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SN Applied Sciences

, 1:524 | Cite as

The influence of bio-optical properties of Emiliania huxleyi and Tetraselmis sp. on biomass and lipid production when exposed to different light spectra and intensities of an adjustable LED array and standard light sources

  • Tim GranataEmail author
  • Patrick Habermacher
  • Vinzenz Härri
  • Marcel Egli
Research Article
Part of the following topical collections:
  1. Earth and Environmental Sciences: Environment, Energy and Engineering Strategies for Sustainability (E3S)

Abstract

The effects of bio-optical characteristics on lipid and biomass production rates for Emiliania huxleyi and Tetraselmis sp. were studied under different irradiances and spectra. Biomass and lipid production increased from irradiances of 50–800 μE m−2 s−1 but differed for each spectrum. The highest biomass and neutral lipid production rates for E. huxleyi occurred under a red-blue LED array, which mimicked the absorption spectra of the cell’s chlorophyll a and accessory pigments, especially fucoxanthins. Biomass and neutral lipid production for Tetraselmis sp. were enhanced under broader spectra of cool white and grow lights. Neutral lipids per cell volume were similar for the two species and inversely proportional to chlorophyll a concentrations. High biomass and neutral lipid production rates were associated with high total quantum absorption and low quantum absorption per cell. Generally, quantum efficiencies were highest for high light treatments with high total quantum absorption and high production rates. Using reconstructed quantum absorption by pigments, the bio-optical model showed that photoprotective pigments were dependent on light intensity and spectra. Quantum efficiencies increased when excluding the contribution of photoprotective pigments to total quantum absorption since they did not directly contribute to photosynthesis, but did promote higher production rates.

Graphical abstract

Keywords

Bio-optical properties Spectral irradiance Biomass and lipid production 

Abbreviations

NPQ

Non-photochemical quenching

PAR

Photosynthetically active radiation

PPFD

Photosynthetic photon flux density

PPP

Photoprotective pigments

PSP

Photosynthetic pigments

PSI

Photosystem I

PSII

Photosystem II

Mathematics Subject Classification

92C99 

JEL Classification

Q42 

1 Introduction

Bio-optics of marine and freshwater phytoplankton have been well studied for spectral properties by aquatic ecologists [1, 2, 3, 4, 5], but few studies in the algal biofuel community have measured spectral characteristics of microalgae, e.g., Kandilian [6]. Yet bio-optics are important since the rate of photosynthesis depends on photon capture by algal cells. This rate can be parameterized for absorption of light by cell pigments and thus is directly regulated to biomass production [7] and efficiency [3].

Bio-optics are most pertinent to biofuel production where large cultures with relatively dense cell concentrations attenuate irradiance as a result of photon absorption by culture medium and by algal pigments. In the outer surface of the mass cultures, where light intensity is high, algae slowly decrease their antenna size (< 12 h) providing the timescales of mixing are sufficient [8]. A smaller antenna size is a result of a reduction in total chlorophyll and accessory pigment concentrations associated with the light reaction centers of photosystems I and II (PSI and PSII). The smaller antenna size causes cells to saturate at higher light intensities with higher biomass production rates [9].

The opposite happens to cells near the bottom of raceways and in the interiors of closed photoreactors. These cells acclimate to low light by producing more pigments relative to their reaction centers, thereby increasing their antenna size. A larger antenna allows cells to saturate at lower light intensities producing biomass at higher quantum efficiency. However, when these low light acclimated cells circulate into high light, they tend to photoinhibit on a very short timescale (seconds to minutes) as excess photon energy reduces photosynthesis and ultimately biomass production. Reduced production has been one of the major problems in mass cultures since cells are exposed to a high irradiance on the surface, but a low photosynthetic photon flux density (PPFD) deeper in the culture. Attenuation of light affects not only the quantum flux of photons but also their spectral distribution. The culture medium absorbs red wavelengths more than blue [10]; however, the spectral absorption by algae depends on the concentration of various pigments in their reaction centers and antenna [1].

Mutants were developed with smaller antenna sizes that efficiently used photons, enhancing productivities per pigment concentration over those of wild types containing more accessory pigments and reaction centers. Although production per antenna size improved using mutants, the production per cell remained about the same as [11, 12, 13] or lower than the wild types [14]. A more recent approach reduced the concentration of chlorophyll b per cell, thereby reducing the antenna size by approximately 50% [15]. While this about doubled production per unit chlorophyll, production per cell was about the same as the wild type.

Another way to achieve higher production rates is to select algal species that: (1) acclimate to high light without severe photoinhibition; (2) have small antenna sizes; and (3) can achieve high production rates. The coccolithophore Emiliania huxleyi can acclimate to high light environments and has high productivity [16], which recent investigations suggest are linked to the cell’s ability to adjust concentrations of accessory pigments [17, 18].

The spectral composition of light has been shown to interact with the spectral properties of accessory pigments to affect biomass production rate [18]. Different blue light spectra were found to give higher rates of primary production than white light for E. huxleyi [19] and the coccolithophore Isochrysis [20]. In contrast, green algae had enhanced production in red light compared to white and green lights [21]. Lipid production for green algae has been shown to be higher in blue light than in white [22], red [23], yellow, green, or purple light [24]. For the cyanobacteria Spirulina, growth in blue light was inferior to red, yellow, green, and white lights [25, 26], which might be expected since these cells possess phycobiliprotein pigments in their antenna that absorb light in longer wavelengths (500–650 nm). However, few of these studies measured the specific absorption or analyzed the bio-optical properties of the cells under these spectrally different light regimes.

In this study, we contrast the bio-optical properties of E. huxleyi, which does not readily photoinhibit, to those of the green algae Tetraselmis sp., a species used for many biofuel studies [27, 28, 29, 30, 31]. Both species produce a suite of photosynthetic pigments (PSP) and photoprotective pigments (PPP) that can exploit different wavelengths in the visible light spectrum. To determine how cells react to different light spectra, treatments were exposed to commercially available light sources and a novel light emitted diode (LED) array with different spectral peaks. Unlike studies that used only narrow band, monochromatic wavelengths, the light sources used here are broader band and therefore more finely tuned to the absorption spectra for antenna pigments. The differences in production rates and optical properties of the two species were parameterized in a bio-optical model and analyzed to determine how quantum efficiencies of biomass and lipid production were dependent on light intensities and spectra.

