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Biotechnol. Prog. 2003, 19, 418427

Cultivation of Microplantlets Derived from the Marine Red Alga Agardhiella subulata in a Stirred Tank Photobioreactor
Yao-ming Huang and Gregory L. Rorrer*
Department of Chemical Engineering, Oregon State University, Corvallis, Oregon 97331

Macrophytic marine red algae are a diverse source of bioactive natural compounds. Microplantlet suspension cultures established from red algae are potential platforms for biosynthesis of these compounds, provided suitable bioreactor configurations for mass culture can be identified. The stirred tank bioreactor offers high rates of gasliquid mass transfer, which may facilitate the delivery of the CO2 in the aeration gas to the phototrophic microplantlet suspension culture. Therefore, the effects of impeller speed and CO2 delivery on the long-term production of microplantlet biomass of the model red alga Agardhiella subulata was studied within a stirred tank photobioreactor equipped with a paddle blade impeller (Di/DT ) 0.5). Nutrient medium replacement was required for sustained biomass production, and the biomass yield coefficient based on nitrate consumption was 1.08 ( 0.09 g dry biomass per mmol N consumed. Biomass production went through two exponential phases of growth, followed by a CO2 delivery limited growth phase. The CO2-limited growth phase was observed only if the specific growth rate in the second exponential phase of growth was at least 0.03 day-1, the CO2 delivery rate was less than 0.258 mmol CO2 L-1 culture h-1, and the plantlet density was at least 10 g fresh mass L-1. Increasing the aeration gas CO2 partial pressure from 0.00035 to 0.0072 atm decreased the cultivation pH from 8.8 to 7.8, prolonged the second exponential phase of growth by increasing the CO2 delivery rate, and also increased the photosynthetic oxygen evolution rate. Impeller speeds ranging from 60 to 250 rpm, which generated average shear rates of 2-10 s-1, did not have a significant effect on biomass production rate. However, microplantlets cultivated in a stirred tank bioreactor ultimately assumed compact spherical shape, presumably to minimize exposure to hydrodynamic stress.

Introduction
Large, sessile marine organisms, including marine invertebrates and seaweeds, produce a diverse array of natural compounds with medicinal potential (Carte, 1996). However, lack of a reliable supply of cell biomass bearing the target compound often limits marine natural product development (Rhouhi, 1995). One division of marine seaweeds, the red macroalgae (Rhodophyta), is a prolific source of bioactive compounds. For example, the temperate red alga Agardhiella subulata contains unusual eicosanoids produced through lipoxygenase metabolism (Graber et al., 1996) and sulfonated galactans with antiviral activity (Witrovouw et al., 1994). The tropical red alga Ochtodes secundiramea contains a variety of halogenated terpenoids (Maliakal et al., 2001), produced via a novel myrcene synthase (Wise et al., 2002) and a marine bromoperoxidase (Rorrer et al., 2001). Unlike microalgae, macroalgae are multicellular, nonvascular, plant-like photosynthetic organisms that are very difficult to culture outside of the marine environment. However, some red macroalgae are amenable to in vitro cell and tissue culture development (AguirreLipperheide et al., 1995; Rorrer et al., 1998; Rorrer, 2000). During our culture development efforts, we found
* To whom correspondence should be addressed. Tel: 541-7373370. Fax: 541-737-4600. Email: rorrergl@che.orst.edu.
10.1021/bp020123i CCC: $25.00

that a novel phototrophic microplantlet culture, consisting of regenerated shoot tissues in liquid suspension, was a particularly promising platform for mass culture, as evidenced by success with two model plants, A. subulata (Huang et al., 1998; Huang and Rorrer, 2002a; Huang and Rorrer, 2002b) and O. secundiramea (Maliakal et al., 2001). Furthermore, since the cell biomass was concentrated into a dense array of branched shoot tissues and not dispersed among single cells, the attenuation of light through this culture suspension was not a limiting factor to biomass production. Consequently, biomass densities of 4 g dry mass L-1 (14 g fresh mass L-1) could be readily achieved during medium perfusion cultivation of Agardhiella microplantlets in a bubble-column photobioreactor (Huang and Rorrer, 2002a; Huang and Rorrer, 2002b). This present work continued our bioprocess engineering analysis of Agardhiella microplantlet cultivation. In particular, we were interested in the growth characteristics of the Agardhiella microplantlets within a stirred tank photobioreactor. The stirred tank bioreactor offers excellent gas-liquid mass transfer, a desired feature for mass cultivation of submerged phototrophic microplantlets that must rely on carbon dioxide in the aeration gas as the sole carbon source for growth. However, hydrodynamic shear forces generated by impeller rotation may have negative impacts on biomass production. Therefore, the first objective of this study was to look at the effect of impeller speed on CO2 delivery and long-term biomass

2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 12/24/2002

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Figure 1. Five hundred milliliter stirred tank photobioreactor.

production of Agardhiella microplantlet cultivated within a stirred tank bioreactor under semicontinuous nutrient medium replacement. The second objective of this study was to determine if increasing the CO2 partial pressure in the aeration gas would enhance biomass production in this bioreactor configuration. We report that the stirred tank bioreactor is a suitable configuration for long-term cultivation of Agardhiella microplantlets. However, biomass production at high plantlet density can be limited by the CO2 input associated with the aeration gas.

