Eng. Life Sci. 2007, 7, No. 2, 127–135
127
I. Perner-Nochta1 A. Lucumi1
Research Article
C. Posten1
Photoautotrophic Cell and Tissue Culture in a Tubular Photobioreactor
1
Institute of Engineering in Life Sciences, Universität Karlsruhe, Karlsruhe, Germany.
An externally illuminated tubular photobioreactor was constructed from 3.4 m stainless steel tubes and 22.1 m glass tubes for the cultivation of photoautotrophic organisms. The 30-L reactor can be equipped with helical static mixers in order to create a uniform radial exchange within the tubes, 40 mm in diameter. A flexible construction of the reactor allows scale-down experiments to be carried out with axial velocities between 0.3–2.5 m/s, gassing-in rates of 0–0.5 L/min, kL a values of 0.002–0.006 s–1 and six metal halide lamps inducing photon flux densities in the range of 70–300 lE/m2s. Two model organisms, the green microalgae Chlorella vulgaris and the bryophyte Physcomitrella patens, were chosen to characterize cell growth and physiology in submerse cultures. Comparative experiments with Chlorella vulgaris in two configurations of the reactor with inserted helical static mixers and plates resulted in maximum growth rates of 1.6 d–1. No growth enhancement was obtained in the case of helical static mixers at a mean PFD of 150 lE/m2s and an axial velocity of 0.4 m/s. No homogenous flow could be obtained in the case of inserted plates. Physcomitrella patens was successfully cultivated in the reactor (l = 0.36 d–1), whereas average axial velocities of ca. 0.6 m/s guarantee favorable gas transport without contributing to cell damage. This makes tubular photobioreactors a promising production system for the production of glycosylated recombinant proteins derived from moss. Keywords: Algae, Bioreactors, Biotechnology, Mixing, Plants Received: November 2, 2006; revised: January 27, 2007; accepted: February 1, 2007 DOI: 10.1002/elsc.200620178
1
Introduction
Until recently, submerse phototrophic tissues, such as bryophytes, have been grown in stirred tank reactors and bubble columns [1–3]. The main problem for a further scale-up is the difficulty in maintaining sufficient light growth conditions, which is also crucial in algal biotechnology. For the cultivation of microalgae, a range of tubular photobioreactors have been developed [4–8]. Besides laboratory tubular photobioreactors which are used for modeling purposes [9], large-scale systems are currently utilized, especially for algal cultures. One example is the world’s largest indoor production plant in Klötze, Germany, with a volume of 500 m3 [10]. Such systems are typically composed by long externally illuminated tubes with diameters of 3–5 cm. Nevertheless, an insufficient light availability in the centre of the tubes has often been reported. Ra-
– Correspondence: I. Perner-Nochta (
[email protected]), Institute of Engineering in Life Sciences, Universität Karlsruhe, D-76128 Karlsruhe, Germany.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
dial mixing could partially compensate the light gradients in the cross-section of the photobioreactor. On the one hand, the homogeneity of the suspension may be improved and, on the other hand, effects of light-dark cycles are generated. Positive effects of regular short-term light-dark cycles on algal growth have been demonstrated on a laboratory scale [11, 12]. Even though algal production processes are operated at flow velocities causing turbulent flow with Reynolds numbers of 9,000–30,000 [5], numeric simulations and experimental data reveal insufficient radial mixing [13]. It has been theoretically shown that helical static mixers are a promising tool to improve radial mixing in photoautotrophic cultures [13]. The influence of typical limitations and cultivation parameters in technical plants on the production process can be studied in scale-down assays [13]. Here, scale-down concerns the illumination of photoautotrohic cells in tubular photobioreactors and its improvement by radial liquid exchange. The experiments were conducted in a flexible and scalable tubular photobioreactor with the model organism Chlorella vulgaris. This green microalgae is a well documented photoautotrophic system with an estimated production of 2,000 t/a worldwide [14].
http://www.els-journal.com
128
Eng. Life Sci. 2007, 7, No. 2, 127–135
I. Perner-Nochta et al.
Furthermore, the aim of the present work is to evaluate this type of reactor for submerse photoautotrophic cell cultures. The advantages and limitations are investigated with the moss Physcomitrella patens which has been studied in plant functional genomics and has recently shown potential for the production of glycosylated recombinant proteins [15, 16].