2 Methods

2.1 Experimental treatments

Parent cultures of E. huxleyi (No. 1210) and Tetraselmis sp. (No. 2604) were purchased from the Roscoff culture collection and grown at a photosynthetic photon flux density (PPFD) of 200 μE m−2 s−1 on a 14:10 light/dark cycle using GloPlus grow bulbs (Hagen T8). Cells were maintained on either on f/2 (E. huxleyi) or h/2 (Tetraselmis sp.) growth medium [32] at a salinity of 33 ppt in artificial seawater (Instant Ocean) [33] and the pH adjusted to 8.3 with 2000 μM Na2CO3. Treatments were run in duplicate in T-flasks (70 ml), inoculated with parent cultures, and filled with media to eliminate headspace. Parent cultures and experimental treatments were maintained in a cold room at 15 ± 1.5 °C.

Treatments were maintained as semi-continuous cultures by halving the volume every 7 days, while the cells were in the exponential growth phase, allowing cells to photoacclimate to the light conditions listed in Table 1. After treatments were photoacclimated, they were diluted to the same concentration and the experiments conducted over a 7-day period. Five different light sources with different spectra were studied: (1) grow lights (Glo); (2) white light (W); (3) blue LEDs (BBB); (4) BBB LEDs with low red (RBBB); and (5) BBB LEDs with high red (RRBBB). The light sources were fluorescent cool white light (Osram 940), fluorescent GloPlus light (Glo), and a variable spectrum, LED array. Spectra for the light sources and for algal absorption are shown in Fig. 1.
Table 1

Spectral characteristics of light sources arranged by increasing percent of blue wavelengths

Light sources

Blue 440–505 nm

% Red 650–680 nm

% Blue–Green–Yellow 505–650 nm

Power demand W

Comments

Sun1, noon cloudless

37

15

48

NOAA, lower atmosphere

Cool white

28

6

66

11403

Company specification

GloPlus

44

11

45

1704

Company specification

LED RRBBB2

77

17

6

21.3

This study

LED RBBB1

87

9

4

21.3

This study

LED BBB1

100

0

0

21.3

This study

1Sun irradiance is spectrally close to GloPlus and cool white light used in this study

2BBB, RBBB, and RRBBB indicated array had 0, 1, and 2 red diode strings plus 3 blue

31500 W based on 40 W per fluorescent bulb plus 20 W per ballast. In all 25 of each are needed for 800 PPFD

4272 W based on 24 W per GloPlus bulb + 10 W per ballast. Eight bulbs and ballasts are needed for 800 PPFD

Fig. 1

Spectra of specific absorption (m2 mg−1) for: a Tetraselmis sp. pigments and b E. huxleyi pigments. c Spectral irradiance of sunlight compared to the light sources used in this study: GloPlus light, cool white light, and LEDs configured for high red and blue (RRBBB), low red and high blue (RBBB), and blue only (BBB). Solar spectral irradiance is only shown as a reference to light sources but was not used in this study. All irradiances were scaled to an integral PAR value of 800 μE m−2 s−1. The peaks in cool white and GloPlus lights are due to the mercury arc of the bulbs. The color scale bar shows the relationship to the quantum visible spectrum

The LED array was adjustable to three different spectra: three blue diode strings for a blue light spectrum (BBB), one red and three blue diodes strings for a blue and low red spectrum (RBBB), and two red and three blue strings (RRBBB). The RBBB and RRBBB spectra were tuned to match the absorption spectrum for chlorophylls and accessory pigments of E. huxleyi, and, to some extent, of Tetraselmis sp.

The GloPlus (grow light, Glo) has peak emissions in the blue, green, and yellow-red bands. Cool white light had a narrow peak in the blue, broader peaks in green to yellow bands, and a temperature rating of 4100 K, which is close to the color of solar irradiance of 5100 K. In comparison with these light sources, solar irradiance is relatively flat with a slight decrease from blue to red wavelengths. Overall, percent blue was higher for the LED array than for other light sources, which complemented the algal absorption spectra for these two species (Table 1).

Treatments were maintained at nominal irradiances of 0, 50, 100, 200, 400 and 800 μE m−2 s−1 (photosynthetic photon flux density, PPFD) to determine production–irradiance (P − I) curves for each spectrum. These represent a range from undersaturated (0–100 PPFD) to near or saturated (200–800 PPDF) intensities. Light intensity was measured as photosynthetically active radiation (QPAR) using a Apogee quantum meter, which integrated visible wavelengths from 400 to 700 nm. Daily cell counts were done on a Zeiss light microscope, and specific growth rate was calculated as μ = ln(N2/N1)/(t2 − t1) where Nt corresponds to cell number at times t2 and t1.

For treatments at 800, 400, 200, and 50 PFFD, neutral lipid content per cell was determined by staining cells with BODIPY [31] and measuring fluorescence at 533 nm on the flow cytometer (BD Accura C6), which, like Nile Red, gives a florescent signal proportional to the mass of neutral lipids [34]. No correction was made to the neutral lipid fluorescence since the background for unstained cells was 103 orders of magnitude lower. Simultaneously, chlorophyll a fluorescence per cell at 675 nm, cell concentration, and forward scatter were also determined using the flow cytometer. Neutral lipid fluorescence was plotted against chlorophyll a fluorescence and cells gated to ensure only algal cells were sampled. Forward scatter height was calibrated to seven sizes of polystyrene microbeads with diameters from 2 to 15 μm (Spherotech Inc).

At the end of each experiment, one of the replicates was dried to determine biomass, and the second replicate was extracted for pigments. For dry weight biomass, treatment volumes of 60 ml were filtered onto pre-dried, pre-weighted GF/C filters using a Millipore filter apparatus and vacuum pump (45 mm Hg). Filtered cells were washed 3 times with DI water to remove salts and each time the vacuum was disconnected so cell would not be damaged. Filters were then dried at 105 °C for 2 h and cooled in a desiccator until a constant weight was attained. The dried biomass was also used to determine neutral lipids as FAMEs, by first freezing filters at − 20 °C, solubilizing biomass in 0.2 ml chloroform/methanol (2:1 v/v) and immediately adding 0.3 ml methylated HCL (Sigma) to complete transesterification [35]. However, the FAME procedure did not give consistent results for standards and samples, even though the water content was 0%. Consequently, neutral lipids are only presented as a relative fluorescence per cell. Fluorescent microscopic images of stained cells showed that neutral lipids were present in lipid bodies.