Materials and Methods

Culture Maintenance. Development, maintenance, and subculture of the axenic, phototrophic Agardhiella subulata microplantlet suspension culture is described in our previous work (Huang et al., 1998; Huang and Rorrer, 2002a). Individual microplantlets consisted of branched, terete shoots emanating from a central core thallus, with growth occurring at apical meristems. Shoot tissues were pigmented red and ranged from 3 to 10 mm in length. Microplantlet suspension cultures were maintained in 250-mL foam stoppered Erlenmeyer flasks. Each flask contained approximately 50 microplantlets in 100 mL of filter-sterilized ASP12 artificial seawater medium of composition described by Huang and Rorrer (2002a), supplemented with 10X nitrate (11.8 mM), 1X phosphate (0.045 mM), 10.0 mM bicarbonate buffer, and 5.0 mM Na-HEPES buffer. Flasks were cultured without agitation at 24 C under 40 mol photons m-2 s-1 cool white fluorescent light within an illuminated incubator with an illumination photoperiod of 10 h light/14 h dark on a 24 h cycle (10:14 LD). The medium was completely replaced every 2 weeks, and microplantlets were subcultured every 4 weeks. Prior to subculture, each microplantlet was cut through its central branch point into three pieces. The excised tissues contained less than 10 shoots per microplantlet. Elongated shoots greater than 10 mm were trimmed to 3-5 mm in length. Stirred Tank Photobioreactor. A schematic of the stirred tank photobioreactor system is presented in Figure 1. A Bellco jacketed spinner flask (model 196500500) with an inner diameter of 10.5 cm, height of 15 cm, working liquid volume of 500 mL, and working liquid height of about 7 cm served as the bioreactor vessel. The vessel jacket was connected to a circulating water bath set at 24 C. The bioreactor was equipped with a vertically pitched, Teflon paddle impeller of 2.5 cm height

and overall diameter of 5.5 cm (Di/DT ) 0.52), which was positioned 1.0 cm above the bottom of the vessel. The impeller was magnetically driven with a single 5.0 cm 1.0 cm Teflon stir bar aligned perpendicular to the paddle assembly. Air was mixed with carbon dioxide gas, sterile filtered, humidified, and then introduced into the liquid suspension by a porous sparger (Applikon Instruments Z811303008,15 m stainless steel mesh). The sparger produced fine bubbles of less than 1.0 mm diameter. External illumination to the vessel was provided by two 9 W Dulux compact fluorescent lamps, one lamp per side. The lamps were positioned 10.2 cm from the vessel centerline to provide an incident light intensity of 80 mol photons m-2 sec-1. The illumination photoperiod was 10 h light/14 h dark on a 24 h cycle (10:14 LD). Two identical 500-mL stirred tank photobioreactors were operated in parallel, each inoculated with microplantlets from a common inoculum. To prepare the inoculum, microplantlets from four randomly selected flask cultures (28 days after subculture) were pooled. Microplantlets were cut and trimmed as described earlier and then rinsed with filter-sterilized ASP12 medium under sterile filtration. The shoot tissues (ca. 0.5 g fresh weight) were then resuspended with 500 mL of fresh ASP12 medium and loaded into each autoclaved bioreactor vessel. The reference process cultivation conditions are given in Table 1. Every 5 days, the medium in the culture suspension was replaced, and the total fresh plantlet weight (FW) within each vessel was determined. Consequently, the medium replacement rate was 500 mL of medium per 500 mL reactor volume every 5 days, or 20% per day. On a given sampling day, the entire suspension within each 500-mL vessel was sterile-filtered under vacuum onto a 20-m nylon mesh filter (50 mm diameter). The filtered microplantlets and nylon mesh were placed into a sterile Petri dish and immediately weighed to precision of (0.001 g. The bioreactor vessel was reloaded with 500 mL of fresh ASP12 medium, and microplantlets were immediately transferred back to the bioreactor vessel. The nylon mesh and Petri dish were then reweighed, and the total fresh cell mass reloaded back to the reactor was determined. The pH of the filtered medium was also measured. At the end of the cultivation, the dry cell mass of the sample was measured as described by Huang and Rorrer (2002a), and the solids content of the biomass was expressed as the ratio of dry cell mass to fresh mass (g DW/g FW). The solids content ranged from 0.21 to 0.29 g DW/g FW. For the batch cultivation experiments, the medium was not replaced. Instead, the microplantlets were resuspended in the filtered medium, and the suspension was added back to the bioreactor vessel. All procedures were carried out using sterile technique within a laminar flow hood. Bubble-Column Photobioreactor. The details of the bubble-column photobioreactor design are detailed by Zhi and Rorrer (1996). Procedures for cultivating Agardhiella microplantlets in this bioreactor are described by Huang and Rorrer (2002b). The reference process cultivation conditions are summarized in Table 1. The medium was completely replaced every 5 days concurrent with fresh biomass weight measurement, as described above for the 500-mL stirred tank photobioreactor. Nitrate and Phosphate Measurements. The nitrate (NO3-) concentration and phosphate (PO4-3) concentration in the medium were assayed by spectrophotometric techniques described by Huang and Rorrer (2002a). For the batch cultivation experiments, the total molar con-