2
Materials and Methods
2.1
Reactor Setup
The constructed tubular photobioreactor presented in Fig. 1 has a volume of 30 L. It consists of a stainless steel section with a length of 3.4 m, where the electrodes and the reactor periphery are connected, and a 22.1 m long glass section used for illumination. The horizontally arranged glass tubes composed
of borosilicate (QVF) with a diameter of 40 mm and a length of 1.5 m are interconnected with U-bends. The glass and stainless steel elements are joined by a sterilizable hose (ThomaFluid EPDM tubing No.12445, Reichelt Chemie Technik), whereas the stainless steel piping (38 mm diameter, steel grade 1.4571) is assembled by 11⁄2 ” triclamp-joints (Inter Technik Elze). The suspension is circulated by means of a sterilizable centrifugal pump with double mechanical seal (Euro-HygiaAD-tronic I/25, Hilge). pH (Easyferm 120, Hamilton) and pO2 electrodes (2P, Applicon), as well as a temperature sensor (PT100) are installed immediately after the glass section of the reactor. A controller (Multigas controller 647B, MKS) and two mass flow-meters (MassFlo 1000 sccm/N2 and 200 sccm/N2, MKS) are used to supply air and CO2 mixtures. The gas transfer of the injected mixture into the suspension is improved by six helical static mixing elements. Data acquisition and control of the pilot plant are carried out by a Biocontroller (ADI 1030, Applicon). Light is provided by six supported metal halide lamps (150 W RX7s-24, BLV) with a color temperature of 10,000 K. Their position relative to the reactor can be individually adjusted. The reactor is surrounded by polished aluminum plates to increase the light reflection. Acids, bases and other media are pumped into the reactor by peristaltic pumps (U1-Midi-D, Alitea) and their flows are monitored by balances (BP 4100S and BP8100S, Sartorius). The reactor sterilization (121 °C, 20 min) is carried out by supplying 3 bar of steam through the heat exchanger with a circulating medium. The reactor periphery is separately sterilized in an autoclave chamber and connected thereafter. The CO2 and O2 contents of the exhaust gas are determined by a Maihak Multor 610 analyzer. The measurements of the PFD are conducted by means of a quantum sensor (Waltz-MQS LI-190).
2.2
Figure 1. Schematic representation of the pilot tubular photobioreactor.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Reactor Configuration
The reactor is operated in three different modes. The glass tubes are either empty or filled with stainless steel elements. In the “mixer” configuration, helical static mixer rods of length 1.5 m are inserted into all straight glass tubes to improve the radial flow. One rod consists of 26 alternating left– and right-twisting elements (4 × 38 × 58 mm). Each element twists through an angle of 180°, while the sequencing elements are at right angles to each other. In the “plate” configura-
http://www.els-journal.com
Eng. Life Sci. 2007, 7, No. 2, 127–135
Tubular Photobioreactors
tion, horizontal plates (4 × 38 × 1500 mm) are inserted into the straight tubes in order to displace the same liquid volume as the helical static mixers. In both cases, the displaced volume adds up to about 13 % of the tube volume.
2.3
Reactor Characterization
Flow velocity was measured by means of 2 mm diameter colored particles of a similar density to that of the medium. Mean axial velocities were calculated according to the residence time of the track particles in the dark (3.4 m) and the illuminated (22.1 m) paths of the reactor during a loop. Liquid holdup was determined by measuring the volume of liquid displaced by gas in equilibrium, at different axial velocities and flow rates. The dynamic “gassing-in” method was used for the determination of the volumetric liquid mass transfer coefficient kLa [17].
2.4
Cultivation of Chlorella vulgaris
Chlorella vulgaris No 211–12 from the culture collection of algae (SAG, University of Göttingen, Germany) was cultivated in 0.5 g/L KNO3, 0.34 g/L KH2PO4, 0.5 g/L MgSO4 · 7 H20, 0.014 g/L FeSO4 · 7 H20 and 0.05 g/L Titriplex®II. Two trace element solutions were added: 0.1 mL/L of solution I (7.4 g/L ZnSO4 · 7 H20, 0.57 g/L H3BO3, 2.38 g/L CoSO4 · 7 H20, 2.36 g/L CuSO4 · 5 H20, 41 g/L MnSO4 · 4 H20) and 0.1 mL/L of solution II (0.92 g/L (NH2)6Mo7O24 · 4 H20). The pH was adjusted to a value of 6.7. Inoculation cultures were cultivated for 20–30 days in 1 L shaking flasks at 23 °C and an average PFD of 18 lE/m2s. For the experiments in the tubular photobioreactor, both the temperature and the pH were maintained at 25 °C ± 1 and 6.7 ± 0.05, respectively. The pH was adjusted by using 4 M NaOH and 1 M HCl. A gas mixture of 5 % CO2 [v/v] and air was supplied at a rate of 0.5 L/min. The PFD at the reactor surface averaged 150 lE/m2s, which corresponded to 10 lE/m2s. Prior to inoculation, an equilibrium was achieved. The biomass concentration, cx, was determined either gravimetrically by drying the centrifuged and twice washed cells of two 30 mL samples at 80 °C until constant weight, or via optical density at 750 nm (OD750), measured in a Varian-Cary 50 Conc UV-Visible Spectrometer, Eq. (1)1): cx = 0.174 · OD750
