AN ABSTRACT OF THE THESIS OF
Sundar Ramanan for the degree of Master of Science in Chemical Engineering presented
on December 17, 1996. Title: Biomass Productivity Enhancement of Laminaria
saccharina Cultures in a Stirred-Tank Bioreactor by Batch and Fed-Batch Nutrient
Delivery.
Abstrac
Gregory L. Rorrer
The biomass productivity of filametous gametophyte cell suspension cultures
derived from the marine brown alga Laminaria saccharina were studied under varying
nutrient concentrations. Two modes of nutrient addition were considered, including
batch addition and fed-batch addition of nutrients. The concentrations of nutrient
components were varied either individually or as a group. A maximum cell density of
1663 mg DCW/L was achieved when the cultures were fed at a rate of 21.5 % day' with
total GP2 nutrients. The culture was not limited by nitrate. In all the elevated nutrient
experiments the chl a concentration increased over the control. The removal of nutrients
at stationary phase ultimately decreased cell density by about 30%.
Biomass Productivity Enhancement of Laminaria saccharin Cultures in a Stirred-Tank
Bioreactor by Batch and Fed-Batch Nutrient Delivery
by
Sundar Ramanan
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented December 17, 1996
Commencement June 1997
Master of Science thesis of Sundar Ramanan presented on December 17, 1996
APPROVED:
Major Professor, representing Chemical Engineering
Head or Chair of Department of Chemical Engineering
Dean of Graduate S, hool
I understand that my thesis will become part of the permanent collecton of Oregon State
University libraries. My signature below authorizes release of my thesis to any reader
upon request.
Redacted for privacy
Sundar Ramanan, Author
ACKNOWLEDGMENT
I would like to extend my gratitude to my major professor Dr. Gregory Rorrer
who taught me the science of doing research.
I also would like to thank my friends in the laboratory at Oregon State campus and
beyond who helped me in various aspects during this program.
I am indebted to my parents and my sister for their love and support.
This work was supported by grant no. NA36RG0451 (project no. R/BT-8) from
the National Oceanic and Atmospheric Administration to the Oregon State University Sea
Grant College Program under the National Sea Grant Program Marine Biotechnology
Initiative.
TABLE OF CONTENTS
Page
INTRODUCTION
1
MATERIALS AND METHODS
Culture Maintenance
Bioreactor Design and Opearation
Nutrient Addition
Nutrient Removal
Culture Growth Measurements
Nitrate Measurements
15-HETE Measurement by EIA
RESULTS
Control Experiments
Batch Addition of Individual Nutrients
Batch Addition of Total Nutrients
Fed-Batch Addition of Individual Nutrients
Fed-Batch Addition of Individual Nutrients
Nutrient Removal
Light Attenuation Estimates
Model Calculations
4
4
7
7
9
10
10
11
14
15
19
19
35
44
60
67
70
DISCUSSION
75
BIBLIOGRAPHY
79
APPENDICES
81
Appendix A: Tabulated Data
Appendix B: Nitrate Analysis
Appendix C: 15 -HETE Measurement by EIA
Appendix D: Nutrient Addition in a Stirred-Tank Reactor
Appendix E: Laminaria saccharina Decontamination Protocol
Appendix F: Nutrient Removal in a Stirred-Tank Reactor
Appendix G: Temperature in Four-Reactor Incubator System
Appendix H: Control Run Repeatability
82
155
163
173
176
178
185
187
LIST OF FIGURES
Figure
1.
Page
Chl a concentration and pH profile in culture vs time for control
cultivation(Run 8-1)
17
Dry cell density and nitrate concentration in culture vs time for control
cultivation (Run 8-1)
18
Chl a concentration in culture vs time for batch addition of individual
nutrients (Run 5)
22
Dry cell density of culture vs time for batch addition of individual
nutrients (Run 5)
23
5.
pH of culture vs time for batch addition of individual nutrients (Run 5)
24
6.
Nitrate concentration in culture vs time for batch addition of individual
nutrients (Run 5)
25
Chl a concentration in culture vs time for batch addition of
total nutrients (Run 6)
26
2.
3.
4.
7.
8.
Dry cell density of culture vs time for batch addition of total nutrients (Run 6) 27
9.
pH of culture vs time for batch addition of total nutrients (Run 6)
28
10.
Chl a concentration in culture vs time for batch addition of
total nutrients (Run 10)
29
Dry cell density of culture vs time for batch addition of
total nutrients (Run 10)
30
12.
pH of culture vs time for batch addition of total nutrients (Run 10)
31
13.
Chl a concentration in culture vs time for batch addition of
total nutrients (Run 9)
32
11.
14.
Dry cell density of culture vs time for batch addition of total nutrients (Run 9) 33
15.
pH of culture vs time for batch addition of total nutrients (Run 9)
34
LIST OF FIGURES (Continued)
Figure
16.
17.
18.
19.
Page
Nitrate concentration in culture vs time for batch addition of
total nutrients (Run 6)
37
Nitrate concentration in culture vs time for batch addition of
total nutrients (Run 10)
38
Chl a concentration in culture vs time for fed-batch addition of
individual nutrients (Run 4)
39
Dry cell density of culture vs time for fed-batch addition of
individual nutrients (Run 4)
40
20.
pH of culture vs time for fed-batch addition of individual nutrients (Run 4)
21.
Chi a concentration in culture vs time for fed-batch addition of
individual nutrients (Run 7)
41
Dry cell density of culture vs time for fed-batch addition of
individual nutrients (Run 7)
42
23.
pH of culture vs time for fed-batch addition of individual nutrients (Run 7)
43
24.
Nitrate concentration in culture vs time for fed-batch addition of
individual nutrients (Run 4)
46
Nitrate concentration in culture vs time for fed-batch addition of
individual nutrients(Run 7)
47
Chl a concentration in culture vs time for fed-batch addition of
total nutrients (Run 6)
48
Dry cell density of culture vs time for fed-batch addition of
total nutrients (Run 6)
49
28.
pH of culture vs time for fed-batch addition of total nutrients (Run 6)
50
29.
Chl a concentration in culture vs time for fed-batch addition of
total nutrients (Run 8)
51
Dry cell density of culture vs time for fed-batch addition of
total nutrients (Run 8)
52
22.
25.
26.
27.
30.
40a
LIST OF FIGURES (Continued)
Figure
Eage
31.
pH of culture vs time for fed-batch addition of total nutrients (Run 8)
53
32.
Chl a concentration in culture vs time for fed-batch addition of
total nutrients (Run 11)
54
Dry cell density of culture vs time for fed-batch addition of
total nutrients (Run 11)
55
34.
pH of culture vs time for fed-batch addition of total nutrients (Run 11)
56
35.
Chl a concentration in culture vs time for fed-batch addition of
total nutrients (Run 9)
57
Dry cell density of culture vs time for fed-batch addition of
total nutrients (Run 9)
58
37.
pH of culture vs time for fed-hatch addition of total nutrients (Run 9)
59
38.
Nitrate concentration in culture vs time for batch addition of
total nutrients (Run 8)
62
Nitrate concentration in culture vs time for batch addition of
total nutrients (Run 11)
63
Dry cell density and pH profile for nutrient removal from
culture vs time. (Run BJ-1)
64
Dry cell density and pH profile for nutrient removal from
culture vs time. (Run BJ-2)
65
42.
Semi-log plot for estimation of p. and lcd. (Run BJ-1)
66
43.
Plot of estimated incident light intensity vs cell density
69
44.
VS S-model predictions for batch addition of total nutrients
73
45.
VSS model predictions for fed-batch total nutrient addition
74
33.
36.
39.
40.
41.
LIST OF TABLES
Table
Elm
1.
GP2 artificial seawater base medium composition
5
2.
GP2 nutrient medium composition
6
3.
Stirred-Tank bioreactor cultivation conditions
16
4.
Batch addition of individual nutrients
20
5.
Batch addition of total nutrients
21
6.
Fed-batch addition of individual nutrients
36
7.
Fed-batch addition of total nutrients
45
8.
Nutrient removal
61
9.
VSS input parameters
72
LIST OF APPENDIX FIGURES
Table
Pate
B-1.
Calibration curve for nitrate standard versus absorbance at 560 nm
158
C-1.
EIA calibration of 15-HETE standards versus abosorbance
at 405 nm by semi-log plot
169
EIA calibration of 15-HETE standards versus abosorbance
at 405 nm by logistic equation
171
C-2.
LIST OF APPENDIX TABLES
Table
Page
A-1.
Laminaria saccharina gametophyte cell cultivation Run BS-4
83
A-2.
Laminaria saccharina gametophyte cell cultivation Run BS-5
92
A-3.
Laminaria saccharina gametophyte cell cultivation Run BS-6
101
A-4.
Laminaria saccharina gametophyte cell cultivation Run BS-7
110
A-5.
Laminaria saccharina gametophyte cell cultivation Run BS-8
119
A-6.
Laminaria saccharina gametophyte cell cultivation Run BS-9
128
A-7.
Laminaria saccharina gametophyte cell cultivation Run BS-10
137
A-8.
Laminaria saccharina gametophyte cell cultivation Run BS-11
146
B-1.
Calibration data of nitrate standard versus absorbance at 560 nm
160
B-2.
Estimation of the yield coefficent for nirate
stirred-tank bioreactor
161
B-3.
in a
Nitrate analysis data summary from stirred-tank cultures
of L. saccharina
162
EIA calibration of 15-HETE standards versus absorbance at 405 nm
by semi-log plot
170
C-2.
Quantification of 15-HETE by EIA
172
F-1.
Nutrient removal studies Run BJ-1
179
F-2.
Nutrient removal studies Run BJ-2
182
G-1.
Temperatures in the four-reactor incubator system
186
H-1.
Control run repeatability
188
C-1.