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Table 1. Process Conditions for Photobioreactor Cultivation of Agardhiella subulata Microplantlets in Liquid Suspension process variable cultivation volume, V vessel inner diameter ASP12 medium replacement rate temperature incident light intensity, Io planes of illumination, R photoperiod aeration rate measured kLa CO2 partial pressure CO2-TR nCO2 ASP12 medium pH 500-mL stirred tank (120 rpm) 500 mL 10.5 cm 500 mL every 5 days (20% per day) 24 C 80 mol photons m-2s-1 2 10 h:14 h LD 150 mL min-1 (0.3 L air L-1 min-1) 41.5 ( 4.2 h-1 0.00035 atm 0.405 mmol CO2 L-1 h-1 0.258 mmol CO2 L-1 h-1 8.7 250-mL bubble column 250 mL 4.5 cm 250 mL every 5 days (20% per day) 24 C 38 mol photons m-2 s-1 2 10 h:14 h LD 100 mL min-1 (0.4 L air L-1 min-1) 89.5 ( 2.3 h-1 0.00035 atm 0.873 mmol CO2 L-1 h-1 0.345 mmol CO2 L-1 h-1 8.7

sumption of nitrate was determined by

Table 2. Fitted Parameters for kLa Correlation Defined by Eq 3a parameter value 700 2.70 10-3 0.51 ( 0.10 1.52 ( 0.13 9 0.92

MN ) CN,0V0 - CN,fVf -

Vs,iCN,i i)1

(1)

where CN,0 and CN,f are the initial and final concentrations of nitrate in the liquid medium, V0 and Vf are the initial and final volumes of liquid medium in the vessel, Vs,i is the volume of liquid medium withdrawn for nitrate and phosphate analysis of the ith sample, and CN,i is the measured nitrate concentration in the ith sample. The biomass yield coefficient based on nitrate limit growth was calculated by the increase of biomass over the total consumption of nitrate:

A B a b n r2
a

Units of kLa are h-1, units of ug are m s-1.

where ug is superficial gas velocity (m s-1) of the aeration gas through the empty vessel, and Re is the impeller Reynolds number, defined as

YX/N )

(Wf - W0)ws MN

Re ) (2)

FLNi D2 i L

(4)

where W0 is the fresh weight of microplantlets in the vessel at inoculation, Wf is the average fresh weight of the microplantlets at the stationary phase of growth, and ws is the solids content of the biomass in the stationary phase of growth. Photosynthetic Oxygen Evolution Rate. In selected cultivation experiments, a 0.25 g fresh weight sample of the filtered microplantlet biomass was used for photosynthetic oxygen evolution rate (OER) measurements. Procedures for OER measurements with Agardhiella microplantlets are described by Huang et al. (1998) and by Huang and Rorrer (2002a). The specific OER was always measured 3 h into the 10:14 LD photoperiod. OER measurements within the OER test cell were carried out at an incident light intensity of 67 mol photons m-2 s-1 (two-sided illumination) and 24 C. Dark-phase oxygen respiration rate measurements were also conducted immediately after oxygen evolution rate measurements by wrapping the OER test cell in aluminum foil to exclude light. Volumetric Oxygen Mass Transfer Coefficient and Average Shear Rate. The volumetric mass transfer coefficient (kLa) for O2 within the stirred tank bioreactor was determined by the dynamic gassing-in method, as described by Huang and Rorrer (2002a). All measurements were conducted at 24 C with 500 mL of filtersterilized ASP12 liquid medium. Values of kLa were obtained at inlet gas flowrates ranging from 50 to 530 mL min-1 and impeller speeds ranging from 60 to 250 rpm. The measured kLa values were fitted into a power law correlation of the form

where Di is the impeller diameter (cm), Ni is the impeller speed (rev s-1), FL is the density of the ASP12 liquid medium with 10X nitrate (1.035 g/cm3 at 24 C), and L is the viscosity of this liquid medium (0.00926 g cm-1 s-1 at 24 C). The fitted parameters (A, B, a, b) obtained from nonlinear regression analysis of kLa vs ug and Re are summarized in Table 2. Figure 2a shows the correlation between the measured and predicted kLa. For an unbaffled, stirred vessel operated in the turbulent regime, the average shear rate Ya (Croughan et al., 1987) is estimated by

112.8Ni r1,8(r0.2 - r0.2) i t i Ya ) r2 - r 2 t i

( )
rc ri

1.8

(5)

where ri and rt are the radii of the impeller and tank respectively, and rc is the radius of the forced vortex zone, estimated by

rc Re ) ri 1000 + 1.6Re

(6)

CO2 Delivery. Carbon dioxide (CO2) in the delivery gas is the sole carbon source for photosynthetic growth. The interphase mass transfer rate (CO2-TR, mmol CO2 L-1 h-1) of carbon dioxide from the aeration gas to the well-mixed submerged culture is

CO2-TR ) kLa

DCO2 PCO2,in DO2 H

- CCO2

(7)

kLa ) Auga + BugaReb

(3)

where CCO2 is the concentration of dissolved CO2 in the

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Table 3. Nitrate-Limited Cultivation of Agardhiella subulata Microplantlets with No Medium Replacementa growth parameters X0 (g FW L-1) CN,0 (mmol NO3- L-1) CP,0 (mmol PO4-3 L-1) YX/N (g DW mmol-1 N) 1 (day-1), day 0-14 Average pH
a

bioreactor 1 1.26 1.18 0.45 1.007 ( 0.101 0.071 ( 0.009 8.8 ( 0.1

bioreactor 2 2.93 1.18 0.45 1.077 ( 0.092 0.062 ( 0.006 8.8 ( 0.1

All errors are ( 1.0 se.

Equation 11 maps the unique set of conditions for the aeration gas flowrate and impeller speed where CO2-TR and nCO2 are equal. This mapping is shown as the solid line in Figure 2b. Above the curve, CO2 delivery is set by eq 10, whereas below the curve it is set by eq 8. Microplantlet Size Distribution. At the end of a given cultivation experiment, 40 microplantlets were randomly selected from the filtered biomass. The longest and shortest axis of each plantlet was measured with a ruler under a dissecting microscope. The plantlet diameter (dp) was estimated by the average of the longest and shortest axes of a given plantlet.

