm then and accumulate enhanced astaxanthinacclimation and to high transform light intowas redassociated akinetes with that protectthis stage cells [26]. in the Cells lower on layers.the top If of the the initial biofilm biomass then (inoculum) accumulate density astaxanthin is very lowand 2 1 (andtransform the light into intensity red akinetes very high,that protect i.e., >600 cellsµmol in the photons lower m layers.− s− ), If reformation the initial biomass of green (inoculum) cells in the lowerdensity layers is very may low be (and delayed the light for days intensity until very astaxanthin high, i.e., accumulation >600 µmol photons in the upper m−2 s− layers1), reformation lowers the of green cells in the lower layers may be delayed for days until astaxanthin accumulation in the upper layers lowers the flux of blue photons to a level suitable for re-greening of akinetes [23,26]. It is, therefore, important to determine the minimum dry biomass (inoculum) density that allows the establishment of both red and green layers quickly after inoculation for a given light intensity. Research results on the vertical TL system determined the appropriate initial density for H. pluvialis

Biology 2019, 8, 68 11 of 14

flux of blue photons to a level suitable for re-greening of akinetes [23,26]. It is, therefore, important to determine the minimum dry biomass (inoculum) density that allows the establishment of both red and green layers quickly after inoculation for a given light intensity. Research results on the vertical TL Biology 2019, 8, x FOR PEER REVIEW 2 11 of 14 system determined the appropriate initial density for H. pluvialis to be 5 g m− and the final biomass of 213 g dry weight m-2 growth area after 16 days of cultivation. On angled TL-PSBRs in Vietnam, four to be 5 g m−2 and the final biomass of 213 g dry weight m-2 growth area after 16 days of cultivation. initial microalgae densities (2.5, 5, 7.5 and 10 g m 2) were tested for selecting the appropriate initial On angled TL-PSBRs in Vietnam, four initial microalgae− densities (2.5, 5, 7.5 and 10 g m−2) were tested density to increase biomass and astaxanthin of H. pluvialis simultaneously but with a 10-day culture for selecting the appropriate initial density to increase biomass and astaxanthin of H. pluvialis period to limit infection. simultaneously but with a 10-day culture period to limit infection. Here, we report that at a light intensity of 400–600 µmol photons m 2 s 1, initial biomass densities Here, we report that at a light intensity of 400–600 µmol photons m−−2 s−1, initial biomass densities of 5–10 g dry weight m 2 yielded astaxanthin productivities of 160–180 mg m 2 d 1. Such biofilms of 5–10 g dry weight m−−2 yielded astaxanthin productivities of 160–180 mg m−−2 d−−1. Such biofilms revealed both an upper red and a lower green layer of cells after six days of growth (Figure 10B and 10C). revealed both an upper red and a lower green layer of cells after six days of growth (Figure 10B and At low initial biomass densities (2.5 g m 2) the biofilm consisted exclusively of red cells (Figure 10A), 10C). At low initial biomass densities (2.5− g m−2) the biofilm consisted exclusively of red cells (Figure and astaxanthin productivities were significantly lower than at higher inoculum densities. Under 10A), and astaxanthin productivities were significantly lower than at higher inoculum densities. the conditions chosen, 5–7.5 g m 2 appears to be an optimal initial biomass density for astaxanthin Under the conditions chosen, 5–7.5− g m−2 appears to be an optimal initial biomass density for production in an angled TL-PSBR. The astaxanthin content in the dry biomass of this study (2–3 %) is astaxanthin production in an angled TL-PSBR. The astaxanthin content in the dry biomass of this in the same range as in many two-phase suspended cultivation systems but lower than the maximally study (2–3 %) is in the same range as in many two-phase suspended cultivation systems but lower reported contents in such systems [9,20,27,28]. This could be due to several factors including strain than the maximally reported contents in such systems [9,20,27,28]. This could be due to several factors type, lack of high-energy (blue) photons in the artificial light source used, mixture of red and green including strain type, lack of high-energy (blue) photons in the artificial light source used, mixture of cells, etc. red and green cells, etc.