NOMENCLATURE
A405
absorbance at light wavelength 405 nm, mAU
A560
absorbance at light wavelength 560 nm, mAU
al
adjustable parameter in logistic equation
a2
adjustable parameter in logistic equation
a3
adjustable parameter in logistic equation
CF
ratio of final nutrient concentration in any culture to the initial nutrient
concentration of the control culture
CI
ratio of initial nutrient concentration to the "lx" nutrient concentration
CN
nutrient concentration in the culture, mmole 1:11
CN,f
final nutrient concentration in the culture, mmole
CN,i
initial nutrient concentration in the culture, mmole
CN,i(1)
initial nutrient concentration at the "lx" level in the culture, mmole
CN,j
predicted nutrient concentration after feed nutrient addition step "j",
mmole L-1
CN,o
feed nutrient concentration, mmole
CN,0(1x)
feed nutrient concentration at "lx" level, mmole
Co
ratio of feed nutrient concentration to nutrient concentration at "lx" level
C15-HETE
concentration of 15-HETE, pg/mL
F
feed inlet flow rate, L day-1
Im
mean light intensity, IJE/m2sec
Io
incident light intensity, RE/m2sec
Ice
Beer's Law constant for biomass suspension, cm2/mg DCW
ka
specific death rate, day1
KN
half-saturation constant, mmole NO3- L-1
L
diameter of culture vessel, m
m
least-squares slope of the nitrate calibration curve, mAU560/mgNaNO3L-1
MDCW
mass of freeze dried sample used for oxylipin extraction, g DCW
RN
cumulative nutrient addition rate, % day-1
RN
cumulative nutrient addition rate, % day-1
rX
volumetric biomass growth rate, mg DCW L-1 day-1
t
cultivation time, days
V
culture volume in the reactor, L
Vbuffer
volume of buffer added to resuspend the dried oxylipin sample, mL
V,
initial culture volume, L
VI
inoculum volume, L
V.
volume of medium used for inoculation, L
Vo
volume of nutrient stock added to the culture, L
Vs
culture sample volume, L
VT,J
current total culture volume after feed nutrient addition step "j", L
X
biomass concentration at any time in the reactor, mg DCW L-1
X,
initial biomass concentration in the reactor, mg DCW L-1
Xf
final biomass concentration in the reactor, mg DCW
Y15-HETE
15-HETE yield in the biomass, pg/g DCW
YXJN
yield coefficient for nitrate, mg DCW/mmole NO3­
µ
specific growth rate, day-1
gmax
maximum specific growth rate, day -1
p
overall of liquid culture in the reactor, mg L-1
A.
density of feed stock solution, mg L-1
APHA
american public health association
ATP
adinosine tri-phosphate
Chl a
chlorophyll a
DCW
dry cell weight
DD
distilled water
EIA
enzyme immuno assay
GC/MS
gas chromatography/mass spectrometry
GP2
general purpose-2
HPLC
high-performance liquid chromatography
15-HETE
15-hydroxyeicosatetraeionic acid
Biomass Productivity Enhancement of Laminaria saccharina Cultures in a
Stirred-Tank Bioreactor by Batch and Fed-Batch Nutrient Delivery
INTRODUCTION
Marine organisms, virtually unexplored as a source of new drugs prior to the late
1960s, have in the last 20 years proved to be an exciting source of structurally unique,
bioactive natural products (Gerwick, 1987). One of the very important groups of
bioactive natural products to find wide distribution in the marine algae are eicosanoids,
which are 20-carbon fatty acids with at least one site of oxidation in addition to the
carboxyl group (Gerwick and Bernart, 1993). It has also long been recognized that
marine plants are abundant producers of polyunsaturated fatty acids, particularly well
known for their elevated levels of 20- carbon (0-3 fatty acids (Stefanov et al., 1988).
Many fatty acids are present in considerable quantities in the marine brown macroalga
Laminaria saccharina (Jamieson and Reid, 1972).
Although chemical synthesis of complex biomedicinal organic compounds seems
to be an obvious path, it is either too expensive or the process is too complicated. Also,
chemically synthesized molecule might not be biologically active. Hence, an alternative
way to get biologically active pharmaceutical compounds is by extraction from field
collected seaweed. This approach has two disadvantages. First, the growth of seaweed in
the field depends on environmental factors which cannot be controlled such as the
chemical composition of the ocean and climatic conditions. Second, bioactive
compounds such as eicosanoids typically constitute less than 5 % of the lipid extract
(Gerwick et al., 1990). Therefore, extraction and purification of biopharmaceutical
products from field-collected seaweed is not economically feasible.
A novel way to circumvent the above problem is by establishing cell cultures of
marine macroalgae for secondary metabolite production. Macroalgal cultures have the
following advantages for this purpose: liquid cell suspension cultures can be grown in
controlled conditions; process parameters can be altered to potentially enhance the
biomass and secondary metabolite productivity; and cell lines can potentially be
2
genetically manipulated to produce higher yields of secondary metabolites. Methods to
establish terrestrial plant cell culture system cannot be fully extended to marine plants.
However, in the past 10 years, few techniques for establishing macroalgal cell
suspensions cultures have emerged (Butler and Evans, 1990).
The bioreactor cultivation of macroalgal suspension cultures is a new area of
marine biotechnology. Previous research on the bioreactor cultivation of macroalgal
suspension cultures for light limited growth of Laminarina saccharina was studied both
in a stirred-tank bioreactor (Qi and Rorrer, 1995) and a bubble column bioreactor (Zhi
and Rorrer, 1996). Various process parameters affecting the growth such as initial cell
density, initial nitrate concentration, aeration rate and the incident light intensity were
also reported.
There are no published reports on the biomass productivity enhancement studies
on macroalgal suspension cultures in bioreactors. The cell suspension cultures derived
from female gametophytes of the brown macro alga Laminaria saccharina is the subject
of current study since the field collected sporophytes are known to contain 15­
lipoxygenase metabolites, including 15-HETE (Proteau and Gerwick, 1993).
Enhancement of biomass within a stirred-tank bioreactor could potentially enhance the
secondary product yield. The enhancement of biomass could be achieved by enhancing
the external nutrient concentration. The nutrient concentration can be increased by
increasing the initial nutrient concentration in the culture (batch process) or by adding the
nutrients incrementally to the culture over the course of the cultivation period (fed-batch
process). The fed-batch addition of all nutrients has the following advantages. First, it
assures that the supply of nutrients for the culture is not limited by a specific nutrient.
Second, it may alleviate problems associated with potential nutrient substrate inhibition at
high initial nutrient concentrations.
The specific objectives of this study are to:
1)
Develop nutrient addition methods to enhance the biomass productivity of
Laminaria saccharina cultures in a stirred-tank bioreactor;
2)
Study the effects of nutrient delivery methods on biomass productivity;
3
3)
Adapt the ELISA technique to measure 15-HETE concentration in cultured
biomass;
4)
Study the effect of nutrient delivery methods on 15-HETE productivity;
5)
Study the effect of nutrient limitation on biomass productivity and 15-HETE
production.
4
MATERIALS AND METHODS
Culture Maintenance
Specific details regarding isolation and characteristics of L. saccharina female
gametophyte suspension cultures are described by Qi and Rorrer (1995). The
composition of the modified GP2 medium (Steele and Thursby, 1988) used for culture
maintenance are given in Tables 1 and 2. Cell suspension cultures were maintained in
250 mL erlenmeyer flasks, each containing 100 mL of culture under 15 gE/m2-sec
illumination, 16h light/ 8h dark photoperiod, and temperature at 12 °C within an
illuminated low-temperature incubator. Erlenmeyer flasks containing the cultures were
not agitated. However, they were gently swirled for 5 seconds once per day to redissolve
atmospheric CO2 into the medium. Gametophyte suspension cultures were subcultured
every 30 days. The cultures were blended in a autoclaved blending cup (Zhi and Rorrer,
1996) on an Osterizer blender at 'liquefy' speed for 5 sec to disperse the clumped
filaments. The blended inoculum was subcultured at 25% v/v into freshly prepared GP2
medium using sterile technique in a laminar flowhood.
Although the cultures were unialgal, they were not truly axenic. In some flask
cultures intense green colonies were observed in the medium, suggesting that
contaminants were present. These contaminants could include green micro-algae and
protozoa which may be normally dormant in the culture. These contaminated cultures
were filtered from the contaminated medium on 20 gm nylon mesh. The cell biomass
was resuspended in a GP2 decontamination medium containing 100 units of penicillin per
mL of culture and 0.2 mL of Tween per 100 mL flask and allowed to incubate for three
days. The decontaminated cells were filtered from the decontamination medium on 20
gm nylon mesh and washed three times with sterile deionized/distilled (DD) and finally
with fresh GP2 medium. The DD water lysed bacterial and microalgal contaminants.
Penicillin was to intended kill the marine bacterial contamination, and Tween was added
to act as a detergent to disrupt cell walls of other micro-contaminants such as protozoa.
5
Table 1.
GP2 artificial seawater base medium composition.
Component
Chemical Formula
Concentration at lx
Level, mg L-1
Sodium chloride
NaCI
Sodium sulfate
Na2SO4
Potassium chloride
KC1
610
Potassium bromide
KBr
88
Sodium tetraborate decahydrate
Na2B4O7
21,030
3,520
101120
34
Magnesium chloride hexahydrate MgC12 61120
9,500
Calcium chloride dihydrate
CaC12
21120
1,320
Strontium chloride hexahydrate
SrC12
61120
20
6
Table 2.
Classification
GP2 nutrient medium composition.
Component
Chemical Formula
Concentration at
lx Level, mg L-1
Macro
Micro
Sodium nitrate
NaNO3
Sodium phosphate
NaH2PO4 H2O
Sodium molybdate (VI) dihydrate
Na2Mo04 2H20
0.012
Potassium iodide
KI
0.042
Zinc sulfate heptahydrate
ZnSO4 71120
0.0112
Sodium orthovandate
Na3VO4.
0.0048
Manganese chloride tetrahydrate
MnC12 4H20
0.0034
Thiamine-HC1
C12H181140SCI HCl
B12
C63H88CoN14014P
0.000125
Biotin
C10 li16N203S
0.000125
Sodium citrate dihydrate
Na3C6H5O7 2H20
63.5
6.4
0.25
0.52
7
Bioreactor Design and Operation
An illuminated stirred-tank bioreactor with a working volume of 900 mL was
used for all the batch and fed-batch cultivation studies. A series of cultivation
experiments were carried out at different process conditions using a common inoculum
source. Four 900 mL bioreactor systems were fabricated. Each bioreactor assembly,
consisting of the bioreactor vessel, external illumination stage (9W dulux fluorescent
lamps), and air delivery system was fitted within an low-temperature incubator
maintained at 12 °C. A Bel lco spinner flask of diameter 10.5 cm and height 20.5 cm
(model 1965-00500) served as the bioreactor vessel. The culture was mixed by a twoblade paddle magnetic impeller (width 5.5 cm, height 2.3 cm) driven by a magnetic stir
plate. The process conditions for growth of gametophyte cultures are listed in Table 2.
A second stirred-tank bioreactor system with a working volume of 1800 mL was
used for nutrient removal studies. For this system a jacketed Bellco spinner flask (model
1965-01000) of diameter 13.0 cm and height 24.5 cm served as a bioreactor vessel. The
bioreactor assembly was mounted to a VirTis Omni-Culture (Model No. 178715)
fermentation unit to provide mixing and aeration. An external illumination stage (9W
dulux fluorescent lamps) provided light symmetrically to each side of the vessel. The
bioreactor culture was mixed by a magnetic impeller (width 7.6 cm, height 0.75 cm). The
temperature was maintained at 12 °C by passing chilled water from a chilling circulator
through the vessel jacket. The operating conditions are listed in Table 2.
Growth of the culture in the bioreactor was measured both as chl a concentration
and dry cell density. A 20 mL sterile syringe connected to the sample tube was used to
withdraw 5 mL samples for chl a measurements and 15 mL samples for dry cell density
measurements. Duplicate samples for both chl a and dry cell density measurements were
obtained.