Pigments were extracted from a sample volume of 50 ml that was filtered onto GF/C at 0.5 atm with 4 drops of MgCO3 added to de-acidify the filters. Filters were placed in 15-ml Falcon tubes with 6 ml of 90% ethanol, vortexed for 5 s, then placed in ultrasonic cold water bath (35 kHz) for 15 min. Pigments were left to extract in the dark overnight in a 5 °C refrigerator, after which 1.5 ml of the well mixed extract was transferred to HPLC vials and immediately processed following the SCOR-UNESCO [36] protocol. Pigments were analyzed on a HPLC Pump PU 2089plus (Jasco System) equipped with an AS2055Plus Autosampler, CO2060Plus column oven, and a MD 2018Plus diode array detector with a resolution from 300 to 900 nm. The column was a stationary phase C18 column (Merck, LiChrospher 100 RP-18/5 μm/250-4). The column temperature was maintained at 30 °C. The starting eluent was 35.0% methanol, 45.0% ethyl acetate, and 20.0% deionized water; the final effluent was 55.0% methanol, 45.0% ethyl acetate, 0% water. The flow rate was 1 ml min−1 and the run time 35 min per sample. Spectral information was consistent with a library of chlorophyll and carotenoid spectra. Pigments were calibrated to standards from DHI (Denmark). The spectral distribution of each pigment, from 400 to 700 nm, was determined by normalizing its spectral area to 1.0 and multiplying the pigment concentration (mg m−3). Pigment absorption, ai (λ), was calculated by multiplying the weight-specific absorption of each pigment (Fig. 1a, b) by the normalized spectral pigment distribution [2, 37].

2.2 The light emitting diode (LED) array

The LED array was designed and built at the Lucerne University of Applied Science and Arts to match the absorption chlorophyll a spectra of E. huxleyi and Tetraselmis sp. with peaks in the red and blue wavelengths [38]. The LED array had to meet not only the spectral requirements, but also requirements for irradiance intensity, energy consumption, and heat emission, to ensure the successful cultivation of the algae.

The irradiance intensity requirements were 0–1600 µE m−2 s−1 (0–391 Wm−2) for an array with a small, 0.03 m2, footprint (20 cm × 15 cm). The power of the array was calculated as 36 W (3 × 391 Wm−2 × 0.03 m2), where the factor 3 accounted for 1/3 of the irradiance at a distance of 10 cm from the array. The online tool Phillips Lumileds was used to specify the number and type of LED diodes for a nominal current of 500 mA. Using the tool LED Spectrum Mixer, which mixes light of different LEDs with given luminous flux to determine the radiated power, the radiated power was iterated to give the luminous flux of the respective waveband. Given the emission of the single LED and the overall emission for each waveband, it was possible to calculate the required type of LED. Based on the algal action spectra (Fig. 1a, b), the blue peak was specified between 420 and 490 nm; therefore, two LED diodes were necessary, blue and royal blue, which accounted for 40% of the radiated power. For the smaller red peak at 670 nm, a deep red LED diode was ideal and accounted for 20% of the radiated power.

The exact color composition was a mix of the three-color diodes, blue, royal blue, and deep red, in a energy ratio of 2:2:1. To meet the specified spectrum and irradiance level at the calculated power output, the array required 20 blue, 20 royal blue, and 10 deep red diodes for a total of 50 LEDs. The 50 LEDs were arranged in 5 strings (rows), 2 red/blue, and 3 blue. Each string could be controlled to reduce the PPFD for any combination of red and blue light. By adjusting the dash pods for spectral quality and the wattage to maintain constant irradiance, light treatments were produced for blue light (3 BBB strings), low red with blue (RBBB strings), and equal red and blue (RRBBB strings).

The final design requirement was to reduce the waste heat given off by the LED array, which was important for two reasons: (1) to prevent algae from being heated by the array so that cultures could be kept at the optimum temperature for growth and (2) to achieve the rated irradiance produced by the LEDs, which is extremely reduced at high operating temperatures. To achieve these criteria, diodes rather than resistors were used, drastically reducing thermal resistance of the board. An aluminum heat dissipater was mounted to the circuit board as a heat sink. The heat dissipater had a thermal resistance of 0.08 K W−1, and its maximum temperature was calculated assuming an ambient temperature of 15 °C and maximum power dissipation for 50 LEDs at 69.5 W (40 × 2.95 V × 0.5 A and 10 × 2.1 V × 0.5 A). The thermal resistance of the thermally conducting tape was 0.01 K W−1, giving a maximum heat dissipater temperature of 21.3 °C (15 °C + [0.01 K W−1 + 0.08 K W−1] × 69.5 W). Using the casing temperature, as well as the thermal resistance of the array, both of which influenced the heating loss of the LEDs, the temperature of the LEDs was calculated. The red LED dissipation loss was 1.5 W and 8 K W−1, giving a maximum operating temperature of 29.7 °C (21.3 K W−1 + [1.05 W × 8 K W−1]). The blue LED loss was 1.45 W and 10 K W−1, resulting in a maximum operating temperature of 35.8 °C (21.3 K/W + [1.45 W × 10 K W−1)]. However, the array was never operated at maximum irradiance, so these temperature maxima were not reached. When the array was tested in a 15 ± 1.5 °C cold room at irradiances from 200 to 1600 μE m−2 s−1, the temperature in 60-ml cultures at a distance of 10 cm from the array was elevated < 0.5 °C.