Results and Discussion

Figure 2. Mass transfer characteristics of the 500-mL stirred tank bioreactor. (a) Measured vs predicted range of kLa by eq 3. (b) Regime where CO2-TR ) nCO2, given by eq 11 with parameters specified by Table 2.

liquid (mmol CO2 L-1), H is Henrys law constant for CO2 in 35 ppt seawater at 24 C (0.0339 atm mM-1), PCO2,in is the partial pressure of CO2 in the inlet aeration gas (3500 ppm or 0. 0035 atm), and DCO2/DO2 is the ratio of CO2 and O2 diffusion coefficients in seawater, equal to 0.93 (Raven, 1984). If CCO2 goes to zero, eq 7 becomes

CO2-TR ) kLa

DCO2 PCO2,in DO2 H

( )

(8)

Alternatively, the CO2 delivery rate per unit volume of culture (mmol CO2 L-1 h-1) can be defined as

nCO2 )

vg (PCO2,in - PCO2,out) V RT

(9)

where PCO2,out is the partial pressure of CO2 in the outlet aeration gas, R is the gas constant (8.206 10-5 L atm mmol-1 K-1), T is the temperature of the aeration gas (K), vg is the volumetric flowrate of the aeration gas (L aeration gas h-1), and V is the liquid culture volume (L culture). If PCO2,out goes to zero, then

nCO2,max =

vg (PCO2,in) V RT

(10)

If the CO2 demand by the culture is high, then CCO2 and PCO2,out will go to zero. Furthermore, if kLa is relatively high and vg is relatively low, then eq 10 will limit the maximum possible rate of CO2 delivery. A criterion for determining if CO2-TR or nCO2 sets the CO2 delivery rate is now needed. Combination of eqs 3, 8, and 10 yields

Auga + BugaReb )

vgH DCO2 RTV DO2

( )

-1/2

(11)

Three limiting factors on sustained biomass production within the stirred tank photobioreactor were studied. First, biomass production with and without medium replacement was compared. Second, the long-term effects of CO2 delivery on sustained biomass production under medium replacement were considered. Third, the effects of impeller speed on CO2-limited biomass production under medium replacement were assessed. Concurrently, the morphological features of the microplantlets under sustained biomass growth within the stirred tank photobioreactor were characterized. Biomass Production without Medium Replacement (Batch Cultivation). The batch cultivation of the Agardhiella microplantlet suspension was studied within the 500-mL stirred tank photobioreactor at an N:P ratio that ultimately depleted nitrate, but not phosphate, from the culture medium. Two cultivation experiments were conducted in parallel, inoculated at 1.26 and 2.93 g FW L-1, respectively. The medium was not replaced over the course of the experiment. Growth parameters are presented in Table 3. The initial nitrate and phosphate concentrations in the medium for each cultivation experiment were 1.28 and 0.45 mM, respectively, to provide an initial N:P ratio of 2.7:1. This N:P ratio was designed to move the cultivation toward nitrate limitation, as typically the N:P ratio within the biomass of benthic macroalgae is centered around 30:1 (Atkinson and Smith, 1983), an order of magnitude higher. Plantlet biomass production, nitrate consumption, and phosphate consumption profiles are presented in Figure 3. Biomass production reached stationary phase when the nitrate concentration fell below 200 , showing the effects of nitrogen limitation on photosynthetic algal growth (Turpin, 1991). The phosphate concentration continued to decrease after culture reached stationary phase, suggesting that phosphate was assimilated and stored without being incorporated into new cell biomass. At the end of the cultivation experiments, the phosphate concentration was at least 60 M. Previous studies on phosphate-limited growth of field-collected Agardhiella plants showed that the specific growth rate was saturated at seawater phosphate concentrations of 10 M, and the phosphorus content of the biomass was saturated at 20 M (Chopin

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Figure 4. Comparison of growth curves for Agardhiella subulata microplantlets in the 500-mL stirred tank and 250-mL bubble-column photobioreactors under a medium replacement rate of 20% per day.

Figure 3. Macronutrient consumption of Agardhiella subulata microplantlets cultivated in the stirred tank photobioreactor with no medium replacement (batch cultivation). (a) Nitrate consumption and biomass production vs time. (b) Phosphate consumption and biomass production vs time. Initial liquid volume was 580 mL.

et al., 1990). Consequently, the microplantlets were not limited by phosphate, and the stationary phase was triggered by depletion of nitrate from the medium. The biomass yield coefficient based on 100% nitrate consumption (YX/N) was nominally 1.01-1.08 g DW mmol-1N. In our previous work, the biomass yield coefficient based on phosphate-limited growth at 100% phosphate consumption (YX/P) was 25.8 g DW mmol-1 P (Huang et al., 2002a). Therefore, the required N:P ratio for Agardhiella microplantlet biomass is 24:1. Assuming an N:P ratio of 24:1 and a biomass yield coefficient of 1.08 g DW per mmol N, the overall photosynthetic biomass stoichiometry is

848CO2 + 872H2O + 24NO3- + PO4-3 f (CH2O)848(NH2)24(PO4) + 896 O2 (12)

Biomass Production with Medium Replacement. Representative biomass growth curves of Agardhiella microplantlets cultivated within the 250-mL bubble column photobioreactor and 500-mL stirred tank photobioreactor at medium replacement rate of 20% per day are compared in Figure 4. There were no significant differences in biomass productivity between these two bioreactor configurations at the process conditions provided in Table 1. Furthermore, under a nutrient replacement rate of 20% per day, biomass production was sustained for 85 days without reaching the stationary phase of growth. This extended growth phase is desirable for the study of the long-term effects of process variables