Nutrient Addition
Chemicals that constitute GP2 nutrient medium are classified as macro-nutrients
or micro-nutrients. The components of macro-nutrients and micro-nutrients, and their
corresponding concentrations at ' 1 x' level are listed in Table 1. The macro-nutrient
8
components are sodium nitrate and sodium phosphate, and the micro-nutrient components
are vitamins and trace metals. The macro-nutrients are essential for the growth of culture
while the micro-nutrients serve as cofactors for enzymes that catalyze the fixing of
exogeneously supplied carbon-dioxide,nitrate, and phosphate into cellular biomass (South
and Whittick, 1987).
Experiments were designed to study the effects of nutrient medium composition
on biomass productivity and 15-HETE concentration in the biomass. The nutrient
composition in the reactor was changed by one of the two methods. In the first method,
the concentration of selected GP2 nutrients was set at the start of the cultivation period
(batch operation). In the second method, a feed stock solution containing GP2 nutrients
at an elevated concentration relative to "lx" medium was added to the culture over the
course of cultivation period in prescribed amounts (fed-batch operation).
The GP2 medium was supplemented with one or more of the nutrient components
at above or below the "lx" level shown in Table 1. When both the macro-nutrients and
micro-nutrients were all changed equally relative to lx level, the feed stock was called the
total nutrient enriched GP2 medium. When one of the nutrient components, e.g. nitrate,
phosphate or micro-nutrient stock as a group was alone increased with all other
components at the lx level, the feed stock solution was called the individual nutrient
enriched GP2 medium.
In the fed-batch culture system, GP2 medium containing nutrients at a known
concentration [CN,o] and known volume [V0] were added to the stirred tank bioreactor at
two day intervals. The predicted concentration of a given nutrient in the culture at a
given time of feed stock nutrient addition [CN, j+il, assuming no consumption of the
nutrient was given by
CN,j+i =
(CN )(VT ) (C N )(Vs ) + (C N,0 )(V0 )
4
.3
(VT
Vs + O)
where CNJ is the current concentration of nutrients, Vs is volume of sample removed
from the reactor due to sampling, and VTJ is the current total culture volume in the
reactor. The cumulative rate of nutrient addition [RN ] was defined as
(1)
9
RN
[CN CND
rl1
(t > 0)
(2)
CN,i
where CN,i represents the initial concentration of nutrients in the culture.
Three dimension less variables were defined to describe the nutrient concentration
in the batch and fed-batch cultivation experiments. The ratio of initial nutrient
concentration of in any culture to the nutrient concentration in the control culture was
defined as
C=
(3)
CN,i (1x)
The ratio of final nutrient concentration in the culture to the initial nutrient concentration
in the culture for fed-batch experiments was given as
CF
CN,j+1
(4)
CN,i (1x)
The ratio of feed nutrient concentration to nutrient concentration at "lx" level for fedbatch experiments was defined as
Co =
CN,0
CN,00x)
(5)
Nutrient Removal
The formation of fatty acids and lipids in algae is controlled by environmental
factors. These include light, temperature and nutrient composition, and especially the
nitrogen level (Pohl and Zurheide, 1979). For example, previous studies with two species
of green algae and four species of blue green algae showed that formation of lipids and
fatty acids depended on the nitrogen concentration in the medium (Piorreck et al, 1984).
When the levels of nitrogen and phosphorous were high in the medium, the biomass
productivity was high. When the nitrogen concentration was lowered, the growth of alga
was poor, but the lipid content increased in the biomass. Hence, in an attempt to
stimulate production of hydroxy fatty acid, 15-HETE in the L. saccharina cultures, all
nutrients were removed from the medium at the onset of the stationary growth phase.
10
The experimental procedure for accomplishing this nutrient removal process is
overviewed below.
The 1800 mL jacketed Bel lco bioreactor was inoculated with gametophyte culture
of L. saccharina with the initial nutrient concentrations at the ' 1 x' level as listed in Table
1. The growth kinetics were followed by measuring the dry cell density as a function of
time. At day 21, near the end of the exponential phase, the reactor was taken to a laminar
flow and the culture was filtered through an autoclaved 20 p.m mesh. The filtered cells
were washed three times with nutrient free GP2 medium. The cells were then back
washed into the reactor vessel with GP2 base medium containing no micro-nutrients or
macro-nutrients. Again, the growth kinetics were followed by measuring the dry cell
density as a function of time. The reactor was shut down after the cells reached a second
stationary phase. The biomass was filtered and stored frozen for future analysis of
oxylipins by EIA or GC/MS.
Culture Growth Measurements
The growth of L. saccharina cultures was measured as bulk chl a concentration in
the culture and dry cell density as described by Qi and Rorrer (1995). Chl a concentration
was measured every 2 days, whereas dry cell density measured every 8 days. The pH of
the bioreactor culture used for dry cell density determination was also measured
immediately after the sample was withdrawn from the reactor. The pH was typically
between 8.0 and 8.5.
Nitrate Concentration Measurement
Nitrate concentration in the culture medium was measured
spectrophotometrically. The procedure given in the APHA standard methods (Method
4500 -NO3 - E, 1992) was used with a nitrate assay kit supplied by Forestry Suppliers Inc.
(Catalog No. 78054). The kit consisted of the following : vacuettes, cadmium foil packs,
dilutor snapper cup and sample cup with top. A 10 mL culture sample was vacuumfiltered on a 20 gm nylon mesh filter. The filtrate was transferred to the sample cup. The
contents of one cadmium foil pack were emptied into the sample cup. The mixture was
11
shaken vigorously for exactly three minutes. The tip of the vacuette was dipped into the
sample cup and allowed to fill completely by capillary action. The vacuette containing
the sample was then immersed into the dilutor snapper cup. The dilutor snapper cup was
filled with 25 mL of deionized/distilled (DD) water. The tip of the ampoule was snapped
open and the contents were thoroughly shaken by inverting the sealed ampoule several
times. The ampoule was allowed to sit at room temperature for 10 minutes. Then 1.5 mL
of solution was transferred immediately to the spectrophotometer cuvette and the total
volume was made up to 3.5 mL with nano-pure HPLC-grade water. The absorbance was
measured at 540 nm. All the absorbance values were corrected for the absorbance of GP2
base medium. Sample assays were performed in duplicate. The concentration for a
culture sample was read directly from the calibration curve. The absorbance increased
linearly with the increasing concentration of nitrate between 0 and 2.42 mmol/L NO3- (0
607.15 mg NaNO3), as shown in Appendix B.
15-HETE Measurement by EIA
The Enzyme Immunoassay (EIA) technique is an accurate method for the
quantitative determination of specific substances in biological fluids.
The oxylipins were extracted from the cell biomass prior to quantitative analysis
by EIA. Frozen wet biomass was first cooled down in a 80°C freezer for two hours and
then freeze-dried. The freezed dried biomass sample was weighed to a precision of ± 0.1
mg (typical weight 15.0 mg) and then extracted in 10 mL of 2:1 v/v CHC13 /Me0H. The
biomass was re-extracted with 10 mL of the same solvent mixture. The lipophyllic
extract was evaporated to dryness under flowing nitrogen. The dry sample was then
resuspended in 20 mL of 0.1M Phosphate buffer solution. The buffer containing lipid
sample was acidified to pH 3.0 with 3 vol % formic acid. An ODS silica cartridge
(Varian Sample Preparation Products, 1211-3027) was flushed with 10 mL of HPLCgrade water followed by 10 mL of ethanol and another 5 mL of HPLC-grade water. The
acidified extract was then loaded to the ODS-silica cartridge. The ODS silica cartridge
containing the non-polar components was washed with 5 mL of HPLC-grade water
followed by 15 vol % ethanol and finally with 5 mL of HPLC grade petroleum ether. The
12
oxylipins, fatty acids, and related compounds in the sample were eluted from the cartridge
with 10 mL of HPLC-grade ethyl acetate. The eluted sample was evaporated to dryness
under flowing nitrogen to remove the ethyl acetate and then reconstituted in 0.2 mL of the
EIA kit buffer.
The EIA assay procedure using a Perseptive Biosystems Titerzyme assay kit
(catalog number 8-6815) is described below. All the reagents were brought-to room
temperature and mixed thoroughly without foaming. The polystyrene EIA well plate was
precoated with goat anti-rabbit antibody. 15-HETE standards were pipetted in 100 !IL
aliquots to each well. Sample aliquots of the same volume were pipetted into each of the
remaining wells. The edge of the holder was gently tapped for at least 15 seconds to
allow thorough mixing of the contents of each well. Next, 100 !IL of 15-HETE antibody
was pipetted to each well except the substrate blank wells and tapped gently for 15
seconds. The entire well plate was covered with a plate sealer supplied by the EIA kit
and incubated at room temperature on an orbital shaker at 150 rpm for 2 hours. Then,
100 !IL of 15-HETE alkaline phosphatase conjugate was added to each well except the
substrate blank wells, and tapped gently. The contents were again incubated for 2 hours
at room temperature on an orbital shaker at 150 rpm. The contents of the wells were
aspirated after removing the plate sealer, and washed three times with 400 AL of wash
buffer with thorough aspiration between washes. The contents were partially dried by
inverting the plate on a paper towel after the final wash. Finally, 300 !IL of p­
nitrophenylphosphate substrate was added to each well and incubated for 2 hours at 37
°C. At the end of incubation, 50 p,1 of EIA stop solution was added to each well and
tapped gently to mix. The absorbance of each well was measured at 405 nm using an
automated microplate reader (l3iotek Instruments, Inc., Model No. EL311SX).
The concentration of 15-HETE in the final sample is quantified by a competitive
binding mechanism. Variable amounts of 15-HETE in the samples compete with a fixed
amount of alkaline phosphatase labeled 15-HETE for a limited number of sites on the
anti-15HETE antibody. The higher the concentration of the compound in the sample, the
less labeled compound is bound to the specific antibody. The green colored product thus
13
formed is inversely proportional to the amount of unlabelled 15 -HETE found. A semilog plot of the absorbance of the standard vs 15-HETE concentration serves as the
calibration curve. The calibration data is fitted to the 3-parameter logistic equation (see
Appendix C).
14
RESULTS
The filamentous Laminaria saccharina gametophyte culture grows by fixing
dissolved CO2, nitrate and phosphate from culture medium into the cellular biomass. The
average C:N:P ratios for four species of Laminaria is 309: 16: 1 (Atkinson and Smith,
1983). The biomass stoichiometry equation based on the above ratio is
309 CO2 + 325 H2O + 16 HNO3 + H3PO4
(CH20)309(NH3)16H3PO4 + 341 02 (6)
L. saccharina gametophyte cultures were cultivated in a stirred-tank bioreactor at
the "lx" GP2 nutrient concentrations listed in Tables 1 and 2. Increasing the levels of
nitrate and phosphate in the base medium could potentially increase the biomass
productivity. Toward this end, nutrient concentrations in the culture medium were varied
from the base level (lx) concentration.