2.3 Bio-optical model

To compare the effects of light spectra on biomass and lipid production for each light intensity, the bio-optical spectral model of Bidigare et al. [3] was used, where the integrated, total quanta absorbed by algae for each light source was defined as:
$$AQ_{ph} = \mathop \int \limits_{400}^{700} Q_{\text{PAR }} \left( \lambda \right) a_{ph} \left( \lambda \right){\text{d}}\lambda$$
(1)
Spectral irradiance, QPAR (λ), for each light source and intensity was interpolated to 2 nm bins. Spectral algal absorption, aph (λ), was measured in visible wavelengths from 400 to 700 nm and resolved to 1 nm on a Cary 100 scanning spectrophotometer. The cuvette path length was 1 cm, and a blank consisting of the medium (i.e. f/2 or h/2), was subtracted from algal absorption. Absorption spectra were also reconstructed from HPLC analyses by summing the specific pigment absorbance multiplied by the pigment concentration [2]:
$$a_{ph } \left( \lambda \right) = \mathop \sum \limits_{i}^{n} a_{i} \left( \lambda \right)C_{i}$$
(2)
To access quantum absorption of the different pigment groups, the carotene pigments containing oxygen (also called xanthophylls) were grouped, either as photoprotective pigments (PPP) or photosynthetic pigments (PSP). The xanthophylls antheraxanthin and beta carotene in Tetraselmis sp. are efficient at quenching fluorescence [39] and hence were grouped as PPP. The xanthophylls lutein, neoxanthin, and violaxanthin all contribute to photosynthesis in Tetraselmis sp. and were grouped with the chlorophyll pigments as PSP. The PPP xanthophylls in E. huxleyi were beta carotene and diatoxanthin, while PSP were chlorophylls, fucoxanthins, and diadinoxanthin. AQPSP and AQPPP represent the quantum absorption by PSP and PPP, respectively, and were calculated by substituting aPSP and aPPP for aph, in Eq. 1.
Biomass production was defined as PB = Bμ, and production data were fitted to the Platt–Jassby model [40]:
$$P_{B} = P_{ \hbox{max} } { \tan }h\left( {\frac{{Q_{\text{PAR}} }}{{I_{k} }}} \right)$$
(3)
where Pmax is the maximum production based on the P − I curve, α is the light-limiting photosynthetic rate measured as the initial slope of a linear regression of the P − I curve, and Ik is the half-saturating constant of light determined as Pmax α−1.
Quantum efficiency of biomass and neutral lipid production of E. huxleyi and Tetraselmis sp. for different light sources and irradiances was parameterized following Bidigare et al. [3] as:
$$\phi_{B} = \frac{{P_{ \hbox{max} } { \tan }h\left( {\frac{{Q_{\text{PAR}} }}{{I_{k} }}} \right)}}{{AQ_{ph} }}$$
(4)
ϕB is proportional to moles carbon fixed per Einstein (moles C Ein−1). Similarly, the efficiency of neutral lipid production was defined as:
$$\phi_{\text{NL}} = \frac{{\left( {\text{NL }} \right)P_{ \hbox{max} } { \tan }h\left( {\frac{{Q_{\text{PAR}} }}{{I_{k} }}} \right) }}{{AQ_{ph} }}$$
(5)
where NL is neutral lipid fluorescence per cell, standardized by the maximum fluorescence to give a relative efficiency.

2.4 Statistical procedures

Linear and nonlinear regressions were preformed using Synergy Software (KaleidaGraph v4.5.3). Analysis of Variance (ANOVA), Chi-squared tests, Tukey’s tests, and Student’s t tests were performed at α = 0.05 or 0.001 significance levels using SPSS v25 software. Chi-squared tests were used to determine normality of distributions. ANOVA data were grouped into species (2), light intensities (3), and light sources (4) with replicates for each treatment to determine overall differences within and among variables. Post hoc Tukey’s tests discriminated which groups differed. For each species, mean and standard deviations for production rates, cell counts, chlorophyll a fluorescence, and neutral lipid fluorescence were calculated from two replicate samples for each treatment. For each replicate, flow cytometer measurements of cell size, neutral lipid fluorescence, and chlorophyll a fluorescence were based on 104 cell counts. At the end of the experiment, one replicate was filtered for dry weight and lipid analysis and the second replicate was used for HPLC analysis of pigments. Thus, pigment data from extracts were based solely on un-replicated treatments. Replicate treatments of chlorophyll a fluorescence measurements, however, had low variability; therefore, it was assumed the extracted pigments were representative of the treatments. Additionally, for ANOVAs, the pooled treatment groups of extracted pigments increased the N and degrees of freedom for comparisons of light intensity and spectra.

3 Results

3.1 Biomass production

Biomass production for both species was highly dependent on the intensity of the light source and its spectral composition, which was characterized by the percent blue, red, and blue-green-yellow wavebands (see Table 1). The highest biomass production rates for both species were at 800 PPFD (Fig. 2a, b), with E. huxleyi achieving over 500 mg L−1 d−1, and Tetraselmis sp. just over 400 mg L−1 d−1. Maximum production rates of Tetraselmis sp. for the 5 light sources decreased linearly with the percent of blue light with BBB < RRBBB < RBBB < Glo < white (Fig. 2a insert). Pmax was also directly correlated with the percent of blue-green-yellow light, but had no correlation to percent red light (Supplement 1a). The maximum biomass production for E. huxleyi was highly nonlinearly related to the light source with BBB < white < RBBB < Glo < RRBBB (Fig. 2b insert). However, Pmax was directly correlated with the percent of red light (Supplement 1b). Generally, Tetraselmis sp. production rates were linear until about 200 PPFD, indicating cells were light saturated at irradiances ≥ 200 PPFD. In contrast, most production rates for E. huxleyi were only saturated at higher light levels (≥ 400 PPFD).
Fig. 2

Biomass production versus QPAR irradiance. a Tetraselmis sp. and b E. huxleyi. Curve fit to biomass is the hyperbolic tangent model of Platt and Jassby [40]. The inset for each species shows the maximum production rate as a function of the percent blue light from each light source. Means ± 2 standard deviations (N = 2)

Parameters for the bio-optical model derived from the P − I curves showed that BBB treatments had the lowest α values for both species (Table 2). Glo and white light gave the highest α values for Tetraselmis sp. compared to RRBBB for E. huxleyi. The average α values for Tetraselmis sp. decreased with the percent of blue light (Supplement 2a), reflecting the same trend as Pmax (Fig. 2a insert), though this is not surprising since α is directly proportional to Pmax and inversely related to Ik. Similarly, α values for E. huxleyi followed the same nonlinear trend as Pmax (Supplement 2a). The average Ik for Tetraselmis sp. (482 PPFD) was lower than for E. huxleyi (540 PPFD).
Table 2

Parameters for bio-optical model of reconstructed cell absorption

 

P max

Intercept, b1

Slope, α1

R value2

I k

P error 3

mg L−1 d−1

(mg L−1 d−1)(μ E m−2 s−1)−1

μE m−2 s−1

σ 2

Tetraselmis sp.