Figure 5. Comparison of long-term growth of Agardhiella subulata microplantlets in the stirred tank photobioreactor at impeller speeds of 140 and 250 rpm under a medium replacement rate of 20% per day. (a) Semilog plot at 250 rpm showing three growth regimes: 1 and 2 denote the exponential phases for 1 and 2, respectively, and 3 denotes the CO2-limited growth phase. (b) Comparison of growth curves at 140 and 250 rpm.

on microplantlet growth, including CO2 delivery and agitation intensity, as described next. Growth Regimes under Cultivation with Medium Replacement. Agardhiella microplantlet suspensions cultivated within the stirred tank photobioreactor with 20% per day nutrient medium replacement passed through three phases of growth, as shown in Figure 5a. These include two exponential phases of growth, followed by a linear, CO2-limited growth phase. In previous work, we showed that two exponential phases of growth were

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Table 4. Effect of Impeller Speed on Growth Parameters for Agardhiella subulata Microplantlets in Stirred Tank Photobioreactora parameter impeller speed impeller Re tip speed (cm s-1) Ya (s-1) 1 (day-1) 2 (day-1) Xo (g FW L-1) Xf (g FW L-1) tf (days) Xc (g FW L-1) tc (days) Im (mol photons m-2 s-1) nCO2 (mmol CO2 L-1 h-1) CO2-TR (mmol CO2 L-1 h-1) culture pH theory (eq 18a or b) (g FW L-1 day-1) measured (g FW L-1 day-1) 60 rpm 3380 17.3 1.86 0.076 ( 0.006 (0-21 days) 0.022 ( 0.005 (21-43 days) 1.12 10.5 43 11.4 105 26 0.258 0.209 8.83 ( 0.06 0.252 b experiment 120 rpm 6759 34.6 4.31 0.067 ( 0.012 (0-15 days) 0.024 ( 0.001 (15-85 days) 0.98 16.3 85 14.0 84 20 0.258 0.394 8.79 ( 0.09 0.310 b 140 rpm 7886 40.3 5.14 0.066 ( 0.010 (0-10 days) 0.030 ( 0.002 (10-47 days) 1.53 17.7 91 10.3 52 19 0.258 0.469 8.83 ( 0.09 0.310 0.221 ( 0.029 (56-91 days) 0.326 ( 0.048 (56-76 days)

423

250 rpm 14082 72.0 9.74 0.070 ( 0.009 (0-10 days) 0.028 ( 0.002 (10-47 days) 1.32 14.7 91 11.0 61 21 0.258 0.976 8.74 ( 0.25 0.310 0.152 ( 0.019 (56-91 days) 0.253 ( 0.014 (56-76 days)

a Aeration rate 0.3 L air L-1 culture min-1, CO partial pressure of 0.0035 atm (350 ppm). All errors are (1.0 se. b CO -limited growth 2 2 phase not observed.

observed during medium perfusion cultivation of Agardhiella microplantlets (Huang et al., 2002a). The first exponential phase of growth was characterized by a burst of photosynthetic activity in the 2 weeks following inoculation, which was the result of cutting and trimming the microplantlet shoot tissues. A second, sustained exponential phase of growth occurred after the cut shoots fully recovered, as evidenced by a stable specific OER vs time profile. During the exponential phases of growth under medium replacement cultivation, the growth curves are given by

Xc )

CO2-TRYX/CO2 f ws2

or Xc )

nCO2YX/CO2 f ws2

(16a,b)

Beyond the critical biomass density (Xc), sustained biomass production is limited by the rate of CO2 delivery, and the growth curve is linear:

Xt ) Xc + (t - tc)

for t > tc

(17)

X(t) ) X0 e1t

for t ) 0 to t1 for t g t1

(13a) (13b)

where is biomass production rate under CO2-limited growth (g FW L-1 day-1). If all the CO2 delivered to the culture is consumed, then the theoretical limit on is given by

X(t) ) Xt1 e2(t-t1)

CO2-TRYX/CO2f ws

or )

nCO2YX/CO2 f ws

(18a,b)

In the first exponential phase of growth, the biomass density is low enough so that the volumetric rate of CO2 demand by the culture suspension is below the maximum possible CO2 delivery rate. However, in the second exponential phase of growth, the biomass density will eventually reach a point where the volumetric CO2 consumption rate reaches the CO2 delivery rate, i.e.

Finally, tc is the cultivation time at which CO2-limited growth begins, which is estimated by

tc ) t1 +

Xc 1 ln 2 Xt1

()

(19)

QCO2 ) CO2-TR or QCO2 ) nCO2

(14)

The volumetric CO2 consumption rate is in turn related to the biomass production rate by

QCO2YX/CO2 f ws

) 2X

(15)

where YX/CO2 is the biomass yield coefficient based on CO2 consumption, equal to 30 mg DW mmol-1 CO2 for Calvin photosynthesis stoichiometry, f is the fractional photoperiod (10 h illumination per 24 h day), and ws is the solids content of the biomass (g DW/g FW). Consequently, the critical biomass density at the point of CO2-limited growth, Xc, is estimated by