Nutrient addition was carried out as either batch or fed-batch process. Two types
of batch experiments were performed. In the first set of experiments, the concentration of
initial nitrate, initial phosphate, or initial micronutrients were alone set at the start of the
experiment relative to the "lx" level. All other components remained at the "lx" level.
In the second set of experiments, the initial concentration of all the nutrients were set at
the start of the experiment relative to the "lx" level.
Two types of fed-batch experiments were performed. In the first set of
experiments, the concentration of nitrat; phosphate, or micronutrients were alone added
to the culture over the course of the cultivation period. In the second set of experiments,
all nutrients were equally added to the culture over the course of the cultivation period.
All the cultivation experiments were compared against a control experiment inoculated
with a common inoculum source. For all cultivation experiments, the initial
concentration of all nutrients were set at "1x" level at the start of the experiment. Tables
4 and 5 shows the nutrient feed concentration for the batch addition of individual
15
nutrients and total nutrients. Tables 6 and 7 shows nutrient feed concentration in the fedbatch addition of individual and total nutrients.
A second set of experiments was carried out to study the effect of nutrient
removal at the onset of stationary phase on biomass productivity and 15-HETE yield.
Control Experiments
Figures 1 and 2 show the growth kinetics of L. saccharina cultures in the 900 mL
stirred-tank bioreactor for a representative control experiment. The initial cell density
was nominally set at 120 mg DCW/L. The environmental process parameters are
provided in Table 3. The initial cell densities and final cell densities are listed in Table 5.
The specific growth rate (p.) was estimated from the least-squares slope of the linear
portion of chl a vs time data on a semi-log plot. The values for p, and the time range over
which it was determined are also provided in Table 5. The pH was measured every eight
days and is shown in Figure 1. The cultivation period ranged from 28 to 48 days
depending on the time required by the culture to reach final cell density.
The measured nitrate concentration in the culture for a typical control experiment
is given in Figure 2. The nitrate concentration at stationary growth phase was finite even
for control culture suggesting that the control culture was not limited by nitrate. This was
observed for all of the control calculations. The yield coefficient for nitrate (Yx/N)was
estimated from the measurements for cell densities and nitrate concentration in the culture
using
Xf
YX/N
CR.f.
CNJ
The average value of Yx/N was 1194.7 ± 236.4 mg DCW/mmol NO3- based on five
control runs (see Appendix B). In comparison, the calculated yield coefficient for
Laminaria sp. sporophytes based on the biomass stoichiometry given in equation (6) is
602.5 mg DCW/mmol NO3-.
The final cell density in L. saccharina cell cultures is affected by the initial cell
density of the culture (Zhi and Rorrer, 1996). Hence, when the initial cell density was
(7)
16
Table 3.
Process Conditions
Set Point Temperature
Stirred-Tank bioreactor cultivation conditions.
Process Parameter
900 mL Bioreactor
1800 mL Bioreactor
11 °C
12 °C
35 - 40 RE ni2 s1
501.1E rn-2 s-1
16 h light/8 h dark
16 h light/8 h dark
Impeller Speed
200 rpm
150 rpm
Cultivation Volume, VT
900 mL
1800 mL
0.42 L air U' culture min-1
0.83 L air U' culture min'
GP2
GP2
8.0 - 8.5
6.9 - 8.5
28-48 days
45 days
Incident Light Intensity
Photoperiod
Aeration rate
Medium
pH
Cultivation time
17
9
8.5 eL
10
15
20
25
30
35
40
45
Cultivation Time (days)
Figure 1.
Chl a concentration and pH profile in culture vs time for
control (lx GP2 Nutrient) cultivation (Run 8-1).
(
) control; ( + ) pH.
18
0
f ..... T
0
5
1
1
10
,
1
T
15
I
1
1
,
1Ti ,,T
20
25
T
30
35
0
40
Cultivation Time (days)
Figure 2.
Dry cell density and nitrate concentration in culture vs time
for control (lx GP2 Nutrient) cultivation (Run 84).
) control; (A ) nitrate concentration.
(
19
fixed at 120 mg DCW/L and the initial nutrient concentration was fixed at "lx" level,
then the stationary phase cell densities achieved for the control culture were
repeatable(see Table 5). The specific growth rate estimated over a given time range (2-10
days) was also repeatable (see Table 5).
Batch Addition of Individual Nutrients
The effect of initial nutrient concentration (nitrate, phosphate or micro-nutrients
as a group) on biomass productivity is shown in Figures 3 - 5. The nutrient concentration
in each bioreactor at the start of the experiment is given in Table 4. Other process
parameters were consistent with the control cultivation (Table 2). The initial cell
densities, final cell densities and specific growth rates are given in Table 4. No
significant difference was observed between the pH of control (lx nutrient concentration)
and other batch cultivation experiments. Although the final chl a culture concentration
for the culture containing "10x" nitrate increased two times relative to the control, there
was no significant increase in the final dry cell density. Cultures initially containing
"10x" phosphate and "10x" micro-nutrients did not show a consistently significant
increase in either chl a concentration or final dry cell density over the control. The
specific growth rate for each of these experiments was also comparable to its
corresponding control experiment. Therefore, increasing the initial concentration of
individual nutrients to the "10x" level did not have a significant impact on the specific
growth rate or the final dry cell density.
Figure 6 shows the nitrate concentration versus time profile for the culture
initially containing "10x" nitrate. The nitrate consumption for culture with "10x" initial
nitrate concentration was three times over control. Although nitrate consumption
increased, the culture was still not limited by nitrate.
Batch Addition of Total Nutrients
The effect of initial concentration of total nutrients on biomass productivity is
given in Figures 7 - 15. The initial cell densities, final cell densities and specific growth
rates are given in Table 5. In this system, the concentrations of all the nutrient
Table 4.
Run #
Nutrient Addition
Component
CN,i
Batch addition of individual nutrients.
Biomass Productivity
C1
Classification
mg U1
te
Xi
Xf.
days
mg DCW I:1
11
mg DCW Ut
day''
28.00
95.2
1390.0
0.157
28.00
258.3
1470.0
28.00
84.5
1020.0
Enhanced Nitrate
BS-5-2
NaNO3
NaH2PO4 H2O
635.0
10.0
6.4
1.0
Vitamins and
trace metals
Enhanced Phosphate
BS-5-3
NaNO3
NaH2PO4 H2O
1.0
Micro
63.5
1.0
Macro
64
10.0
Vitamins and
trace metals
Enhanced Micro-nutrients
BS-5-4
NaNO3
NaH2PO4 H2O
Vitamins and
trace metals
Macro
1.0
Micro
63.5
1.0
Macro
6.4
1.0
10.0
Micro
.
0.190
0.167
Table 5.
Batch addition of total nutrients.
Nutrient Addition
Run #
C1
Results
tf
days
Xi
mg DCW
X1
11
tp to tr
mg DCW
day-1
days
BS-6-4
0.00
30.00
90.4
307.0
0.030
2-10
BS-10-2
0.50
47.25
107.0
516.0
0.051
10-20
BS-4-1
1.00
27.70
118.0
1037.0
0.120
2-10
BS-5-1
1.00
28.00
94.0
1070.0
0.172
2-10
BS-6-1
1.00
30.00
110.0
889.0
0.219
2-10
BS-7-1
1.00
41.00
97.9
538.0
0.167
2-9
BS-8-1
1.00
37.70
125.0
751.0
0.237
2-10
BS-9-1
1.00
37.92
127.0
832.0
0.120
2-10
BS-10-1
1.00
47.25
108.0
690.0
0.062
10-20
BS-11-1
1.00
46.93
183.0
748.0
0.117
2-10
BS-10-3
2.35
47.25
119.0
747.0
0.081
10-20
BS-6-2
3.50
30.00
93.2
1300.0
0.179
2-8
BS-9-4
3.60
37.92
144.0
1350.0
0.150
2-10
BS-10-4
9.10
47.25
166.0
938.0
0.196
10-20
22
16:
5
10
15
20
25
30
Cultivation Time (days)
Figure 3.
ChI a concentration in culture vs time for batch
addition of individual nutrients (Run 5). ( ) control;
(t) nitrate, C1 = 10; ( * ) phosphate, CI = 10;
( 0 ) micro-nutrients, C1 = 10.
23
1600
oo
10
15
20
25
30
Cultivation Time (days)
Figure 4.
Dry cell density of culture vs time for batch
addition of individual nutrients (Run 5). ( ) control;
( +) nitrate, CI = 10; ( * ) phosphate, CI = 10;
( 0 ) micro-nutrients, CI = 10.
24
10
15
20
25
30
Cultivation Time (days)
Figure 5.
pH of culture vs time for batch addition of individual
nutrients (Run 5). ( ) control; (t) nitrate, CI = 10;
( * ) phosphate, C1 = 10; ( 0 ) micro-nutrients, CI = 10.
25
70Cc
60(ii"
J
50Ck
O 40(k
cti
30CE
20CE
1
5
10
15
20
25
30
Cultivation Time (days)
Figure 6.
Nitrate concentration in culture vs time for batch
addition of individual nutrients (Run 5). ( ) control;
(+ ) nitrate, C1 = 10.
26
12
10
15
20
25
Cultivation Time (days)
Figure 7.
Chl a concentration in culture vs time for batch
addition of total nutrients (Run 6). ( ) control;
(t)
= 3.5; ( 0 )
= 0.
30
27
1400.0.
fr 1200.0:
o
1000.0
cr)
E
800.0
>G
.7."
600.0:
Cl)
C
to
400.0­
cu
0 200.0­
0.0
0
5
10
15
20
25
30
Cultivation Time (days)
Figure 8.
Dry cell density of culture vs time for batch
addition of total nutrients (Run 6). ( ) control;
(+)CI = 3.5;(0)C1 = 0.
35
28
9.0
8.5­
8.0­
7.5­
7.0
0
5
10
15
20
25
30
Cultivation Time (days)
Figure 9.
pH of culture vs time for batch addition of total nutrients
(Run 6). ( ) control; ( +) CI = 3.5; ( 0 ) CI = 0.
29
14
5
10
15
20
25
30
35
40
45
Cultivation Time (days)
Figure 10.
Chl a concentration in culture vs time for batch
addition of total nutrients (Run 10). ( ) control;
(+)CI = 0.50; ( * )
= 2.35; (
)CI = 9.10.
50
30
1200­
1000­
0
D)
X
800­
600
Ft)
0
75
C.)
400­
00­
0
0
5
10
15
20
25
30
35
40
45
Cultivation Time (days)
Figure 11.
Dry cell density of culture vs time for batch
addition of total nutrients (Run 10). ( ) control;
(+)
= 0.50; ( * )
= 2.35; ( 0 )
= 9.10.
50
31
9.0
7.5­
7.0
0
10
15
20
25
30
Cultivation Time (days)
Figure 12.
pH of culture vs time for batch addition of total nutrients
(Run 10). ( ) control; ( + ) CI = 0.50; ( * ) CI = 2.35;
(D)CI = 9.10.