Glo

428 (25.0)

18.8

1.03

0.96

416

0.30

White

271 (17.4)

− 9.33

0.66

0.95

410

0.84

BBB

120 (28.0)

7.77

0.21

0.95

579

0.35

RRBBB

176 (20.5)

− 5.62

0.36

0.94

482

1.8

E. huxleyi

      

Glo

173 (38.0)

− 14

0.28

0.83

616

4.6

White

300 (49.1)

− 11.6

0.52

0.95

578

1.0

BBB

135 (26.0)

3.88

0.28

0.95

482

1.6

RRBBB

505 (60.1)

− 3.68

0.94

0.96

537

0.14

Mean Pmax (standard deviations, N = 2)

1Linear regressions of P − I curves. P = b + αQPAR for 0 ≤ QPAR ≤ 200 μE m−2 s−1 to determine α and Ik

2Regression coefficients for linear fits of α, where p < 0.0001 for all treatments except E. huxleyi Glo, where p = 0.001

3Residual error: P − I curve minus measured biomass production as variance over QPAR

3.2 Pigments, neutral lipids, and cell size

Chlorophyll a concentrations per cell for Tetraselmis sp. were twice as high as E. huxleyi concentrations (Fig. 3a, b). For both species, there was a negative trend between chlorophyll and irradiance. Chlorophyll a per cell decreased with increasing irradiance, although there was variability for the 200 PPFD treatments (Fig. 3a, b). The opposite trend was found for lipid fluorescence per cell, which increased with increasing irradiance (Fig. 3c, d).
Fig. 3

Chlorophyll a concentration per cell and neutral lipid fluorescence per cell for 3 irradiances. a Tetraselmis sp. chlorophyll a, b E. huxleyi chlorophyll a, c Tetraselmis sp. neutral lipids, and d E. huxleyi neutral lipids. Means ± 2 standard deviations (N = 2). Colors same as Fig. 2

Cell diameters increased with increasing irradiance (Fig. 4a) for both Tetraselmis sp. (df = 14, F = 17.804, p < 0.001) and E. huxleyi (df = 11, F = 15.729, p = 0.001), which was not a result of growth cycles since the growth phase was synchronized by the light/dark cycle. Only E. huxleyi under BBB light had the same diameters at all irradiances. No size dependence was found for spectral irradiance. The maximum volume for E. huxleyi was 247 μm3 compared to the larger Tetraselmis sp. cells, which had a volume of 397 μm3. Neutral lipid fluorescence also increased with cell size (Fig. 4b). Müller et al. [41] showed cell size decreased with light and nitrate limitation but increased with phosphate limitation. Since our experiments had 25 times more N and P than those of Müller et al., we suggest the changes in the cells size in our treatments were a result of light treatments.
Fig. 4

Average cell size as a function of QPAR irradiance for: a Tetraselmis sp. and E. huxleyi. Neutral lipid fluorescence per cell as a function of: b QPAR and c chlorophyll a concentration per cell; and d neutral lipid fluorescence per cell volume versus chlorophyll a concentration per cell volume. Means ± 2 standard deviations (N = 2)

There was a negative log–log trend between chlorophyll a concentration per cell and neutral lipids per cell (Fig. 4c) for Tetraselmis sp. (log neutral lipids = − 1.29 log chlorophyll a + log 9.24 × 10−3, R2 = 0.69) and E. huxleyi (log neutral lipids = − 0.41 log chlorophyll a + log 12,341, R2 = 0.60); however, the slopes were significantly different (N = 15, |t| = 3.02, p = 0.005). When lipid concentrations were normalized by cell volume (Fig. 4d), the slopes for Tetraselmis sp. and E. huxleyi were only significantly different at the 0.05 level (R2 = 0.41, N = 15, |t| = 2.245, p = 0.034). Although there was considerable scatter in the data, the log slope of − 0.411 showed for every 1000-fold reduction in chlorophyll a there was a 2.5-fold increase in neutral lipids.

Increases in chlorophyll a concentrations at lower light levels were accompanied by increases in concentrations of certain accessory pigments (Fig. 5a, b). For Tetraselmis sp., the accessory pigments lutein and chlorophyll b had the highest concentrations and their concentrations increased with chlorophyll a levels (Fig. 5a). For E. huxleyi, the accessory pigments chlorophyll c and fucoxanthins had the highest levels and increased with chlorophyll a concentrations (Fig. 5b). Along with lutein, other carotenoids found in Tetraselmis sp. cells were beta carotene, neoxanthin, violaxanthin, and antheraxanthin, all in low concentrations. Generally, at high light levels, beta carotene, neoxanthin, and violaxanthin decreased in concentration, while antheraxanthin increased (Fig. 5c). Beta carotene, diadinoxanthin, and diatoxanthin were found in low concentrations in E. huxleyi, and the major carotenoid was 19’hexanoyloxyfucoxanthin. The amounts of these carotenoids varied with light levels, where high light caused fucoxanthins, diadinoxanthin, and beta carotene concentrations to decrease and diatoxanthin to increase (Fig. 5d).
Fig. 5

Accessory pigments. a Tetraselmis sp. accessory pigments as a function of chlorophyll a, b E. huxleyi accessory pigments as a function of chlorophyll a, c Tetraselmis sp. accessory pigments as a function of light sources, and d E. huxleyi accessory pigments as a function of light sources for one sample per treatment

3.3 Bio-optics

The bio-optical effects, on a per cell basis and for total biomass, were accessed for quantum absorption (AQph), which is aph, the sum of the spectrally weighted pigment absorption, multiplied by QPAR (λ), the spectral light intensity of the light source (Eq. 1). On cellular basis, AQPSP and AQPPP per cell (i.e., normalized by biomass) increased as light level decreased (Fig. 6a, b); however, this was only significant between the 50 PPFD and 800 PPFD treatments for AQPSP (p ≤ 0.012) and AQPPP (p ≤ 0.034). AQPSP per cell was always higher than AQPPP per cell, since the photosynthetic pigments were more concentrated in the cells than the PPP xanthophylls. The opposite trend occurred in AQph, AQPSP, and AQPPP for total quanta absorbed by the biomass. Biomass had the highest total quantum absorption at the highest light levels, and least at the lowest for each light intensity and spectrum (Table 3). For Tetraselmis sp., the major absorption was by the PSP system, especially for the three Glo treatments, where AQPSP absorbed 77–96% of the total quanta. In contrast, AQPPP only contributed 4–23% (Table 3). For E. huxleyi, the percentages of AQPSP and AQPPP were quite different and AQPPP played a more important role, especially for the RRBBB treatment. An ANOVA substantiated a significant difference in AQPSP versus AQPPP for Tetraselmis sp. (df = 46, |t| = 4.022, p < 0.001). E. huxleyi, however, showed no statistical difference in AQPSP and AQPPP (p = 0.689). Thus, AQPPP contributed to more photon absorption for E. huxleyi, but contributed less to Tetraselmis sp. (Table 3). This was substantiated by taking the ratio of AQPPP to AQPSP, which was significantly different for the two species (df = 23, F = 7.889, p = 0.012).
Fig. 6