Using eq 17, the measured is estimated from the slope of X vs t data at t > tc. Likewise, specific growth rates 1 and 2 are estimated by fitting growth curve data to time windows of 0 < t < t1 and t1 < t < tc, respectively. Representative growth curves for Agardhiella microplantlets over extended cultivation times of 91 days (13 weeks) are presented in Figure 5. These cultivation experiments were carried out at a medium replacement rate of 20% per day to sustain microplantlet growth so that all three phases of biomass production could be readily observed. Table 4 compares the growth parameters for each growth phase. The predicted growth curve lines in Figure 5 were generated from eqs 13 and 17, using estimates for 1, 2, and obtained by experiment. Measured values for were at or below the theoretical limit on as specified by eq 18. The type of CO2 delivery limitation was addressed by comparing values for CO2-TR defined by eq 8 to values

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Table 5. Effect of pH Environment on Long-Term Biomass Production of Agardhiella subulata Microplantlets in Stirred Tank Photobioreactor at 120 rpma process variable CO2 partial pressure (atm) cultivation pH aeration rate (L air L-1 culture min-1) 1 (day-1) 2 (day-1) Xc (g FW L-1) tc (days) bioreactor 1 0.00035 8.79 ( 0.09 0.300 0.067 ( 0.012 (day 0-15) 0.024 ( 0.001 (day 15-85) 14.0 84 bioreactor 2 0.0072 7.88 ( 0.09 0.584 0.078 ( 0.002 (day 0-15) 0.028 ( 0.001 (day 15-85) b b

Figure 6. Comparison of long-term growth of Agardhiella subulata microplantlets in the stirred tank photobioreactor at pH 7.8 (0.0072 atm CO2 in aeration gas) and pH 8.8 (0.00035 atm CO2 in aeration gas). The medium replacement rate was 20% per day.

a All errors are (1.0 se. b CO -limited growth phase not ob2 served.

(HCO3-) and carbonate (CO3-2) according to the equilibrium expressions

for nCO2 estimated by eq 10, and by use of Figure 2b. Ifthe impeller speed was above 60 rpm, then CO2 delivery was ultimately limited by the molar flowrate of CO2 in the aeration gas (nCO2) and not interphase mass transfer (CO2-TR). Light transfer limitations on photosynthetic growth of Agardhiella microplantlets in the 500-mL stirred tank photobioreactor are considered below. In previous work, we characterized the effects of light intensity and photoperiod on the intrinsic growth kinetics of Agardhiella microplantlets (Huang et al., 1998; Huang and Rorrer, 2002b) and developed a mean light intensity criterion to assess the importance of light attenuation through the microplantlet suspension (Huang and Rorrer, 2002a). The mean light intensity (Im) is estimated by

CO2(g) T CO2(aq) CO2(aq) + H2O(g) T H2CO3 T

(21a)

HCO3- + H+ and HCO3- T CO3-2 + H+ (21b,c)

where the pKa values for H2CO3 and HCO3- dissociation in seawater at 24 C are 6.05 and 9.10, respectively (Raven, 1984). Consequently, increasing the CO2 partial pressure will decrease the pH at a given bicarbonate buffer concentration. Concurrently, the Na-HEPES buffer (5.0 mM, pKa ) 7.5) in the ASP12 medium buffers the pH increase in response to dissolved CO2 demand by the suspension culture at pH 8-9. The long-term cultivation of Agardhiella microplantlets in the stirred tank photobioreactor at aeration gas partial pressures of 0.00035 and 0.0072 atm are presented in Figure 6. Each cultivation experiment was carried out at a medium replacement rate of 20% per day. Increasing the aeration gas CO2 partial pressure from 0.00035 to 0.0072 atm decreased the culture pH from 8.8 to 7.8. At the elevated CO2 partial pressure of 0.0072 atm and pH of 7.8, the cumulative biomass production after 85 days was 25 versus 16 g FW L-1 for the control cultivation at pH 8.8. During the 85 day cultivation period, the CO2limited growth phase was not clearly observed. However, a comparison of the growth parameters in Table 5 shows that increasing the CO2 partial pressure still increased the specific growth rates. Consequently, the growth curves shown in Figure 6 reflect the effect of pH environment on biomass production. On the basis of the results obtained in Figure 6, the coupled effect of aeration gas CO2 partial pressure on culture pH and specific oxygen evolution rate (OER) of the Agardhiella microplantlets was investigated. The results are presented in Figure 7 and Table 6. The specific OER is indicative of the photosynthetic growth activity of the culture. The experiment had two stages. In the first stage, from day 0 to day 41, the aeration gas CO2 partial pressure was fixed at 0.00035 atm to provide a culture pH of nominally 8.8-8.9. In the second stage, from day 42 to day 60, the CO2 partial pressure was incrementally increased then decreased with time according to the profile shown in Figure 7a to provide the pH profile shown in Figure 7b. The culture pH was bounded within the nominal physiological range of 6.59.0, and the culture pH equilibrated with the new CO2 partial pressure within 2 h following the step change in CO2 aeration gas level. In the first stage, the specific

Im(X) )

RIo (ko + kcX)DT

(1 - e-(ko+kcX)DT)

(20)

where Io is the incident light intensity to the culture measured at the inner surface of the vessel, R is the number of planes of illumination (R ) 2 for two-sided illumination), ko is the light attenuation constant for the liquid medium (0.241 cm-1), and kc is the light attenuation constant for the microplantlets (0.033 L cm-1 g-1 FW). Estimates for Im within the stirred tank photobioreactor at the end of the cultivation are presented in Table 4. In previous work, we determined that the light intensity at 63.2% of light-saturated growth (Ik) for Agardhiella microplantlets ranged from 14 to 19 mol photons m-2 s-1. The Im values at the end of the cultivation are above this value. Cultivation Under Supplemental CO2 Addition and Nutrient Replacement. Figure 5 showed that cultivation of Agardhiella microplantlet suspensions in the 500-mL stirred tank photobioreactor at a CO2 partial pressure of 0.00035 atm (350 ppm) in the aeration gas and a medium replacement rate of 20% per day were ultimately subject to CO2-limited growth. If the CO2 partial pressure in the aeration gas is increased, then the CO2 delivery rate is increased by eq 8, and hence the exponential phases of growth will be prolonged, as proposed by eqs 16 and 19. Furthermore, the CO2 partial pressure affects the culture pH. Specifically, at pH 8-9, dissolved CO2 in seawater speciates into bicarbonate

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425

Figure 8. Photographs of photobioreactor cultured Agardhiella subulata microplantlets. (a) Microplantlets cultivated for 43 days in the stirred tank photobioreactor without medium replacement. (b) Microplantlets cultivated in the bubble column photobioreactor.