Figure 10. TheThe H.H. pluvialis pluvialis biofilmbiofilm at at initial initial dry dry biomass biomass density density of of2.5 2.5 g m g–2 m (A–2),( A7.5), g 7.5 m g–2 m(B–2) and(B) 10 and g 10m–2 g (C m)–2 after(C) 6 after days 6 daysof cultivation. of cultivation. 4.2. Influence of Algal Storage Time 4.2. Influence of Algal Storage Time In the bench-scale vertical TL-PSBR setup of Kiperstok [18], if all 12 TL modules are operated In the bench-scale vertical TL-PSBR setup of Kiperstok [18], if all 12 TL modules are operated at at the same time, the total area of biofilms is about 12 24 2,5434 = 732.50 cm2 (each module has the same time, the total area of biofilms is about 12 × 24× × 2,5434× = 732.50 cm2 (each module has 24 24 plates, each plate has a diameter of 18 mm) so the amount of algae biomass to reach a density of plates, each plate has a diameter of 18 mm) so the amount of algae biomass to reach a density of 5 g 5 g m 2 will be only 0.366 g. This amount of biomass was easily collected by centrifuging the algal m−2 will− be only 0.366 g. This amount of biomass was easily collected by centrifuging the algal suspension for inoculation of the TL-PSBR. However, upon upscaling, the initial algae biomass needs suspension for inoculation of the TL-PSBR. However, upon upscaling, the initial algae biomass needs to be large (about 30 g if two large-scale angled TL-PSBRs are simultaneously operated with a total to be large (about 30 g if two large-scale angled TL-PSBRs are simultaneously operated with a total inoculation area of 4 m2 to reach a density of 7.5 g m 2) and storage of algal biomass before inoculation inoculation area of 4 m2 to reach a density of 7.5 g m−2) and storage of algal biomass before inoculation becomes an issue. Therefore, the effect of algal storage time after centrifugation on subsequent growth becomes an issue. Therefore, the effect of algal storage time after centrifugation on subsequent and astaxanthin accumulation of H. pluvialis in TL-PSBRs was investigated in this study. growth and astaxanthin accumulation of H. pluvialis in TL-PSBRs was investigated in this study. The most interesting result was obtained when the initial biomass was stored for 24 h at 4 C before The most interesting result was obtained when the initial biomass was stored for 24 ◦h at 4 °C inoculation of the TL-PSBR. This yielded higher biomass and astaxanthin productivities compared to before inoculation of the TL-PSBR. This yielded higher biomass and astaxanthin productivities initial biomass that had not been stored. It is well known that upon exposure to low temperature (4 C) compared to initial biomass that had not been stored. It is well known that upon exposure to low◦ Haematococcus predominantly exists in the non-motile palmella stage (e.g., as reported previously for temperature (4 °C) Haematococcus predominantly exists in the non-motile palmella stage (e.g., as an arctic strain by Klochkova et al. [29]). Non-flagellate green palmella stages of Haematococcus have reported previously for an arctic strain by Klochkova et al. [29]). Non-flagellate green palmella stages a stronger tolerance to photooxidative stress [30] and could be more resistant to cell immobilization of Haematococcus have a stronger tolerance to photooxidative stress [30] and could be more resistant and/or may transform more successfully into akinetes. Longer storage times (>24 h) of the initial to cell immobilization and/or may transform more successfully into akinetes. Longer storage times (>24 h) of the initial biomass, however, led to decreased biomass and astaxanthin productivities on TL-PSBRs presumably because of increased rates of cell death in the highly concentrated suspension.

4.3. Influence of Light Conditions and CO2 Supplement

Light intensities > 300 µmol photons m−2 s−1 increased dry biomass and astaxanthin accumulation of autotrophic H. pluvialis in TL-PSBRs in the presence of 5% (v/v) of CO2 added to the gas phase [23].

Biology 2019, 8, 68 12 of 14 biomass, however, led to decreased biomass and astaxanthin productivities on TL-PSBRs presumably because of increased rates of cell death in the highly concentrated suspension.

4.3. Influence of Light Conditions and CO2 Supplement 2 1 Light intensities > 300 µmol photons m− s− increased dry biomass and astaxanthin accumulation of autotrophic H. pluvialis in TL-PSBRs in the presence of 5% (v/v) of CO2 added to the gas phase [23]. The results obtained in this study using different light intensities with the angled TL-PSBR generally corroborated the results of Kiperstok [18]. The somewhat lower biomass productivities (6-8 g biomass 2 1 2 1 m− s− compared to 11-12 g biomass m− s− in Kiperstok et al. [23] under comparable conditions 2 1 (500 µmol photons m− s− and 6 days of growth)) obtained in this study could be related to one or several of the following factors: the application of CO2 to the culture medium instead of the gas phase, slower medium flow in the angled PSBR compared to a vertical setup, the different substrate and source materials, the frequency of culture medium exchange, different methods of inoculation of the biomass to the TL-PSBR. Similarly, the astaxanthin productivities in the angled TL-PSBR were slightly 2 1 2 1 lower (0.15–0.2 g m− d− compared to 0.2–0.3 g m− d− ) than those obtained by Kiperstok et al. in a vertical system [23]. However, astaxanthin productivity was higher than in most previous studies both in suspended and immobilized systems [7,9,19,20,27,28]. 2 1 Under a light intensity range of 300–1000 µ mol photons m− s− , the dry biomass productivity and astaxanthin accumulation were not significantly different between intensity points after 10 days of cultivation. This contrasts with the results of Kiperstok et al. [23], who showed that at 1000 µmol 2 1 2 2 photons m− s− , the total biomass content was ~130 g m− (compared to ~70 g m− in this study). This could be related to the higher CO2 concentrations used in Kiperstok et al. [23] and the different mode of CO2 application (in the gas phase vs. in the culture medium; see also discussion by Li et al. [31]). Further efforts should be made to optimize CO2 application to the algal biofilm. It should also be systematically investigated whether exchange of the culture medium with a nutrient (N and P)-depleted culture medium for the final 3 days of the 10 day cultivation period presents an advantage with respect to astaxanthin productivity compared to a longer (e.g., 16 day) cultivation period under nutrient-replete conditions (as in Kiperstok et al. [23]).

5. Conclusions In conclusion, this study has shown that H. pluvialis can be grown successfully in a horizontal/low-angled TL-PSBR with acceptable biomass and astaxanthin productivities. Storage time and thus the physiological status of the initial biomass (inoculum) appears to have a signiﬁcant eﬀect on later growth and astaxanthin productivity of H. pluvialis in the TL-PSBR. A larger-scale angled TL-PSBR was successfully tested for pilot-scale applications.

Author Contributions: Conceptualization, H.-D.T. and M.M.; methodology, T.-T.D. and H.-D.T.; formal analysis, T.-T.D.; investigation, T.-T.D and B.-N.O.; resources, M.M.; data curation, M.-L.N.-T. and T.-T.D.; writing—original draft preparation, T.-T.D. and T.-L.L.; writing—review and editing, H.-D.T., M.-L.N.-T. and M.M.; supervision, H.-D.T. and M.M.; project administration, H.-D.T.; funding acquisition, D.N. and H.-D.T. Funding: This research was funded by Ministry of Industry and Trade, grant number ĐT03.16/CNSHCB. Acknowledgments: We want to thank the Ministry of Industry and Trade for sponsoring this study, ĐT03.16/CNSHCB. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Biology 2019, 8, 68 13 of 14

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