Quantum light absorption per cell for 3 irradiances levels and the 4 light sources. a Tetraselmis sp. and b E. huxleyi, where PSP are photosynthetic pigments and PPP are photoprotective pigments for one sample per treatment

Table 3

Results of the bio-optical model for total biomass

Treatment

PPFD

AQ ph 1

AQ PSP 1

% Error2

ϕ B

ϕ B-PSP 3

ϕ NL 4

ϕ NL-PSP 3,4

μE m−2 s−1

μE m−3 s−1

 

moles C E−1

Relative

Units

Tetraselmis sp.

Glo

50

1.01

0.777

22.8

0.005

0.007

0.0021

0.003

 

200

4.03

3.64

9.9

0.023

0.026

0.0099

0.011

 

800

18.3

17.6

4.1

0.011

0.012

0.0097

0.010

White

50

0.673

0.553

17.8

0.005

0.006

0.0019

0.002

 

200

2.14

1.47

31.1

0.029

0.042

0.0141

0.020

 

800

2.23

1.61

27.9

0.057

0.080

0.0450

0.062

BBB

50

4.58

2.70

41.0

0.001

0.002

0.0006

0.001

 

200

4.79

3.32

30.7

0.004

0.006

0.0029

0.004

 

800

4.73

3.51

25.7

0.012

0.016

0.0117

0.016

RRBBB

50

1.35

0.840

37.9

0.005

0.008

0.0022

0.004

 

200

6.20

4.32

30.3

0.006

0.008

0.0034

0.005

 

800

21.7

12.8

41.2

0.004

0.006

0.0037

0.006

E. huxleyi

Glo

50

0.537

0.489

8.8

0.037

0.041

0.001

0.001

 

200

1.97

1.49

24.2

0.017

0.023

0.001

0.001

 

800

42.5

24.7

42.0

0.002

0.003

0.001

0.001

White

50

0.594

0.378

36.3

0.007

0.011

0.000

0.001

 

200

0.658

4.13

37.2

0.008

0.012

0.014

0.022

 

800

8.74

6.04

30.9

0.016

0.024

0.005

0.007

BBB

50

3.57

1.47

58.8

0.002

0.006

0.000

0.000

 

200

2.82

1.23

56.5

0.010

0.024

0.001

0.002

 

800

7.84

3.98

49.2

0.008

0.016

0.002

0.004

RRBBB

50

0.336

0.252

25.0

0.042

0.056

0.005

0.007

 

200

2.39

1.24

48.3

0.035

0.068

0.004

0.009

 

800

5.48

2.04

62.8

0.044

0.119

0.044

0.119

1AQph and AQPSP for reconstruction photosynthetic pigment absorption of total biomass

2The percent PSP to total pigments, which represents the contribution of PPP pigments

3Efficiency calculated without the contribution of PPP

4Relative lipid production efficiency derived by normalizing by the maximum lipid fluorescence

Spectral algal absorption per cell, aph, was higher for Tetraselmis sp. (Fig. 7) than for E. huxleyi (Fig. 8) given the larger cell size of Tetraselmis sp. and thus its larger cross-sectional area. For both species, cell absorption was greatest at 50 PPFD and least at 800 PPFD, which corresponded to the highest pigment concentrations at the lowest light levels and reduced cellular pigment concentrations at the highest light levels (also see Fig. 5). The two highest absorption peaks occurred in the blue wavebands, caused by the combination of chlorophyll and carotenoid pigments while smaller peaks in the orange-red were only a result of chlorophylls a, b, and c. Nearly 77% of absorption peaks in the blue wavebands of Tetraselmis sp. (Fig. 7) were accounted for by photosynthetic pigment concentrations and only 23% by the photoprotective pigment concentrations (i.e., antheraxanthin and beta carotene). These pigment concentrations resulted in 91% of the blue light absorption by aPSP in high light and even higher absorption per cell in low light. E. huxleyi followed the same pattern with high absorption in the blue wavebands (Fig. 8), a result of high photosynthetic pigment concentrations, which contributed to 90% of the aPSP absorption per cell. In low light, aPPP for Tetraslmis increased as a result of increased beta carotene absorption, even though antheraxanthin decreased when it was epoxidized to violaxanthin. For E. huxleyi in low light, aPPP also increased with increasing beta carotene levels, despite the reduction of diatoxanthin, which was converted to diadinoxanthin.
Fig. 7

Tetraselmis sp. spectral absorbance per cell, aph, for the 4 light sources. a Glo, b white, c BBB LEDs, and d RRBBB LEDs. aph was measured by spectrometry and may include pigments other than those in Fig. 5

Fig. 8

E. huxleyi spectral absorbance per cell, aph, for 4 light sources. a Glo, b white, c BBB LEDs, and d RRBBB LEDs. aph was measured by spectrometry and may include pigments other than those in Fig. 5

Quantum efficiencies of biomass production were very low for all light sources and intensities with an average of 1.4% for Tetraselmis sp. and 1.9% for E. huxleyi. The exceptions were for Tetraselmis sp. biomass in the Glo 800 PPFD treatment with up to 5.8% efficiency and E. huxleyi biomass in the RRBBB 800 PPFD treatment with up to 4.4% efficiency. Although photoprotective pigments contribute to overall absorption of light by the cells, they do not directly contribute to the biomass production. Consequently, their contribution was removed by substituting AQPSP for AQph, in Eq. 4. This resulted in high biomass efficiency. The highest biomass efficiency of E. huxleyi was for the RRBBB 800 PPFD treatment, which ranged from 5.6 to 11.9%, near the theoretical maximum efficiency of 12.5% Bidigare et al. [3]. For all treatments, except Glo 50 PPFD for E. huxleyi, efficiencies for neutral lipid production were highest in the high light regimes. For Tetraselmis sp., the highest lipid production efficiency was 4.5% in the white light treatment; however, the efficiency increased to 6.2% when substituting AQPSP in Eq. 5. Neutral lipid production for E. huxleyi was highest in the RRBBB treatment at 4.4%, but increased to 11.9% using AQPSP to calculate efficiency.