Figure 7. Effect of aeration gas CO2 partial pressure on photosynthesis of Agardhiella subulata microplantlets within the 500-mL stirred tank photobioreactor. The medium replacement rate was 20% per day for days 0-24 and then 50% per day for days 24-66. (a) CO2 partial pressure vs time profile. (b) Specific OER and culture pH vs time profile.
Table 6. Effect of pH on Photosynthetic Growth Characteristics of Agardhiella subulata Microplantlets in Stirred Tank Photobioreactor at 120 rpma process variable CO2 partial pressure (atm) cultivation pH av specific growth rate (day-1) specific OER, PO (mmol O2 g-1 DW h-1) respiration rate, QO (mmol O2 g-1 DW h-1)
a

bioreactor 1 0.00035 (day 0-41) 8.84 ( 0.07 (day 0-24) 0.055 ( 0.007 (day 0-24) 0.106 ( 0.021 (day 19-41) 0.028 ( 0.013 (day 19-41)

bioreactor 2 0.0072 (day 0-30) 7.84 ( 0.23 (day 0-24) 0.074 ( 0.010 (day 0-24) 0.193 ( 0.014 (day 19-32) 0.048 ( 0.007 (day 19-32)

All errors are (1.0 se.

oxygen evolution rate decreased slowly during CO2limited growth from day 19 to 41. In the second stage, as CO2 partial pressure was incrementally increased to 0.18 atm and the pH was incrementally lowered from 8.9 to 6.7, the specific OER went through a sharp optimum at pH 7.8 and partial pressure of 0.0072 atm. Past day 50, the specific OER was relatively insensitive to increasing the pH back up from 6.7 to 8.0. These results, combined with photosynthetic measurements within the first 41 days of cultivation (Table 7) concur that 0.0072 atm CO2 partial pressure and culture pH of 7.8 maximized the growth of Agardhiella microplantlets. Effect of Impeller Speed on Cultivation with Medium Replacement. If the impeller Re exceeds 1000, then flow field is turbulent (Nagata, 1975). As shown in Table 4, all stirred tank experiments were agitated in

the turbulent regime with the impeller Re ranging from 3380 (60 rpm) to 14083 (250 rpm). The minimum impeller speed to uniformly suspend the microplantlet suspension was 60 rpm, whereas the fastest impeller speed achievable in the 500-mL Bellco stirred tank bioreactor vessel was 250 rpm. Under these agitation conditions, average shear rates ranging from 2 to 10 s-1 at impeller tip speeds of 27 to 72 cm s-1 were obtained. The specific growth rates for the first and second exponential growth phases were not significantly affected by impeller speed (Table 4). However, if culture growth reached the CO2-limited growth phase, then the measured value of at 250 rpm was only 70% of that at 140 rpm (Table 4, Figure 5). This suggests that biomass production in this growth regime decreased as impeller speed increased. Even so, no physical damage to the shoot tissues was observed at the end of these stirred tank cultivation experiments. The average shear rate range of 2-10 s-1 is considered harmful for many other cell cultures. The critical shear rate for mammalian cell cultures grown in a stirred tank bioreactor similar to this study is 3-7 s-1 (Croughan et al., 1987). Furthermore, the flow regime map developed by Doran (1999) for stirred tank bioreactors suggests that the cultivation conditions used in this study, Di/DT ratio of 0.5, aeration rate of 0.3 L air L-1 min-1, and impeller speed of 250 rpm, would lie in the damage region for plant cell suspension cultures. Growth Morphology. Photographs of Agardhiella microplantlets cultivated in the stirred tank photobioreactor are presented in Figure 8. For microplantlets cultivated within the bubble-column photobioreactor, primary shoots and their secondary branches tended to elongate freely, resulting in an asymmetric shape. However, for microplantlets cultivated within the stirred tank bioreactor, linear nonbranched shoots emanated from the central core, and the overall shape of the plantlets was spherical and compact. The fluid mixing pattern and physical contact of impeller with the plantlets within the stirred tank may have forced microplantlets to grow into this compact and spherical shape. Previously, we reported that Agardhiella microplantlets cultivated within a bubble column photobioreactor were not friable, and the plantlet number density remained constant during the cultivation period (Huang and Rorrer, 2002a). Microplantlets cultivated in the stirred tank photobioreactor also did not break apart. Clearly, bioreactor hydrody-

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CO2 partial pressure of 0.0072 atm and cultivation pH of 7.8. Impeller speeds ranging from 60 to 250 rpm, which generated average shear rates of 2-10 s-1, did not have a significant effect on biomass production rate. Microplantlet tissues cultivated in a stirred tank bioreactor ultimately assumed a compact spherical shape in order to minimize their exposure to hydrodynamic stresses generated by impeller rotation and aeration.