32
14
J
12­
io­
C
8­
4-FI;
c.)
0
ccs
-
2:
5
10
15
20
25
30
Cultivation Time (days)
Figure 13.
Chl a concentration in culture vs time for batch
addition of total nutrients (Run 9). ( ) control;
(D)CI = 3.60.
40
33
oo
5
10
15
20
25
30
35
Cultivation Time (days)
Figure 14.
Dry cell density of culture vs time for batch
addition of total nutrients (Run 9). ( ) control;
(D)CI = 3.60.
40
34
9.0
Q
8.0­
7.5­
7.0
0
5
10
15
20
25
30
35
40
Cultivation Time (days)
Figure 15.
pH of culture vs time for batch addition of total nutrients
(Run 9). (
) control; ( 0 ) CI = 3.60.
35
components were equally set at the start of the cultivation experiments relative to the
control. The concentration of nitrate served as a marker since all the other components
were altered proportionately. The initial concentration of all nutrients ranged from "Ox"
no macro or micro-nutrients, (C1 = 0) to "9.1x" (CI = 9.1). The dry cell density for the
bioreactor containing no macro-nutrients or micro-nutrients in the base GP2 base medium
(Ox) increased from 90 mg DCW/L at inoculation to 307 mg DCW/L by day 30, and the
specific growth rate was only 0.03 day-1. No culture growth was expected by this
experiment. However, modest culture growth did occur, and was probably due to the
presence of unconsumed nutrients (e.g. nitrate) carried over with the inoculum as shown
by the nitrate concentration vs. time data presented in Figure 16. For cultures inoculated
with "0.5x" initial nutrient concentration, the final dry cell density increased to 516 mg
DCW/L, but the specific growth rate was only 0.051 day I. The concentration of nitrate
went to zero at day 10 as shown in Figure 17 , demonstrating that this cultivation was
nitrate limited.
Growth curves from the cultures initially containing "2.35x' and "9.10x" total
nutrient concentration are given in Figure 17. Increasing the total nutrient concentration
to the "9.1x" level resulted in only a 135% increase in the final cell density relative to the
control, although the consumption of nitrate was 4.5 times greater than the control. The
nitrate concentration in the culture for this experiment did not reach zero at the stationary
growth phase demonstrating that the culture was not limited by nitrate at this nutrient
level.
Fed-Batch Addition of Individual Nutrients
Nutrient addition parameters defined in the Materials and Methods section are
listed in Table 6. The initial and final dry cell densities for a fed batch individual nutrient
addition are given in Table 6. The effect of fed-batch addition of individual nutrients on
the growth kinetics of bioreactor cultures are given in Figures 18 23. The cumulative
rate of individual nutrient addition (RN) is given by equation 2. The final chl a
concentration for cultures fed with nitrate enriched feed stock at a RN of 8.83 % day -1
over the 28 day cultivation period was 13.40 mg chl a/L. The chl a concentration for the
Table 6.
Run #
Classification
Nutrient Addition
Chemical Formula
Fed-batch addition of individual nutrients. (CI = 1)
Biomass Productivity
CN,0
mg 1:1­
CO
Cr
RN
% day -1
tf
Xi
X1
days
mg DCW L4
mg DCW L-1
day.'
Enhanced Nitrate
BS-4-2
BS-7-2
Macro
NaNO3
Micro
NaH2PO4 H2O
Vitamins and trace metals
Macro
NaNO3
Micro
Enhanced Phosphate
BS-4-3
BS-7-3
BS-7-4
635.0
6.4
10.0
1.0
1.0
3.45
1.0
1.0
8.83
0.03
0.03
28
123.0
941.0
0.201
635.0
6.4
10.0
1.0
1.0
4.42
8.34
0.03
0.03
41
98.6
894.0
0.207
0.03
8.83
0.03
28
139.0
877.0
0.145
0.03
8.34
0.03
41
127.0
923.0
0.172
0.03
0.03
8.83
28
134.0
944.0
0.219
0.03
0.03
8.34
41
122.0
905.0
0.201
-­
Macro
NaNO3
Micro
NaH2PO4 H2O
Vitamins and trace metals
63.5
64
-­
Macro
NaNO3
Micro
Enhanced Micro-nutrients
BS-4-4
NaH2PO4 H2O
Vitamins and trace metals
NaH2PO4 H2O
Vitamins and trace metals
Macro
NaNO3
Micro
NaH2PO4 H2O
Vitamins and trace metals
Macro
NaNO3
Micro
NaH2PO4 H2O
Vitamins and trace metals
N,
1.0
1.0
1.0
1.0
10.0
1.0
3.45
1.0
63.5
1.0
1.0
64
10.0
1.0
4.42
-­
1.0
63.5
6.4
-­
1.0
1.0
1.0
1.0
10.0
3.45
63.5
6.4
1.0
1.0
1.0
-­
10.0
4.42
1.0
.
37
100
75­
0
z
-
50­
25
10
15
20
25
Cultivation Time (days)
Figure 16.
Nitrate concentration in culture vs time for batch
addition of total nutrients (Run 6). ( ) control;
(+)
= 0.
38
10
15
20
25
30
35
40
45
Cultivation Time (days)
Figure 17.
Nitrate concentration in culture vs time for batch
addition of total nutrients (Run 10). ( ) control;
(
)CI = 0.50; (* ) CI = 2.35; (0 )CI = 9.10.
50
39
14­
10
15
20
25
Cultivation Time (days)
Figure 18.
Chl a concentration in culture vs time for fed-batch
addition of individual nutrients (Run 4).
) control; (+ ) nitrate, RN = 8.83 % day-I;
(
( * ) phosphate, RN = 8.83 % day-1;
( 0 ) micro-nutrient, RN = 8.83 % day-I.
30
40
1200
1000:
0
c)
800­
X 600
0
400­
200­
0
0
10
15
20
25
Cultivation Time (days)
Figure 19.
Dry cell density of culture vs time for fed-batch
addition of individual nutrients (Run 4).
( ) control; (+ ) nitrate, RN = 8.83 % day-1;
( * ) phosphate, RN = 8.83 % day-1;
( 0 ) micro-nutrient, RN = 8.83 % day-1.
30
40a
9.0
8.5
Q 8.0­
7.5­
7.0
0
5
10
15
20
25
30
Cultivation Time (days)
Figure 20.
pH of culture vs time for fed-batch addition of individual
nutrients (Run 4).
(
) control; ( ) nitrate, RN = 8.83 % day-1;
( * ) phosphate, RN = 8.83 % day-1;
( 0 ) micro-nutrient, RN = 8.83 % day 1.
41
12­
5
10
15
20
25
30
35
40
Cultivation Time (days)
Figure 21.
Chl a concentration in culture vs time for fed-batch
addition of individual nutrients (Run 7).
(
) control; (+ ) nitrate, RN = 8.34 % day-1;
( * ) phosphate, RN = 8.34 % day-I;
( 0 ) micro-nutrient, RN = 8.34 % day-1.
r-45
42
1200
1000­
800­
X 600­
c7)
ct)
400­
TD
0
200­
5
10
15
20
25
30
35
40
Cultivation Time (days)
Figure 22.
Dry cell density of culture vs time for fed-batch
addition of individual nutrients (Run 7).
(
) control; (+ ) nitrate, RN = 8.34 % day-1;
( * ) phosphate, RN = 8.34 % day-1;
( D ) micro-nutrient, RN = 8.34 % day-1.
45
43
5
10
15
20
25
30
35
40
45
Cultivation Time (days)
Figure 23.
pH of culture vs time for fed-batch addition of individual
nutrients (Run 7).
(
) control; ( ) nitrate, RN = 8.34 % day 1;
( * ) phosphate, RN = 8.34 % day-1;
( ) micro-nutrient, RN = 8.34 % day-1.
44
control cultivation at the same process conditions was 6.35 mg chi a/L. Previous work
with sporophytes of Laminaria saccharina (Chapman et al., 1978) showed that the
photosynthetic capacity and the chlorophyll content increased with increasing external
NO3- concentrations in the 0 to 20 gm NO3- range. The final chl a concentrations for fed-
batch addition of phosphate enriched GP2 medium and micro-nutrient enriched GP2
medium under the same RN of 8.83 % day -1 were 8.62 mg chl a/L and 9.59 mg chl a/L
respectively. The fed-batch addition of any individual nutrient at a rate of 8.83 % day-1
resulted in a comparable final cell density relative to control.
The nitrate concentration vs time profile for cultures fed with nitrate at a rate of
8.83 % day -1 are shown in Figures 24 and 25. The concentration of nitrate in the culture
increased over the cultivation period showing that the rate of nitrate addition was greater
than the rate of nitrate consumption.
Fed-Batch Addition of Total Nutrients
Nutrient addition parameters defined in Materials and Methods section are listed
in Table 7. The cumulative nutrient addition rate (RN) is defined by equation 2. Each
culture was fed at two day intervals with a 20 mL feed-stock solution at nutrient
concentrations ranging from "5x" (Co = 5) to "25x" (Co = 25) to provide RN values
ranging from 3.6 % to 21.5 % day-1. Additionally, one experiment was carried out at
"lx" (Co = 1 and RN = 0.036% day-1). For this experiment the rate of nutrient addition
was calculated by neglecting the volume of culture removed for sampling. The initial
nutrient concentrations were all fixed at the "lx" level. Although nutrient feed stock
solution was added to the culture at a rate of 10 mL per day, the final culture volume did
not vary much from that of the initial volume since a 20 mL culture sample was
withdrawn every two days for sampling. The effect of fed-batch addition of total
nutrients on the biomass productivity is given in Figure 26 37. The biomass
productivity for cultures fed at 0.036 % day -1 was comparable to the control cultivation
experiment. Increasing the cumulative nutrient addition rate (RN) by increasing Co
increased the chl a culture concentration and the final dry cell density. A final dry cell
density of 1663 mg DCW/L was obtained for cultures fed at a rate of 21.5% day -1 vs. 751
Table 7.
Fed-batch addition of total nutrients. (C1 = 1)
Nutrient Addition
Run #
CO
CF
Biomass Productivity
RN
tf
Xf
% day'l
days
mg DCW 1,4
BS-6-3
1.00
1.00
0.036
30.00
86.1
Xf
mg DCW 1,-1
970.0
BS-8-2
5.00
2.35
3.58
37.70
151.0
1400.0
0.233
2-10
BS-11-2
5.00
2.57
3.35
46.93
173.0
1410.0
0.102
2-10
BS-9-2
10.00
4.20
8.44
37.92
127.0
1220.0
0.177
2-10
BS-9-3
10.00
4.82
10.17
37.92
157.0
1400.0
0.131
2-10
BS-8-3
15.00
5.73
12.54
37.70
112.0
1130.0
0.163
2-10
BS-11-3
15.00
6.51
11.73
46.93
138.0
1360.0
0.109
2-10
BS-8-4
25.00
9.10
21.50
37.70
175.0
1660.0
0.197
2-10
BS-11-4
25.00
10.44
20.11
46.93
179.0
1820.0
0.131
2-10
11
to to tr
days'
days
0.18
2-10
46
300
250­
--I 200­
co
0
crs
Z
cy)
E
4..