– 1)
List of symbols at the end of the paper.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Cultivation of Physcomitrella patens
P. patens wild type was grown in 250 mL shake flasks containing modified Knop medium [3]: 1,000 mg/L Ca(NO3) · 4 H2O, 250 mg/L KCl, 250 mg/L KH2PO4, 250 mg/L MgSO4 · 7 H2O and 12.5 mg/L FeSO4 · 7 H2O. The flasks were externally illuminated by means of halogen lamps in day-night cycles (LD 16/8) with an average PFD of 20 lE/m2s. The moss suspensions from the flasks were disrupted weekly with a rotor stator device (Ultraturrax, IKA, Germany) and 50 % of their medium was also renewed weekly. For the experiments in the tubular photobioreactor, 0.20–0.40 g DW of an inoculation culture from the shake flasks and a diluted Knop medium (6:10) were used. The pH and temperature were maintained at 5.8 ± 0.05 and 21 ± 1 °C. Additionally 0.4 L/min of a mixture of air and CO2 was continuously supplied. The CO2 content of the feed was held at 1.95 % [v/v]. The centrifugal pump was operated at a low velocity, according to the limit values proposed by Vandanjon et al. [19] in experiments with microalgae. Therefore, the average axial speed of the suspension in the reactor was maintained at 0.6 m/s. No helical static mixers or plates were introduced in the glass tubes throughout the moss cultures. An average PFD of 98.7 lE/m2s was maintained on the external surface of the glass pipes. LD (16/8) was performed up to the fourth day of culture, synchronized with the rhythm of the shake flasks. After a one-day LD (20/4) transition, continuous illumination (LL) was applied until the end of the culture. The concentration of the suspension was determined gravimetrically by drying the cell material of two 20 mL samples at 80 °C until constant weight. The content of chlorophyll-a and b was analyzed from acetone extracts. For this purpose, the samples were mechanically disrupted and the extinction of the extracts was measured at 647, 660 and 750 nm. The extinction values were correlated with the concentration of pigments after the calibration with standards. Cell damage was determined by inspection of offline pictures and by measuring free pigments from supernatants (data not shown).
3
Results and Discussion
The 30-L tubular photobioreactor was operated in three configurations: without helical static mixers or plates, with helical static mixers, and with plates. These configurations influenced the reactor performance.
(1)
The content of chlorophyll-a was analyzed in vivo by means of the optical density at 680 and 750 nm [18] using Eq. (2): cchla = 12.87 · (OD680 – OD750)
2.5
129
(2)
3.1
Flow Velocity
The mean flow velocity was measured at different pump frequencies, with and without gassing-in. Significantly higher flow velocities were attained if the reactor was completely filled with liquid compared to the usual cultivation conditions with gassing-in. However at low pump frequencies and a gas flow rate of 0.5 L/min, the axial velocity was slightly higher than in the reactor without gas. This effect was reversed at higher frequencies and was probably caused by an inefficient energy transfer of the pump to the fluid, due to stalling at high velocities.
http://www.els-journal.com
130
Eng. Life Sci. 2007, 7, No. 2, 127–135
I. Perner-Nochta et al.
Pump frequencies of 10–80 Hz resulted in velocities of 0.37–2.6 m/s with the “plate” geometry whereas in the “mixer” case, the velocities reached only 0.21–0.85 m/s, most likely because of the flow resistance originated by the helical static mixers (see Fig. 2). The flow velocities within the dark and the illuminated reactor sections demonstrated a linear correlation with the frequency of the pump. However, at high pump fre-
Figure 3. Gas proportion in the reactor depending on the pump frequency, fP, at different gassing-in rates in the “mixer” (-) and “plate” (- -).configurations.
Figure 2. Mean flow velocity versus the pump frequency in the photobioreactor with helical static mixers (~) and plates (~), filled only with water (-), with 0.5 L/min air gassing-in (- -), and with reactor valves partially closed (䉫).
quencies, the tracking particle was exposed to achieve an intense and irregular flow in the pump head. Thus, the ratio rtd/t (residence time in the dark section, td, to the total residence time) increased, e.g., in the “mixer” case at fp = 30 Hz, residence times were measured to be tl = 110 s and td = 18 s, with rtd/t = 0.14 and at fp = 70 Hz, tl = 25 s; td = 6 s, with rtd/t = 0.19. Both rtd/t and the difference between the mean velocity in the dark and the light sectors of the photobioreactor were augmented by raising the pumping frequency. This velocity fluctuation comprised up to 13 % of the mean axial velocity of the suspension.