The differences in the antenna pigments of the two species played a major role in the differences in AQph, AQPSP, and AQPPP for each light regime. Of all the pigments in Tetraselmis sp. cells, only beta carotene absorbed highest in the blue blue-green wavelengths (i.e., up to 500 nm). For E. huxleyi, all pigments, except for chlorophyll a, strongly absorbed into the blue-green band and fucoxanthins even higher, up to 530 nm in the green wavebands. Yet, there were no significant correlations between neutral lipid efficiencies and AQph, AQPSP or AQPPP, either on a per cell basis or per surface area (not shown). However, when near to saturating light levels (i.e., 200 and 800 PPFD) were compared, the trend in AQPPP for E. huxleyi showed quantum absorption increased with increasing blue light (Fig. 9a) and decreased with increasing blue-green-yellow (Fig. 9b). This trend, however, was not significant for Tetraselmis sp., nor were there significant correlations for the percent AQPPP to the percent red light. Further, there was no correlation of AQPPP to the ratio of red/blue for either species (Supplement 3a, b).
Fig. 9

Percent total quanta absorbed by photoprotective pigments, AQPPP, for four light sources as a function of: a QPAR percent blue light and b QPAR percent blue-green-yellow light. Means ± 2 standard deviations for each light source (N = 3)

4 Discussion

4.1 Biomass production

Chromatic adaptation of different species to the light environment has been shown to influence biomass production [7, 42, 43, 44, 45, 46]. The higher production rates of Tetraselmis sp. in Glo and white light treatments and lower production rate in the BBB light treatment are supported by other studies on green algae [30, 47, 48]. For the E. huxleyi, the highest biomass production rate was for the RRBBB treatment, which has been demonstrated for many microalgae rich in blue and blue-green, light absorbing fucoxanthins [19, 20, 28, 46]. The higher biomass production rate of E. huxleyi may be the result of photoacclimation to high light and PPP absorption in the blue to blue-green bands, relative to PSP absorption. The fact that the highest production rates for Tetraselmis sp. and E. huxleyi occurred for spectra having both red and blue wavebands is reminiscent of the Emerson effect for algae, where the maximum photosynthetic rate occurs when red and blue light are combined [19].

4.2 Pigments, neutral lipids, and cell size

Previous studies show low, non-photochemical quenching (NPQ), in cells adapted to high white light (> 1200 PPFD), coincided with the high lipid production [49] and high biomass production [50], which are similar to our finding for Tetraselmis sp. Saturating levels of blue light have been shown to produce higher lipid content than white light [22], which was true for our treatments. Further, our results support the findings that lipid content is lowest in low light conditions, no matter which spectrum is applied [51, 52]. Low chlorophyll a and accessory pigment concentrations at high irradiances are an adaptation by algae to decrease light harvesting pigments in high light environments [53, 54]. Our spectral data support that reaction centers of microalgae, chlorophyll a protein complexes, carotenoids, and chlorophylls b, c all decrease as light levels increase [55]. In contrast to the chlorophyll a trend, high neutral lipid concentrations occurred in high irradiance treatments, which has been also demonstrated by Wu et al. [56]. The 38% increase in Tetraselmis sp. cell volume accounted for the near doubling of chlorophyll a concentrations in Tetraselmis sp. cells and confirmed that larger volumes stored more neutral lipids.

Since lipids are produced in the thylakoid membrane, this may be a physical phenomenon with a trade-off between the volume of lipid droplets produced, the number of reaction centers, and the volume of chloroplasts. Interestingly, neutral lipid globules in stressed microalgae sequester beta carotene and are thought to act as a sunscreen layer [57]. Since lipid bodies are pervasive in many species of microalgae, a high lipid concentration may be an adaptation to light stress.

Photoprotective pigments absorb excess quantum energy that would inhibit the photosystem [58, 59]. Similar to our findings, the increases in photoprotective xanthophylls at high light levels have been attributed to diatoxanthin-diadinoxanthin cycle for E. huxleyi [17, 18] and violaxanthin-antheraxanthin-zeaxanthin cycle for green algae [55]. Although no zeaxanthin was detected in Tetraselmis sp., violaxanthin (PSP) and antheraxanthin (PPP) were present. Beta carotene also contributed to NPQ and dominated PPP quantum absorption for both species and at most light levels in this study. Beta carotene concentrations increased approximately 50% from high to low light for both Tetraselmis sp. and E. huxleyi, which compares well with the 33% and 50% increases in beta carotene for E. huxleyi measured by Garrido et al. [18] and Mcknew et al. [17], respectively.

4.3 Bio-optics

The uptake of photons by algae was dependent on the specific absorption of the pigments and the concentration of pigments. PPP xanthophylls for the two species have 2.7–3.2 times the specific absorption capacity of chlorophyll a [37]. For the PSP light harvesting pigments in E. huxleyi, chlorophyll c has 4 times higher specific absorption than chlorophyll a. In comparison, only lutein (PSP) in Tetraselmis sp. has 3 times the specific absorption capacity of chlorophyll a [37]. Higher specific absorption is important since, even at low concentrations, pigments can contribute significantly to light absorption in specific wavebands. For example, the high specific absorption of beta carotene is why it dominated PPP absorption over antheraxanthin, even though these pigments had similar concentrations in high light. The dual role of xanthophylls in photoprotection and photosynthesis is regulated by the pH gradient in the thylakoid membrane. De-epoxidization of xanthophylls to photoprotective pigments only occurs if the pH in the thylakoid membrane is below a critical concentration [60]. The strength of the pH gradient depends on the flux of excess irradiance (i.e., above light saturation). However, in this study Tetraselmis sp. and E. huxleyi treatments saturated at higher light levels and may not have reached the critical pH. This is supported by data in Fig. 10, suggesting Ik may be a good indicator of high light stress and promotion of de-epoxidized xanthophylls.
Fig. 10

Concentrations of antheraxanthin and diatoxanthin in the high light treatments as a function of Ik

Larger Ik value indicates a smaller antenna size, which was true for E. huxleyi. The linear decrease in Ik with the percent of blue light for E. huxleyi (and increase for Tetraselmis sp., see Supplement 2b) illustrates a spectral dependence of antenna size. Many studies use short-term photosynthesis measurements of O2 and 14C, obtaining Ik values that are lower than our values for biomass production. This may be a methodological artifact since the former measurements do not account for higher respiration at lower light levels. High respiration would metabolize more carbon, therefore reducing biomass production and shifting Ik to higher values.