150­
I
c"
lay
10
I'
e.
-
go.
so.
0".
15
20
25
30
Cultivation Time (days)
Figure 24.
Nitrate concentration in culture vs time for fed-batch
addition of individual nutrients (Run 4).
(
) control; ( ) nitrate, RN = 8.83 % day-1;
(--) nitrate predicted, RN = 8.83 % day-1.
47
300
250­
0
6
150­
.0
cr)
E 100.
50:
5
10
15
20
25
30
Cultivation Time (days)
Figure 25.
Nitrate concentration in culture vs time for fed-batch
addition of individual nutrients (Run 7).
(
) control; (+ ) nitrate, RN = 8.34 % day 1;
(--) nitrate predicted, RN = 8.34 % day-1.
48
Cultivation Time (days)
Figure 26.
Chl a concentration in culture vs time for fed-batch
addition of total nutrients (Run 6).
(
) control; ( * ) RN = 0.036 % day-1.
49
1400.0.:
1200.0:
0
CI 1000.0
a)
800.0­
$
(7)
600.0:
400.0:
a)
200.0:
0.0
0
5
10
15
20
25
30
Cultivation Time (days)
Figure 27.
Dry cell density of culture vs time for fed-batch
addition of total nutrients (Run 6).
(
) control; ( * ) RN = 0.036 % day1.
35
50
10
15
lllllll
20
25
30
Cultivation Time (days)
Figure 28.
pH of culture vs time for fed-batch addition of total nutrients
(Run 6). ( ) control; ( * ) RN = 8.34 % day-1.
51
14­
12­
co
ca)
8­
o
6­
c.)
co
4­
5
10
15
20
25
30
35
40
45
Cultivation Time (days)
Figure 29.
Chl a concentration in culture vs time for fed-batch
addition of total nutrients (Run 8).
(
) control; (+ ) RN = 3.58 % day-I;
(*)RN = 12.54 % dayI; (0)RN = 21.50 % day-1.
52
oo
5
10
15
20
25
30
35
40
Cultivation Time (days)
Figure 30.
Dry cell density of culture vs time for fed-batch
addition of total nutrients (Run 8).
(
) control; ( ) RN = 3.58 % day-1;
( *)RN = 12.54 % day-1; ( ) RN = 21.50 % day-1.
53
0
5
10
15
20
25
30
35
40
45
Cultivation Time (days)
Figure 31.
pH of culture vs time for fed-batch addition of total nutrients
(Run 8). ( ) control; ( ) RN = 3.58 % day 1;
( * ) RN = 12.54 % day-1; ( )RN = 21.50 % day-1.
54
18
16­
14­
c 12_
o
10:
8­
6:
4­
2­
0
0
5
10
15
20
25
30
35
40
45
50
Cultivation Time (days)
Figure 32.
Chl a concentration in culture vs time for fed-batch
addition of total nutrients (Run 11).
(U) control; ( ) RN = 3.35 % day-1;
( * ) RN = 11.73 % day1; ( ) RN = 20.11 % day1.
55
00
5
10
15
20
25
30
35
40
45
50
Cultivation Time (days)
Figure 33.
Dry cell density of culture vs time for fed-batch
addition of total nutrients (Run 11).
( ) control; ( ) RN = 3.35 % day-1;
(*)RN = 11.73 % day-1; (0 ) RN = 20.11 % day-1.
56
9.0
8.5­
7.5­
7.0
0
10
15
20
25
30
35
40
45
50
Cultivation Time (days)
Figure 34.
pH of culture vs time for fed-batch addition of total nutrients
(Run 11). ( ) control; ( ) RN = 3.35 % day-1;
(*)RN = 11.73 % day-1; (0 )RN = 20.11 % day-1.
57
14
-3 12:
10:
0
a)
0
c
as
6
4­
:cCD
2­
0
5
10
15
20
25
30
35
Cultivation Time (days)
Figure 35.
Chi a concentration in culture vs time for fed-batch
addition of total nutrients (Run 9).
( ) control; (+ ) RN = 8.44 % day-1;
(*) RN = 10.17 % day1.
-r-40
58
oo
I
10
15
20
25
30
35
Cultivation Time (days)
Figure 36.
Dry cell density of culture vs time for fed-batch
addition of total nutrients (Run 9).
(
) control; (
RN = 8.44 % day-I;
(*)RN = 10.17 % day-1.
40
59
9.0
7.5­
7.0
0
5
10
15
20
25
30
35
40
Cultivation Time (days)
Figure 37.
pH of culture vs time for fed-batch addition of total nutrients
(Run 9). ( ) control; (+ ) RN = 8.44 % day-1;
( * ) RN = 10.17 % day-1.
60
mg DCW/L for the control culture (Table 7). This result is supported by the previous
work of Conolly and Drew (1985) for sporophytes of Laminaria saccharina, where they
obtained marked stimulation in growth when the nutrient medium was replaced at the rate
of 50 % day' and contained both nitrate (151.1.M) and phosphate (31.1,M). The specific
growth rates varied from 0.163 day' to 0.237 day-1 but showed no discernable trend.
The measured nitrate concentration versus time profile in cultures fed at different
rates (RN) are given in Figures 38 and 39. The concentration of nitrate in the culture
increased over the cultivation period showing that the rate of nitrate addition was greater
than the rate of nitrate consumption.
Nutrient Removal
The effect of nutrient removal on growth kinetics for stirred-tank bioreactor
cultures of L. saccharina is given in Figures 40 and 41. The process parameters listed in
Table 2 were maintained both before and after the removal of nutrients from the culture.
As described in the Materials and Methods section, nutrient removal was accomplished
by filtering the cells from the nutrient medium and resuspending cell biomass in nutrient
free GP2 base medium. The culture growth parameters, initial cell densities, final cell
densities, specific growth rates and culture death rates are given for two repeat
experiments in Table 8. A Cell density of 740 mg DCW/L was obtained at day 18, near
the end of the exponential growth phase. The final cell density decreased to 450 mg
DCW/L by day 28 and remained the same until shutdown at day 44. The pH was 7.9 to
8.0 during the growth phase of the culture. However, the pH dropped from 8.1 at day 28
to 6.9 at day 30, and then increased to 7.5 by day 44.
The rate of biomass productivity decrease in response to the removal of nutrients
by replacement with nutrient free GP2 base medium can be described the specific death
rate, lcj. The specific death rate is defined as
kd
1 dX
X dt
(8)
Values for kj were estimated from the least square slope of the linear portion of the ln(X)
vs. time data during the period following nutrient removal (Figure 42). As shown in
61
Table 8.
Nutrient removal. Time of nutrient change over was day 21.
Xi
Xmax
X1
t
to to tr
kd
mg DCW/L
mg DCW/L
mg DCW/L
day"1
days
dafi
BJ-1
120
731
528
0.115
0-14
- 0.037
t to td
days
21-33
BJ-2
158
534
358
0.116
2-14
- 0.064
26-33
Run #
62
200
150­
co
0
as
z
loft
C)
E
0
0
5
10
15
20
25
30
35
Cultivation Time (days)
Figure 38.
Nitrate concentration in culture vs time for batch
addition of total nutrients (Run 8).
1;
) nitrate, RN = 3.58 % day
( ) control; (
(--) nitrate predicted, RN = 3.58 % day-1.
40
63
10
Figure 39
20 25 30
Cultivation Time (days)
15
Nitrate concentration in clture
u
vs time for batch
addition of total nutrients (Run 11).
( X ) control; ( ) nitrate, RN = 3.35 % day l;
( 0 ) nitrate, RN = 20.11 % dayi;
() nitrate predicted, RN = 3.35 % dal;
day'1;
((~-) nitrate predicted, RN = 20.11 % day
64
800_
8.5
700::
0 600:
-8.0
cm 500­
E
=
406_:
"c7
c
a)
300­
-7.5
ca.
­
200:
-7.0
100­
00
5
10
15
20
25
30
35
40
6.5
45
Cultivation Time (days)
Figure 40.
Dry cell density and pH profile for nutrient removal from
L. saccharina culture vs time. (Run BJ-1).
()X, (0)pH.
65
800­
8.5
a 700-­
(..)
600­
8.0
CD 500­
E
X 400­
-7.5
a
( 300­
a)
CI 200:.
-7.0
TC)
C.)
100:
0
o
5
10
15
20
25
30
35
40
45
6.5
Cultivation Time (days)
Figure 41.
Dry cell density and pH profile for nutrient removal from
L. saccharina culture vs time. (Run BJ-2).
) X, ( ) pH.
(
66
a)
10
15
20 25
30
35 40
45
Cultivation Time (days)
Figure 42.
Semi-log plot for estimation of 1.t and kd. (Run BJ-1).
67
Table 9, although the specific growth rates prior to nutrient removal were very repeatable,
the specific death rates following nutrient removal were variable but still were
consistently less than 50 % of the specific growth rate.
Light Attenuation Estimates
All the cultivation experiments showing enhanced biomass productivity were not
limited by nitrate at the stationary phase of growth. Therefore, a second important growth
parameter, light, was considered as a potentially controlling factor. The following
assumptions were made in estimating the mean light intensity:
1) Uniform and one-dimensional light flux.
2) Culture is symmetrically illuminated from both sides of the culture vessel.
3) The biomass suspension is well mixed.
4) Beer's law describes the attenuation of light through the biomass suspension.
Based on the above assumptions, the mean light intensity (Im) in the culture as a function
of incident light intensity (Io) and cell density (X) was estimated using the model
proposed by Rabe and Benoit (1962)
I
2 I0
=
m
lc
XL
[1
exp(-1cc X L)]
(9)
where L is the is the culture vessel diameter and lc, is the Beer's law constant for the
biomass suspension. The value of lc, for 30 day-old Laminarina saccharina female
gametophyte clumped biomass suspensions blended to 400 gm pellet size was 0.153 ±
0.014 cm2/mg DCW (Hayden, unpublished results). The calculated Im vs. X profile for
vessel diameter of 10.5 cm (900 mL bioreactor) is shown in Figure 43. At a cell density
2000 mg DCW/L, the mean light intensity in the culture was calculated to be 22.7
gE/m2sec. Qi and Rorrer (1995) reported that the apparent half-saturation constant for
light limited growth of L. saccharina in a 900 mL bioreactor was 18.4 gE/m2sec. In this
68
study, no final cell densities exceeding 2000 mg DCW/L were achieved. Hence, the
cultures were not limited by light, since I. was greater than the half-saturation constant
for light-limited growth.
69
80
70
60
fg 50
40
j 30=
20
10,
0
-1
0
500
r1
1­
1000
llll
1500
I
2000
I
I
1
2500
Xf (mg DCW/L)
Figure 43.
Plot of estimated incident light intensity vs cell density.