3.2
Liquid Holdup
The liquid holdup in the photobioreactor is dependent on the geometrical configuration, flow velocity and bubble size. In the “mixer” configuration, the bubbles were small (lm scale) and well transported within the liquid phase. In the “plate” configuration the bubbles had a size of 2–5 cm (at 30 Hz) and evident separation between the liquid and gas phases occurred. At a higher pump frequency, some small bubbles were formed on a lm-scale. However, they had a very wide size distribution. Bubble size and distribution also determines the kinetics of the degassing and gassing-in in the reactor. Small bubbles improve the gas transfer to the cells but are not easily separated at the end of the circulation loop. The liquid holdup at various frequencies and gas flow rates is presented in Fig. 3. In the “mixer” configuration, the amount of liquid replaced by gas showed a minimum of
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ca. 50 Hz. For example at 0.5 L/min, the gas fraction, egas, was 2.8 % at 30 Hz, 0.9 % at 50 Hz, and 1.7 % at 70 Hz. This phenomenon can be explained by a size-dependent bubble strip off from the reactor. Small bubbles on the upper side of the tubes were barely transported at low velocities. However, above a critical velocity, the bubbles could be removed and further separated. At very high velocities (fp > 50 Hz), the performance of the degassing was unsatisfactory, and egas increased. In the “plate” configuration, egas increased linearly with the pump frequency. In order to obtain the same working point as in the “mixer” configuration, at 30 Hz of egas = 1.9 % and a gassing-in rate of 0.5 L/min, valve V1 (compare to Fig. 1) was partially closed. This induced a pressure loss in the reactor and a decrease of egas.
3.3 Volumetric Liquid Mass Transfer Coefficient The volumetric liquid mass transfer coefficient, kLa, measures the velocity of the gas exchange within the reactor, where high kL a values indicate a good mass transfer between the liquid and the gas phase. The resulting kLa values for oxygen ranged from 0.002–0.0055 s–1 (see Tab. 1). As expected, the values in the “mixer” configuration are higher than in the “plate” configuration. The kL a values of the tubular photobioreactor are in the same order of magnitude as those of stirred tank reactors. By means of kL a, the oxygen mass transfer rate (OTR) can be determined. Assuming a maximally allowable oxygen concentration of 200 % in the medium at the end of the illuminated zone, the maximal achievable OTR will be ca. 5.2 mmol/L h, for kLa = 0.0055 s–1 with air.
3.4
Light Distribution on the Reactor Surface and Radial Light Profiles
The surface to volume ratio in the reactor was 89 m–1, whereas the dark volume made up 11 %. The net PFD varied according to the number of metal halide lamps and their distance to the reactor surface. However, the light distribution on the reactor surface was not homogenous and its variation was strongly de-
http://www.els-journal.com
Eng. Life Sci. 2007, 7, No. 2, 127–135
Tubular Photobioreactors
131
Table 1. Experimental determination of kLa for the “mixer” and “plate” reactor configurations at different axial velocities (mean values from repeated determinations). Configuration
Flow Velocity vmean [m/s]
Plate Mixer
kL a [s–1]
0.94 0.87 0.0039
0.83 0.0044
0.49
0.0054
–1
kL a [s ]
0.0052
0.38
0.35
0.0027*
0.0019*
0.0055
* The reactor valves were partly closed (see Fig. 1).
pendent on the distance and the focus of the lamps (see Fig. 4). The inclusion of helical static mixers also affected the light transfer to the suspension. The PFD distribution on the surface of the reactor with mixers, for a gap of 700 mm can be seen in Fig. 5, and may be used to calculate the volumetric light intensity, IV (11 lE/L s), or its equivalent value related to the surface of the reactor (333 lE/m2s).
The Lambert-Beer law may be applied to allow a simplified characterization of the radial variation of the PFD in photobioreactors (Eq. (3)). I(k,r) = Io(k) · e–K(k)r
(3)
Here, I is the PFD as a function of the radius, r, and wavelength, k. Io represents the PFD at the inner side of the reactor wall and K is the apparent light extinction coefficient. K can be described as a linear function of the cell density, cx, in the reactor, Eq. (4). In this expression, kc is the specific light extinction due to the biomass and ko due to the medium [20]: K = ko + kc · cx
(4)
The average PFD, Im, across a one-dimensional path inside the tube can be approximated with the aid of the following expression: Zr a a Io Im
1 e Kr I dx (5) r Kr 0
where r is the radius of the tube, a is a geometric constant with a value of 2 for symmetric illumination from both sides of the reactor wall. In Fig. 6, the radial profiles of the PFD for different biomass concentrations of P. patens are plotted at 80 lE/m2s, measured at the outer wall of the tubes. Under these conditions, the measured PFD at the inner side decayed up to 73 lE/m2s. The reduction of available light in the center of the reactor was acceptable for biomass concentrations up to 0.5 g/L. Furthermore, the experimental data supported the linear dependence between the cell concentration and the apparent extinction coefficient from Eq. (4). The calculated light extinction coefficient of the Knop medium, ko, was 0.0027 mm–1 and the corresponding kc value for the moss was 0.0654 L/g mm. The values of Im for the tested cell concentrations, according to Eq. (5), are shown in Fig. 6.