Notation
CN CP
Figure 9. Size distribution of Agardhiella subulata microplantlets after 43 days of batch cultivation in the 500-mL stirred tank photobioreactor at two different inoculum levels. The distribution frequency was based on 1.0 mm increments in plantlet diameter from 2.5 to 14.5 mm. The solid lines are the predicted Gaussian distributions.
Table 7. Size Distribution of Agardhiella subulata Microplantlets in Stirred Tank Photobioreactor after 43 Days of Cultivation at Impeller Speed of 120 rpma growth parameters Xo (g FW X at 43 days (g FW L-1) av dp (mm)
a

CO2-TR dp Di DT f H kc kLa (kLa)CO2 ko Ik Im Io nCO2 Ni PCO2 PO QO QCO2 R Re t t1 tc T ug vg V ws X Xc Ya YX/CO2

bioreactor 1 1.26 6.58 7.5 ( 1.9

bioreactor 2 2.93 9.25 5.3 ( 1.4

L-1)

Sample size was 40 plantlets.

namics play a role in biomass production of microplantlet tissues derived from the red algae, as higher agitation intensities within a stirred tank bioreactor drive the microplantlet growth morphology to a compact spherical shape in order to minimize exposure to hydrodynamic stress. Figure 9 compares final size distribution of microplantlets from the nitrate-limited batch cultivation experiments at two initial biomass densities of 1.26 and 2.93 g FW L-1 after 43 days of cultivation. The average diameters are presented in Table 7. The inoculum of 2.93 g FW L-1 had approximately twice as many plantlets as the inoculum of 1.26 g FW L-1. As a result, the final size of the spherical microplantlets increased when the inoculum density was decreased, because the number of plantlets in the vessel was smaller, but the total nutrient loaded to each vessel was the same. Consequently, for batch cultivations, the final plantlet size is determined from the inoculum density, the initial plantlet size, and initial loading of nutrients.

Conclusions
The limiting factors on the biomass production of phototrophic Agardhiella subulata microplantlet suspension cultures within a stirred tank photobioreactor have been assessed. A stirred tank photobioreactor equipped with a paddle blade impeller was suitable for the longterm cultivation of A. subulata microplantlets in liquid suspension. Nutrient medium replacement was required for sustained biomass production. Biomass production went through two exponential phases of growth, followed by a CO2 delivery limited growth phase. If the impeller speed was greater than 60 rpm, then the interphase mass transfer rate for CO2 (CO2-TR) was sufficiently high so that CO2 delivery was ultimately limited by nCO2, the molar flowrate of CO2 in the aeration gas per unit volume of culture. The specific growth rate and photosynthetic oxygen evolution rate were maximized at an aeration gas

nitrate concentration in culture liquid (mmol L-1) phosphate concentration in culture liquid (mmol L-1) interphase mass transfer rate of CO2 (mmol CO2 L-1 h-1) diameter of spherical shaped microplantlets (mm) diameter of impeller assembly (cm) inner diameter of bioreactor vessel (cm) illumination portion of the 24 h photoperiod (h day-1) Henrys law constant for dissolution of CO2 gas in seawater (atm L mmol-1) specific light attenuation constant (L g-1 FW cm-1) volumetric mass transfer coefficient for O2 (h-1) volumetric mass transfer coefficient for CO2 (h-1) light attenuation constant of liquid medium (cm-1) light intensity at 63% of photosynthetic saturation (mol photons m-2 s-1) mean light intensity within microplantlet culture suspension (mol photons m-2 s-1) incident light intensity to vessel surface (mol photons m-2 s-1) CO2 delivery by aeration gas per unit culture volume (mmol CO2 L-1 h-1) impeller speed (rpm) partial pressure of CO2 in the aeration gas (atm) specific O2 evolution rate of microplantlet biomass (mmol O2 g-1 DW h-1) specific dark-phase O2 respiration rate of microplantlet biomass (mmol O2 g-1 DW h-1) volumetric rate of CO2 consumption in suspension culture (mmol CO2 L-1 h-1) gas constant (0.08206 L atm mol-1 K-1) impeller Reynolds number (dimensionless) cultivation time (days) cultivation time for first log phase of growth (days) cultivation time at which microplantlet growth becomes CO2-limited (days) cultivation temperature (C) superficial gas velocity of aeration gas through empty bioreactor vessel (m s-1) volumetric flowrate of aeration gas (mL min-1) culture volume (mL or L) solids content of microplantlet biomass (g DW/g FW) biomass density of microplantlets in liquid suspension (g FW L-1) biomass density at which microplantlet growth becomes CO2-limited (g FW L-1) average shear rate in stirred tank (s-1) biomass yield coefficient based on CO2 consumption (mg DW mmol-1 O2)

Biotechnol. Prog., 2003, Vol. 19, No. 2 YX/N R 1 2 L FL 0 f i biomass yield coefficient based on N consumption (mg DW mmol-1 N) number of planes of illumination to photobioreactor vessel surface CO2-limited growth rate (g FW L-1 day-1) specific growth rate of microplantlets in first log phase of growth (day-1) specific growth rate of microplantlets in second log phase growth (day-1) viscosity of liquid medium (g cm-1 s-1) density of liquid medium (g cm-3)

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Additional Sub- and Superscripts initial, at beginning of cultivation final, at end of cultivation ith sample

Abbreviations DW FW OER dry cell mass wet (fresh) cell mass oxygen evolution rate

Acknowledgment
Support for this research was provided by the National Science Foundation, Division of Bioengineering and Environmental Systems (BES-9810797).

References and Notes

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Accepted for publication October 25, 2002. BP020123I