The increase in algal absorption per cell as irradiance decreased was the result of increasing cellular pigment concentrations. This also accounted for the increased quantum absorption per cell as light levels decreased. However, total quantum absorption was higher at higher irradiances as a result of higher biomass and the increased photon flux. Even though cells that adapted to high light had low quantum absorption per cell, more total quanta were absorbed in high light treatments because of their higher biomass. In contrast, low light cells, with high quantum absorption per cell, could not compensate for the lower biomass, and so these treatments always absorbed fewer photons. The consequence for mass cultures, with large volumes and non-homogeneous cell distributions, is that cells trapped in low light dead zones will have lower quantum absorption and production rates than cells in high light regions.

The increased quantum flux of blue light for the LEDs, relative to white and Glo treatments, demonstrates how the unique LED array was tuned to pigment absorption of E. huxleyi. Although the percent AQPPP increased in high light for the LED treatments, the total quanta absorption, AQph, remained relatively low, thereby increasing the quantum efficiencies of biomass and neutral lipid production. This was not as evident in Tetraselmis sp. cells. The fact that AQPPP contributed to more photon absorption for E. huxleyi and less to Tetraselmis sp. corresponds to the highest biomass production rates for Tetraselmis sp. in GloPlus and for E. huxleyi in RRBBB.

From a practical standpoint, the LED array was a more efficient light source since it generated only the wavelengths that the algae could absorb and did not produce wavelengths that were under-utilized, as did the GloPlus and cool white fluorescent bulbs. This is crucial in cases where biofuel production is augmented by electric lighting since operating expenses and carbon footprints are larger. We estimate the lifetime of our LED array is 5 years (based on a 800 PPFD, 14:10 day/night cycle), more than most efficient cool white and growth fluorescent bulbs of the same intensity. Other advantages of using LEDs tuned to algal action spectra for biofuel bioreactors are that they provide the right mix of spectral irradiance for biomass production, can optimize lipid production, and have lower energy consumption than other commercial light sources.

Our study has confirmed that spectral absorption by algae is a major factor contributing to biomass and lipid production rates for cultures under different light intensities and spectra. By tuning the light spectrum to the absorption spectrum of the algae, both biomass and lipid production can be optimized. Therefore, knowing the spectral properties of microalgae could enhance biofuel production by producing more biomass and lipids, although this will depend on improving photobioreactor designs. Models of photobioreactor mixing that have incorporated scalar irradiance [61, 62, 63, 64] and spectral irradiance [65] have shown that continuous mixing in strong light is needed to achieve high biomass production [66]. To enhance biomass and lipid production, the light spectrum could be matched to algal absorption in a well-mixed photobioreactor so that algal cells would experience a high, time-averaged light field with no dead zones to trap cells in suboptimal light levels. Thus, more efficient mixing would reduce extreme highs and lows in light intensities. This, plus the rapid reversibility of PSP to PPP and vise versa, means cells could adjust to light levels without high metabolic costs [8, 67].

5 Conclusions

Spectral quality and intensity of light sources directly influenced the shape of biomass production curves for Tetraselmis sp. and E. huxleyi. Cells adapted to high irradiance levels had decreased pigment concentrations and spectral absorption per cell, but still exhibited high total quantum absorption as a result of high biomass. High levels of neutral lipids per cell were associated with low, cellular chlorophyll a concentrations. The highest lipid production occurred for E. huxleyi under a high red-blue light spectrum using LEDs, even though the LEDs had the same photon flux density as the other light sources.

This study infers that another way to achieve the efficacy of high biomass and lipid production may be to optimize optical properties of the microalgae. This can be accomplished by tuning light sources to pigment distributions in high light regimes, such that cells are not severely photoinhibited or have extreme NPQ. One way to achieve this is to improve mixing conditions in photobioreactors so microalgae adapt to a light regime that, averaged over short timescales, is high and constant.

Notes

Acknowledgements

We wish to thank Dr. Jeannine Winkler and Christian Rösch for helping with the flow cytometer analyses. The authors are indebted to Daniel Steiner and Blake Mathews at the EAWAG laboratory, Kastanienbaum, Switzerland, for processing the HPLC samples. We also thank Drs. Ulrike Zika and Fabian Ille for proofreading the revised manuscript. Lastly, the comments and suggestions of three reviewers greatly improved this manuscript and for that, we thank them.

Authors’ contributions

Dr. Granata designed and conducted experiments and wrote 80% of the manuscript. Drs. Egli, Härri, and Habermacher all contributed the remaining text and optimization of the LED arrays.

Funding

This project was funded by a grant to Drs. Granata and Egli from the Office of Research, Hochschule Luzern. Special thanks to Dr. Andrea Weber Marin, vice-dean of the Lucerne School of Engineering and Architecture and Head of Research and Development, for supporting this work.

Compliance with ethical standards

Conflict of interest

The authors declare this work has no conflict of interest either personally or financially.

Consent for publication

The authors approved the consent for publishing the manuscript.

Data availability

Data analyzed in this study are presented in the main manuscript and in supplemental files. Other data files are available in the Lucerne Open Repository (LORY) zenodo.org/communities/lory/.

Ethics approval

The authors have read and agreed to the ethics for publishing the manuscript.

Supplementary material

42452_2019_529_MOESM1_ESM.pdf (24 kb)
Supplement 1. The percent blue, red, and blue-green-yellow light for each light source in relation to Pmax for: a) Tetraselmis sp. and b) E. huxleyi. (PDF 24 kb)
42452_2019_529_MOESM2_ESM.pdf (35 kb)
Supplement 2. Trends in α and Ik for each light source, relative to their percent blue light emission, for: a) Tetraselmis sp. and b) E. huxleyi. (PDF 34 kb)
42452_2019_529_MOESM3_ESM.pdf (23 kb)
Supplement 3. The trends in percent of AQPPP for Tetraselmis sp. and E. huxleyi as a function of: a) percent red light and b) the ratio of red:blue light for each light source. (PDF 22 kb)

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Bioscience and Medical TechnologyLucerne University of Applied Science and Art, (Hochschule Luzern)HergiswilSwitzerland
  2. 2.IIES, Lucerne University of Applied Science and Art, Lucerne (Hochschule Luzern)HorwSwitzerland

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