3.5 Figure 4. Average distribution of the PFD on the illuminated surface of the tubular reactor. (The measured PFD values are averaged in nine reactor sections with an identical exposed surface. The gap between light sources and reactor were: (A) 380 mm and (B) 1030 mm.)
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Growth Experiments with Chlorella vulgaris
Comparative experiments with helical static mixers and plates were conducted in the tubular photobioreactor with axenic cultures of C. vulgaris at an axial velocity of 0.4 m/s for 11 days. The effects of regular mixing caused by helical static mixers on the algal metabolism were investigated.
http://www.els-journal.com
132
Eng. Life Sci. 2007, 7, No. 2, 127–135
I. Perner-Nochta et al.
growth from rchla = 16 mg/g DW to 28 mg/g DW, and then dropped on day five to 8 mg/g DW.
3.5.2 Plates
Figure 5. PFD distribution on either the reactor surface for three lamps per side at a distance of 700 mm. (x-axis reactor width; y-axis reactor height; helical static mixers are between positions 15 cm and 165 cm; vertical lamp positions are indicated by ([])
Again a biomass concentration of cx = 2.3 g/L was obtained at the end of the cultivation. After a very short lag phase, exponential growth occurred with l = 1.4 d–1 (days 1–4) followed by a phase of reduced growth. During days 8–11, growth occurred with l = 0.08 d–1. The maximal specific carbon dioxide uptake rate, rCO2 = 2.2 mg/g DW min was obtained on the third day of cultivation, and then diminished quickly to 0.4 mg/g DW min (day 6), and slowly until the end of the culture. As expected, the oxygen production rate was synchronized with rCO2. The chlorophyll-a specific content presented maximal values at the early stages of the exponential growth phase. The chlorophyll content of the algae started at rchla = 20 mg/g DW and dropped to 12.4 mg/g DW, before increasing to a value of 28 mg/g DW during days 2–4. The specific chlorophyll concentration decayed to 9 mg/g thereafter.
3.5.1 Helical Static Mixers The batch cultivation of C. vulgaris in the pilot photobioreactor equipped with helical static mixers resulted in a final biomass concentration of 2.3 g/L (see Fig. 7). After a lag-phase of two days, cell growth occurred in two stages: Exponential growth with a specific growth rate, l, of 1.6 d–1 lasted for two days. Then growth decreased to rates of ca. l = 0.2 d–1 during days 6–8, and to l = 0.06 d–1 during days 9–11. Since cell growth of phototrophic organisms is connected to carbon fixation, it can be tracked by the specific carbon dioxide uptake rate, rCO2, which was calculated from the difference between the inlet and exhaust gases. During the whole experiment, rCO2 decayed from 4 mg/g DW min to 0.44 mg/g DW min. The oxygen production rate only increased during the exponential growth phase. During the first three days, pO2 was ca. 90 % and then rose to 111 % on day five and decreased to ca. 100 % during days 7–11 (data not shown). The specific chlorophyll-a content, rchla, increased during the exponential
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 6. Radial variation of the PFD at different biomass concentrations in a 40 mm diameter tubular photobioreactor. (The cell suspension consisted of Physcomitrella patens protonemal tissue in Knop medium. Estimated values based on the Lambert-Beer law (solid lines) and the average PFD along a one-dimensional path are illustrated).
http://www.els-journal.com
Eng. Life Sci. 2007, 7, No. 2, 127–135
Tubular Photobioreactors
133
Fourthly, mass transfer can be a problem in tubular photobioreactors [24]. Here, the oxygen mass transfer is not likely to have limited growth towards the end of the exponential growth phase. This reveals the following short estimation (see Eqs. (6–8)) with ro2, the specific oxygen uptake rate, l, the growth rate and yx,o2, the yield coefficient for oxygen:
Figure 7. Time course of biomass concentration, cx, as well as the calculated growth rates, l, the specific CO2 uptake rate, rCO2, and the specific chlorophyll-a content, rchla, in the experiment with helical static mixers at v = 0.4 m/s.