70
Model Calculations
The following are the assumptions for both the batch and fed-batch cultivation systems:
1.
Cell growth was limited by one nutrient, e.g. nitrate.
2.
Cell growth was not limited by light.
3.
The cell culture suspension was homogeneous and well mixed.
4.
Balanced growth.
The cell and nutrient balance equations for batch culture are given by
d(X V)
= rx V
dt
d(CN V)
dt
(10)
rR
YX/N
The dependence of nutrient concentration on specific growth rate is given by the Monod equation
maxC N
( 12 )
KN + CN
The volumetric biomass productivity (rx) is given by
= µX
Tx
(13)
If the total volume (V) is constant, then rearrangement of equations (10) to (13) yields the
following system of first order differential equations:
dX
=
dt
,u
max N X
KN CN
(14)
dC N
YmaxC N X
dt
(KN + CN )YX/N
(15)
The initial conditions are X = Xi and CN = CN,;.
The cell, nutrient and total balance equations for the fed-batch culture system are given by
d(X V)
dt
d(CN V)
dt
(16)
rx
x
= F CN,0 +
-r V
YX/N
(17)
71
d(p V)
dt
= F po
(18)
Rearragement of equations (16) to (18) yields the following system of first order differential
equations:
dX
dt
PmaxCN
dCN
F
V
dt
KN +CN X
X
(19)
(VF)
it
CN X
(20)
N'
(KN +CN)YX /N
dV
dt
(21)
The initial conditions are X = Xi , CN = CN,i and V = Vi. Definitions for all of the variables
given in equations (1) to (12) are provided in the Nomenclature list.
The Virtual Systems Simulator (VSS) version 2.2, was used to solve the system of first
order differential equations posed by the batch and fed-batch cultivation models. This sofware
was developed by the Bioresource Systems Engineering Group at Oregon State University (Bolte,
1996). The model input parameters for the batch and fed-batch addition of nutrients are listed in
Table 9. The half saturation constant (KN) was taken as 0.0014 mM NO3" (0.12 mg NaNO3/L)
based on previous studies of Laminaria saccharina cultured sporophytes (Chapman et al.,1978).
The yield coefficient (Yx/N) for nitrate was 1.194 g DCW/mM NO3 , as detailed earlier in the
Results section (Control experiments). The specific growth rate (g) estimated from the growth
culture data was assumed to approximate the the maximum specific growth rate since KN was
much smaller than CN. Equations 3 to 6 (batch model) and 10 to 12 (fed-batch model) were
integrated by the fourth-order Runge-Kutta method with a step size of 0.01 day. The results from
these simulations are given in Figures 44 and 45 for batch and fed-batch operation respectively.
Table 9.
Run #
VSS input parameters
CNJ
CN,O
F
Vi
MM NO3
Xi
MM NO3
L day''
L
gmax
g DCW/L
day'
10-1
1.035
n/a
n/a
0.9
0.107
0.062
10-2
0.488
n/a
n/a
0.9
0.106
0.051
10-3
1.901
n/a
n/a
0.9
0.119
0.084
10-4
7.413
n/a
n/a
0.9
0.166
0.196
8-1
0.945
n/a
n/a
0.9
0.124
0.236
8-2
0.898
3.735
0.01
0.9
0.233
0.233
8-3
0.898
11.205
0.01
0.9
0.163
0.163
8-4
0.898
18.675
0.01
0.9
0.197
0.197
Batch Addition
Fed-Batch Addition
73
10000
9000
8000 :­
= 0.05 x
C1= 2.35 x
---- CI= 9.10 x
7000
6000
5000
4000
3000
2000
1000
0
0
10
20
30
40
50
60
Cultivation Time (days)
Figure 44.
VSS-model predictions for batch addition of total nutrients.
74
0
5
10
15
20
25
30
35
40
Cultivation Time (days)
Figure 45.
VSS model predictions for fed-batch total nutrient addition.
75
DISCUSSION
The bioreactor process conditions listed in Table 3 were kept constant for all the
nutrient addition studies with the 900 mL stirred-tank bioreactor. These process
conditions were based on previous bioreactor studies with cell suspension cultures
established from Laminaria saccharina (Qi and Rorrer, 1995; Zhi and Rorrer, 1996). In
these studies, incident light intensity and initial cell density significantly affected culture
growth at the "lx" nutrient level shown in Table 2. Therefore, the light intensity was
fixed at 40 pE/m2sec since the apparent half-saturation constant for L. saccharina was 18
RE/m2sec in a stirred-tank bioreactor. Also, the initial cell density was set at 120 mg
DCW/L as recommended by Zhi and Rorrer (1996).
The three major components that make up the GP2 nutrient medium (nitrate,
phosphate, and micro-nutrients) were increased in the GP2 base medium for the
following reasons: nitrogen constitutes the compounds which form the building blocks in
the biomass; phosphorus acts a primary energy transfer agent; and the vitamins and trace
metals play a vital role in ion transport and enzyme activation (South and Whittick,
1987). The biomass productivity improved significantly only when all the nutrients were
added to the culture in either batch and fed-batch systems. This could be explained by the
following stoichiometric equation for nitrate assimilation into protein by photosynthetic
macroalgae (Turpin, 1991)
NO3 + keto-acid + 6 ATP + 10 e"
protein
(22)
The synthesis of energy transfer agent ATP is controlled by the concentration of
phosphorous, and the ion transport is supported by micro-nutrients (South and Whittick,
1987). Hence, an elevated level of both of these nutrients in the medium potentially
enhances the fixation of nitrate into cellular biomass as protein. Since algal chloroplasts
have high protein content (Piorreck, 1984), the elevated chl a content observed for all the
76
nitrate addition experiments indicates that protein is being synthesized according to
equation 22. Furthermore, the yield coefficient based on nitrate for the gametophyte cell
suspension culture in the stirred-tank bioreactor was about twice that of Laminaria sp.
sporophytes based on the biomass stoichiometry given by equation 6. This suggests that
gametophytes assimilate nitrate better than sporophytes.
Simulations for the batch cultivation process based on Monod kinetics predicted a
final cell density of 1123 mg/L at day 45 for cultures with initial nutrient concentrations
at "lx" level. At higher initial nutrient concentrations, the predicted cell density was
much higher than the measured cell density. Also, simulations for the fed-batch nutrient
addition cultivation process predicted final cell density which greatly exceeded the
measured cell density. Although the general trend of the growth curve was observed for
both experimental data and model predictions of the fed-batch process, the model vastly
overpredicted the biomass yield. The model calculations assumed that all the nitrate was
consumed. However, this was not observed experimentally. Hence, assuming that nitrate
was the limiting nutrient was the reason for the differences between the experimental data
and the model predictions.
The batch and fed-batch addition of individual nutrients did not significantly
increase the final biomass density. This suggests that the GP2 nutrient medium is
balanced and no one compound is critically limiting. The subtle enhancement in the
biomass productivity was within the repeatability of the data.
Although the enhancement in biomass productivity was observed for the total
nutrient addition in either batch or fed-batch mode, no significant improvement in the
specific growth rate was observed. This results suggests that the cultivation period must
be extended to achieve the observed enhanced final cell densities, and explains why the
time scale observed to reach the final cell density for both the batch and fed-batch
addition of total nutrients was always longer than that for the control experiments.
77
For most nutrient addition cultivation experiments, the chl a concentration
reached a stationary phase at day 21 for most cultivations, whereas the final stable dry cell
density was not observed until after 40 days. This observation could be explained as
follows: the bulk chl a concentration is an indirect method of measuring the growth
conditions. Shuler and Kargi (1992) reported that similar indirect growth measurements
such as protein or DNA content level off in late exponential growth phase before final
biomass density is attained.
According equation 6, dissolved CO2 also gets fixed into the biomass. Although
the concentration of other nutrient components were varied, the CO2 concentration in the
gas phase was constant at an ambient level of 350 ppm. However, from previous studies
stirred-tank bioreactor studies with Laminaria (Hayden, 1995) and Acrosiphonia (Rorrer
et al., 1996) showed that adding increasing the CO2 concentration in the gas phase did not
improve the culture growth. Also, Qi and Rorrer (1995) showed that the cultures were
not limited by CO2 mass transfer at "lx" nutrient conditions in a stirred-tank bioreactor.
From this study we can conclude that the concentration of the nutrient medium
plays a vital role in the biomass productivity, even above saturation levels. The mode of
nutrient addition is another significant factor for biomass productivity enhancement.
Batch cultures were limited by nitrate for nutrient concentrations less than "lx" level.
However, both batch and fed-batch cultures at nutrient concentrations above the "lx"
level were not limited by nitrate. Modest improvement in biomass productivity was
observed when nutrients (nitrate, phosphate) or nutrient groups (micro-nutrients) were
added individually, either in batch or fed-batch mode. However, improvements were on
the order of the repeatability of the control experiments. Therefore, one cannot conclude
that individual nutrient addition had an biomass productivity enhancing effect, at least at
the experimental conditions considered. However, the biomass productivity increased 2.4
times over the control when cultures were fed with total nutrients at a rate of 20 % day-1.
This suggest that amongst all the paramaters considered, fed-batch addition of all the
nutrients enhanced biomass productivity.
78
L. saccharina gametophyte cultures nominally grow at a rate of 0.15 day-1. The
doubling time based on the above growth rate is 6.67 days. In the fed-batch nutrient
addition process, the nutrient concentration was fortified every two days, which is one-
third the doubling time. Therefore, the discrete addition of nutrients can be approximated
as a continuous operation (Westgate and Emery, 1989). Consequently, problems related
to perfusion, such as pumping the nutrients at a very slow rate to achieve the same
nutrient replenishment, could be eliminated by simple fed-batch addition of nutrients
once every two days. Also, the fed-batch addition of nutrients is an intrinsic method for
enhancing the biomass productivity. This concept could be easily extended to other
reactor configurations and could be easily scaled-up with out compromising the sterility.
Fed-batch nutrient addition finds application where: 1) biomass productivity or
secondary metabolite productivity is affected by nutrient substrate inhibition; 2) the
culture growth is limited by the depletion of the limiting nutrient in the reactor; 3)
balanced growth needs to be approximated over the batch cultivation period to extend the
time of the exponential growth phase; and 4) the nutrient is labile over extended time
periods at the cultivation conditions.
Based on the current research, the maximum biomass productivity was observed
for cultures fed with 20.11 % day-1 of total nutrients. No significant change in specific
growth rate was observed for all the nutrient addition experiments, the culture was
probably not limited by light or CO2 concentration in the gas phase.
The following are the recommendations for future research:
1)
Study the effect of fed-batch addition of total nutrients coupled with enhanced
CO2 levels in the gas phase on biomass productivity to determine if CO2 mass
transfer limitations exist;
2)
Add sugars to the medium in the attempt to trigger mixotrophic growth and
enhance biomass productivity.
79
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Introduction to Applied Phycology (Akatsuka, I). The Hague: SPB Academic
Publishing. 1990. Pp 629-645.