3.6
Discussion
Successful batch cultivations revealed only small differences in growth for the experiments with and without helical static mixers. Cell attachment to the walls and accumulation or agglomeration of the algae in the reactor did not occur in either configuration. Algal cells as well as gas bubbles were properly transported by the liquid stream in the “mixer” configuration. The distinctively larger bubbles only separated and formed two phases in the “plate” configuration. The maximum biomass concentration of 2.3 g/DW L was low compared to the documented values ranging from 2–8 g/DW L [5]. There are four reasons for the low impact of regular mixing, which is created by helical static mixers, on growth rates. Firstly, in the less mixed “plate” reactor setup, no homogenous flow could be achieved. Here, lower gas flow rates are required to improve the flow behavior. Secondly, the PDF of ca. 150 lE/m2s might have been too low. The effect of intermittent light is probably more detectable at higher light intensities above 800 lE/m2s. It is only in this case that the cells that were in the wall near the layers may undergo enough photosynthesis to store additional energy for further reactions in the dark [12, 21]. According to Morita et al. [22], the growth of C. vulgaris is limited at low intensities below 25 lE/m2s. In the experiments performed, this critical PFD is reached at a low penetration depth of 10 mm at cx = 0.2 g/L (4 mm for 1 g/L) [23]. During the exponential growth, cx was below 0.3 g/L, and therefore, minor shading effects are expected during this phase rather than in the latter growth phases at higher cx values. Thirdly, nutrient limitation may have occurred. Both experiments showed decreasing rchla which indicates either a changing metabolism in C. vulgaris during the cultivation or a lack of nutrients for the generation of chlorophyll-a. Most likely a nitrogen limitation has controlled the chlorophyll production, and therefore, was responsible for the early end of the exponential growth phase. In a later phase, the cells were increasingly restricted to maintenance alone. The specific carbon dioxide uptake rate between the exponential and linear growth phases coincided with the behavior of rchla.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
l = yx,o2 · ro2
(6)
yx,o2 ≈ 1
(7)
and OTR = ro2 · cx
(8)
In the experiments, the pO2 values did not exceed 112 %, which is expected if all CO2 (5 % CO2 at the inlet) is metabolized. In that case, the maximum OTR is still 1.25 mmol/L h and the corresponding maximum growth rate would be 4.2 d–1, a value higher than the maximum growth rate obtained. The volumetric production rates, PV, were maximally 0.7 g/L d and are low compared to the reported PV values of 1–5 g/L d in enclosed systems [5], which is probably due to the low PFD. A photosynthetic efficiency of about 2–9 % was calculated, whereas values of up to 20 % have been reported [25]. In experiments conducted at higher light intensities excluding nutrient limitation, a prolonged exponential growth phase, and also a higher growth rate in the case of helical static mixers are to be expected.
3.7
Growth Experiments with Physcomitrella patens
Assays in the reactor without mixers or plates at different flow regimes were performed to characterize stress responses in moss. An axial velocity of 0.8 m/s produced an elongation of the moss threads, a relaxation of moss agglomerates, an adequate gas-liquid exchange (kL a = 0.0042 s–1), but induced cell damage. Furthermore, cells collapsed at velocities over 0.9 m/s. On the other hand, at 0.2 m/s the formation of cell knots was facilitated and the adherence of threads to tube walls increased. The average diameter of the gas bubbles increased and consequently kL a dropped to 0.002 s–1. Therefore, a middle velocity of 0.6 m/s should be used for cell culture purposes. The time course of the cell concentration in a standard batch of P. patens is shown in Fig. 8. During the first 5 days of cultivation, the specific growth rate of cells, l, reached 0.361 d–1, which corresponds to the values reported for semi-continuously cultured P. patens in stirred glass vessels, whereas a correction factor is introduced to compare data from the LL regimes [26]. Therefore, a reduction of the dark period on day 5 (LD, 20-4) caused an increase in l of up to 0.45 d–1. As the biomass level increased and light scattering became dominant, the specific growth rate fell to 0.3 d–1, although the LL regime was introduced. This transition between exponential and light limited growth is characteristic at biomass concentrations ranging from 0.5–1.0 g/L. The pigment concentration of the moss, which illustrates the capacity of the cells to absorb light, showed constant values until the lower growth phase appeared. The highest chlorophyll content of 14.0 mg/g DW was detected during the exponential growth phase. In the later days
http://www.els-journal.com
134
Figure 8. Time course of dry weight (grey dots), chlorophyll content (empty squares) and specific oxygen production rate (solid line) during a model batch of the Physcomitrella patens wild type in a 30-L tubular photobioreactor. (After a biomass concentration of ca. 0.5 g/L, the growth rates and pigment production decreased as well as the photosynthesis. The late phase of the culture was characterized by accelerated cell differentiation. Values of dry weight and chlorophyll were obtained as averages from duplicates, whereas the standard deviations were always lower than 6 %).
of cultivation, the moss had only ca. 50 % of the total chlorophyll from the previous phase of cultivation. The specific O2 production rate, ro2, showed patterns which were similar to the pigment plots. In the early stages of cultivation, rO2 achieved 30–40 mg/g DW h. However, from day 8 of culture, it decreased rapidly. During the dark periods, cell respiration achieved ca. 14 % of the oxygen production due to photosynthesis. Similar developmental variations of P. patens in suspension were observed as reported by several authors [27–29]. During the exponential growth phase, the moss threads were predominantly chloronemal, i.e., short, deep green and with cross-cell walls perpendicular to the growth direction. As the biomass concentration increased, the moss threads became longer and branched. Later, the proportion of cells corresponding to the next developmental stage increased. These cells, the so called caulonema, are longer than the chloronema and contain a small amount of chloroplasts.
3.8
Eng. Life Sci. 2007, 7, No. 2, 127–135
I. Perner-Nochta et al.
Discussion of the Growth Experiments
The novel pilot tubular photobioreactor proved to be an effective alternative for cultivation of the bryophyte P. patens. The usual problems associated with insufficient mixing and adherence to the cells at the walls of the reactor in traditional plant cell cultures, were overcome. Moreover, the axial velocity of the suspension may be used as a process parameter to tune turbulence and/or cell stress in the cultivation. The decay of the specific growth rate in the batches can be explained by means of the reduction of the light availability at high biomass concentrations, together with the moss differentiation [3, 27]. Cell differentiation implies caulonemal proliferation, and therefore, a reduction of the photosynthetic activity and chlorophyll content. For biotechnological applications, it may be possible to maintain the chloronemal stage for long-term cultivations,
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
when the differentiation factors are controlled [30]. Adequate strategies including cell disruption, phytohormone dilutions, and addition of differentiation antagonists may be easily performed in scalable tubular photobioreactors. The day-night cycles reduced the specific growth rate and were not required in the first part of the cultivation. However, the critical dark period and the maximal duration of LL still has to be determined. Additional experiments using different illumination frequencies (light and dark cycles in s or ms) may be conducted to improve the knowledge required for scale-up. A moss tissue propagation based on tubular photobioreactors has shown several advantages compared to classical suspension cultures in stirred reactors. High specific growth rates and carbon dioxide fixation rates were achieved as a result of the improved mixing and light transfer to the cells in the pilot photobioreactor. Moreover, fouling and adherence of moss rhizoids to the inner walls of the reactor were successfully avoided by means of an appropriate suspension turbulence. This may allow for long term cultures with a tight control of environmental conditions in the future. Experiments with recombinant P. patens lines expressing complex glycosylated proteins have shown promising results in molecular farming [3]. For the production of high amounts of moss biomass and target proteins required for preclinical studies, a scale-up strategy has been proposed based on parallel reactor modules of ca. 150 L with a similar geometry to the introduced pilot reactor [15]. This flexible set up may facilitate further capacity enlargement without reducing the homogeneity of the suspension.
4
Conclusions
An appropriate mixing strategy for the cell and tissue culture of photoautotrophic organisms is necessary to assure homogeneity in the bio-suspension and utilize the positive effects of light-dark cycles. Regular mixing patterns caused by helical static mixers do not improve algal growth compared to irregular mixing at low PFD (under 150 lE/m2s). The effects at high PFD still have to be investigated. The use of tubular photobioreactors for the culture of photoautotrophic cell tissues such as moss is promising, especially when scalability and a tight control of the process is required, e.g., in the case of pharmaceuticals.
Acknowledgement The authors would like to thank the BMBF for funding this work.
Symbols used LD LL PFD cx cchla
light-dark cycles continuous illumination photon flux density biomass concentration chlorophyll-a concentration
http://www.els-journal.com
Eng. Life Sci. 2007, 7, No. 2, 127–135
fp I Im Io IV K kc ko kLa r rCO2 rchla rO2 rtd/t td tl yx,O2
pump frequency light intensity mean light intensity light intensity at the inner side of the reactor wall volumetric light intensity apparent light extinction coefficient specific light extinction due to the biomass specific light extinction due to the medium volumetric liquid mass transfer coefficient radius specific carbon uptake rate specific chlorophyll-a content specific O2 production rate ratio of the residence time in the dark to the total residence time residence time in the dark reactor section residence time in the illuminated reactor section yield coefficient for oxygen
Greek symbols a egas k l
geometric constant gas fraction in the photobioreactors wavelength growth rate
References [1] P. J. Boyd, J. Hall, D. Cove, An airlift fermenter for the culture of the moss Physcomitrella patens, in: Methods in Bryology, Proc. Bryology Methods Workshop Mainz (Ed: J. M. Glime), Hattori Botanical Laboratory, Nichinan, 1988, 41–45. [2] E. Decker, R. Reski, The moss bioreactor, Current Opin. Plant Biol. 2004, 7, 166–170. [3] G. Gorr, Biotechnologische Nutzung von Physcomitrella patens (hedw.) B.S.G., Ph.D. Thesis, University of Hamburg 1999. [4] M. Borowitzka, Commercial production of microalgae: ponds, tanks, tubes and fermenters, J. Biotechnol. 1999, 70, 313–321. [5] O. Pulz, Photobioreactors: Production systems for phototrophic microorganisms, Appl. Microbiol. Biotechnol. 2001, 57, 287–293. [6] P. Carlozzi, Hydrodynamic aspects and Arthrospira growth in two outdoor tubular undulating row photobioreactors, Appl. Microbiol. Biotechnol. 2000, 54, 14–22. [7] E. Molina Grima, F. Acién Fernández, F. Garciá Camacho, F. Camacho Rubio, Y. Chisti, Scale-up of tubular photobioreactors, J. Appl. Phycol. 2000, 12, 255–368. [8] M. Morita, Y. Watanabe, H. Saiki, Investigation of photobioreactor design for enhancing the photosynthetic productivity of microalgae, Biotechnol. Bioeng. 2000, 69, 693–698. [9] Z. Csögör, M. Herrenbauer, I. Perner, K. Schmidt, C. Posten, Design of a photobioreactor for modelling purposes, Chem. Eng. Process. 1999, 38, 517–524. [10] P. Spolaore, C. Joannis-Cassan, E. Duran, A. Isambert, Commercial applications of microalgae, J. Biosci. Bioeng. 2006, 101, 87–96. [11] N. Gerbsch, Grundlegende Untersuchungen zur Entwicklung eines Photobioreaktors auf Basis seitabstrahlender Lichtwellenleiter, Ph.D. Thesis, TU Berlin 1997.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Tubular Photobioreactors
135
[12] J. Degen, A. Uebele, A. Retze, U. Schmid-Staiger, W. Troesch, A novel airlift photobioreactor with baffles for improved light utilization through the flashing light effect, J. Biotechnol. 2001, 92, 89–94. [13] I. Perner, Scale-Down von Photobioreaktoren, Ph.D. Thesis, Shaker, Aachen 2003. [14] O. Pulz, W. Gross, Valuable products from biotechnology of microalgae, Appl. Microbiol. Biotechnol. 2004, 65, 635–648. [15] A. Hohe, R. Reski, From axenic spore germination to molecular farming, Plant Cell Rep. 2005, 23, 513–521. [16] D. Schaefer, J.-P. Zryd, The moss Physcomitrella patens, now and then, Plant Physiol. 2001, 127, 1430–1438. [17] M. Sobotka, A. Prokop, I. J. Dunn, E. Einsele, Review of methods for the measurement of oxygen transfer in microbial systems, Annu. Reprod. Ferment. Processes 1982, 5, 127–135. [18] Z. Csögör, Untersuchungen zur Modellierung des Wachstums und der Produktbildung bei Mikroalgen, Ph.D. Thesis, Shaker, Aachen 2000. [19] L. Vandanjon, N. Rossignol, P. Jaouen, J. M. Roberts, F. Quemeneur, Effects of shear on two microalgae species: Contribution of pumps and valves in tangential flow filtration systems, Biotechnol. Bioeng. 1999, 63, 1–9. [20] Y. Huang, G. Rorrer, Dynamics of oxygen evolution and biomass production during cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor under medium perfusion, Biotechnol. Prog. 2002, 18, 62–71. [21] F. Rubio, F. Camacho, J. Sevilla, Y. Chisti, E. Grima, A mechanistic model of photosynthesis in microalgae, Biotechnol. Bioeng. 2003, 81, 459–473. [22] M. Morita, Y. Watanabe, H. Saiki, High photosynthetic productivity of green microalgae Chlorella sorokiniana, Appl. Biochem. Biotechnol. 2000, 87, 203–218. [23] Y. S. Yun, J. M. Park, Attenuation of monochromatic and polychromatic lights in Chlorella vulgaris suspensions, Appl. Microbiol. Biotechnol. 2001, 55, 765–770. [24] R. W. Babcock, J. Malda, J. C. Radway, Hydrodynamics and mass transfer in a tubular airlift photobioreactor, J. Appl. Phycol. 2002, 14, 169–184. [25] E. Molina Grim, F. García Camacho, A. Contreras Gómez, J. A. Sánchez Pérez, F. G. Acién Fernandez et al., Evaluation of photosynthetic efficiency in microalgal cultures using averaged irrradiance, Enzyme Microb. Technol. 1997, 21, 375– 381. [26] A. Hohe, R. Reski, Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts, Plant Sci. 2002, 163, 69–74. [27] D. J. Cove, N. W. Ashton, The hormonal regulation of gametophytic development in bryophytes, in The Experimental Biology of Bryophytes (Eds: A. F. Dyer, J. G. Duckett), Academic Press, London 1984, 177–196. [28] G. Schween, G. Gorr, A. Hohe, R. Reski, Unique tissue-specific cell cycle in Physcomitrella, Plant Biol. 2003, 5, 50–58. [29] R. Reski, Development, genetics and molecular biology of mosses, Botanica Acta 1998, 111, 1–15. [30] A. Hohe, E. Decker, G. Gorr, G. Schween, R. Reski, Tight control of growth and cell differentiation in photoautotrophically growing moss (Physcomitrella patens) bioreactor cultures, Plant Cell Rep. 2002, 20, 1135–1140.
http://www.els-journal.com