Chapman, A. R. 0., Markham, J. M., and Liining, K. 1988. Effect of Nitrate
Concentration on the Growth and Physiology of Laminaria saccharina
(Phaceophyta) in culture. Journal of Phycology, Vol 14, 195-198.
Conolly, N. J., and Drew, E. A. 1985. Physiology ofLaminaria. IV. Nutrient
Supply and Daylength, Major Factors Affecting Growth of L. Digita and L.
Saccharina. Marine Ecology, Vol 6, 299-320.
Gerwick, W. H., Bernart, M. W., Moghaddam, M. F., Jiang, Z. D, Solem, M. K., and
Nagle, D. G. 1990. Eicosanoids from the Rhodophyta: A New Metabolism in the
Algae. Hydrobiologia, 204/205, 621.
Gerwick, W. H., Bernart, M. W., and Moghaddam, M. F. Eicosanoids and Related
compounds from Marine Algae. In Marine Biotechnology, Vol 1:
Pharmaceutical and Bioactive Natural Products (D. H Attaway and 0. R.
Zaborsky, eds). New York: Plenum Press. 1993, 101-152.
Hayden, C. 1995. Summer research work on L. saccharina. Unpublished results.
Jamieson, G. R., and Reid, E. H. 1972. The Component Fatty Acids of Some Marine
Algal Lipids. Phytochemistry, Vol. 7, 1565-1572.
Method 4500-NO3 - E. 1992. APHA Standard Methods., 18th ed. P 4-89.
Piorreck, M., Baasch, K-H., Pohl, P. 1984. Biomass Production, Total Protein,
Chlorophylls, Lipids and Fatty Acids of Freshwater Green and Blue-Green Algae
Under Different Nitrogen Regimes. Phytochemistry, Vol 23, No. 2, 207-216.
Pohl, P., and Zurheide, F. 1979. Fatty Acids and Lipids of Marine Algae and the
Control of their Biosynthesis by Environmental Factors. Marine Algae in
Pharmaceutical Science. Berlin: Walter de Gruyter.
Proteau, J. A., and Gerwick, W. H. 1993. Divinyl ethers and Hydroxy Fatty Acids from
Three Species of Laminaria (Brown Algae). Lipids, Vol 28, 783-787.
80
Qi, H., and Rorrer, G. L. 1995. Photolithotrophic Cultivation ofLaminaria saccharina
Gaemetophyte cells in a Stirred-Tank Bioreactor. Biotechnology and
Bioengineering, Vol 45, 251-260.
Rabe, A. E., and Benoit, R. J. 1962. Mean Light Intensity-A Useful Concept in
Correlating Growth Rates if Dense Cultures of Microalgae. Biotechnology and
Bioengineeing Vol 4, 377-390.
South, R. G., and Whittick, A. 1987. Introduction to Phycology. Oxford: Blackwell
Scientific Publications.
Steele, R. L., and Thursby, G. B. 1988. Laboratory Culture of Gametophytic Stages of
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saccharina (Phaeophta). Environmental Toxicology and Chemistry, Vol 7, 997­
1002.
Stefanov, K., Konaklieva, M., Brechany, E. Y., and Christie, W. W. 1988. Fatty
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3495-3497.
Turpin, D. 1991. Effects of Inorganic N availability on Algal Photosynthesis and
Carbon Metabolism. Journal of Phycology, Vol 27, 14-20.
Zhi, C., and Rorrer, G. L. 1996. Photolithotrophic Cultivation of Laminaria saccharina
Gametophyte Cellsin a Bubble-Column Bioreactor. Enzyme and Micorbial
Technology, Vol 18, 291-291.
81
APPENDICES
82
Appendix A
Tabulated Data
Table A-1. L aminaria saccharina gametophyte cell cultivation Run 4.
Run Identification
Bionrector Run ft
Culture Loading
BS-4-1
Process Parameters
Culture:
L. saccharine female gametaphytes
Ls-D-13-910,5,9(dated 04/2305)
Ls-D-14-52,10(dated 05/2995)
71,41
days
Inoculum Resler
Blomactor Desaidisn:
1-L Bella, Stiffed Tank Bioreador
Aged Inoculum:
inocaltn Cal Density
Tine Staffed:
6:00 pm
Date Started:
07/04/95
805.0 rnL
900.0 mL
118.3 mg DOW&
Run#
Semple*
Day:WT.4n
4-1 1
19600 cm
Sande
CH a
klev1
Mid
RIAU
(m01-1
00D
8.54
4.21
2-600 om
200
474
45.1
4.5.91 re.
A 55
44-1
6-800om
608
45-1
R..19tYrn
a17
E116
4-5-2
Dr ASK
441-1
1n.A. Mom
47.1
12-1991An
17 17
A-74
0:/115165
4k11-1
141(1/Y15
1A17
1 x 9 Watt cool-white Dulux lamp, two sides
16 hr ON/
Wt. Dried
VA. Dried
Average
Chia
(mot)
FP
FP+Cells+GP2
(01
lot
X
X
X+/-1s
(ma °MU (tea DCW/L1 (ma DCWA.
0.0768
0 0793
00839
00510
118 5
150
007633
15 11
rant
00844
n nirts
31
0571
it nano
063
5.0
57
57
0 93
50
63
1.03,
n
7A
1 24
S0
DA
1 AA
5.0
78
1.27
50
104
1.69
n n
inn
1889
5n
50
153
7 17
16.00
173
190
252
310
50
n
993
251
380
5 11
979
358
5.0
50
1875
1114
178.0
95
3818
3527,
VP
0.5
141
1 93
:51
AR
41$
In
517
/10
06761
am
356
333
02
1.13
155
16-6130vm
118 3
115 1
063
1511
4-9-1
4-9-2
Average
249
50
50
443-2
0720'95
two-blade paddle, H 2.5 an, W= 53 cm
6
Mere:
200 rpm
33 uE/nY2-sec
14 an from vessel centerline
8.5 an from vessel surface
Avemge
15.0
599
4.6-2
07/1995
Mutilator Design:
Pholoperioct
15n
4,11-2
07/17/15
0.0693 Oiler
AU 0
935 nn
4-3-2
07/10/95
0 X Base Median
Volume
pH
n
p74 9141A
Illuminator lo:
Illuminator Position:
None - Control
Tme
Cultivation
4.1.7
0705
timelier Speed:
5.80
542
5.61
0.44
12 C
13 C
11 C
Culture Growth
Tnal
07154195
Mixer Setkng:
635 mgt.
3490 mot
Na11033 Conc. 0P2 Medium
Nutrient Loaded
Lowing Ratio
GP2 salts per Stec
Data
Gas Terry:mature:
Vessel Temperature:
Setpcint Trircarature:
Impeker Design:
95.0 mL
GP2 Medurn Volume (Vm):
Trial Culture Volume (VT):
Initial Ce8 Density ()(0):
NeNO3 Conc. GP2 Medium
Samets
Al Ammeter S/N:
Al Row:
1075.0 mg DCWA_
inoculum Volurne (VD:
4,15mm Holes under impeller
50
059393
397
mLhnin
SP09,31 Design:
Air Row Setting:
8 hr OFF
win
Table A-1.(cont.) Laminaria saccharina gametophyte cell cultivation Run 4.
07/22/95
4-10-1
18-5:45pm
17.99
4-10-2
07/25195
4-11-1
4-11-2
21-10.00cm
21.17
8.49
5.0
5.0
517
8.42
51$..,
345.
5.0
5.0
556
9.06
591
9.63
8.44
20.0
20.0
820.0
2.32
9.76
9.341
40.0
20.0
800.0
2.48
915
15.0
0.0760
:
15.0
I /....: it,­
..
-
07/30/95
4-13-1
4-13-2
26-11:45pm
26.24
08401/95
4-14-1
28-10,35am
27.70
4-14-2
8.46
828.4
0.5
I
III
9.48
9.53.
9.51
20.0
20.0
820.0
2.91
9.32
9.63
9.56
9.59
50 0
00
770 0
2.91
8 83
5.0
592
9.64
5.0
5.0
582
585
5.0
5.0
591
587
828.9
828.2
::.,
a
4-12-2
0.0937
0.0636.
20.0
20.0
0.0755
0.0749
0.0996
0.1933.
943.4
1160.5
1051.9
108.5
211.0
20.0
0.0756
0.1013.
78 8
0.1051
1023 6.,
865.5
944.3
0.0821
;,..,1
I
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I
A
I
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11
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II
sLit=1077,,,
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tItAII
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MINIM MIMI MET,
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1111111111111 EIM:Fra Emir?,
mourn ismv,
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rpm..
Im:ra
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517.7,7M [MIMI Ern7.11, I =WM MINN =WTI =NM IMIKEI =1E71
11111
VATZTAIIETTMII =MN
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rinrimi F
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mem
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MITITIrTna
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rms. 1111111
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1111111
mrt... I menpr,
INIIIETI
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111111
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nom
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III
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ET611111111
[11.
IL
11
111Tirirn, MEM 111111M1 MEM NMI
MOM=
111.1=1
MEM
"NMI
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MI=
MIME
Norm
=137.1Elfrri. MIllrTIMIIIF111NIMPFI
limw;r7rJ EllE71
MIMEO
imormwoomm.mr.m
1lI 1 awn
Table A-1.(cont.) Laminaria saccharina gametophyte cell cultivation Run 4.
07/22/95
4-10-1
18-5:45pm
17.99
07/25/95
4-11-1
.21-10:000m
21.17
374
370
6.09
6 03
6.06
5 a.
5.0
5.0
378
365
6.16
5.95
6.05
5.0
4-10-2
8.50
4-11-2
0.0760
0.0755
0.0896
0.0895
555.5
584.5
570.0
14.5
20.0
20.0
0.0773
0.0742
0.0981
772.2
872.9
822.5
50.4
0.0968
20.0
20.0
0.0758
0.0768
0.1030
0.1014
1097.4
963.9
1030.6
66.7
15.0
15.0
07/28/95
4-12-1
24-5:35Dm
23,98
5.0
5.0
376
382
6.12
6.22
6.17
26-11:45pm
2624
5.0
5.0
387
383
6.30
627
5.0
5.0
396
384
6.45.
4-12-2
07/30/95
4-13-1
4-13-2
08/01/95
4-14-1
4-14-2
28-10:4,5am
2720.
8.54
624
635
6.26
O.
I
".`
I
- . .
1.1r irric=11
II. ...fa
II
ralyrm 11131MI 5r7MII 11=171 =NM 11711
MOM
MINIM
rms.
memo
It
It.,
111177-1 MEN15771 MEM MIMTI 1111111F1 INNIC71
=11=11
momom.
EINIM
N/morn
Mir, mom
limy!
517"47M RTINNI FP111117 111111I9
[MIME =1111=
1111111MINIIII
1
RIM= IIMI!"1
11=11111
rmon NW" 1111117"1
1111M71 MEM NEM
MNIEN NIIM1111111II NM=
OMNI NONNI MEM
MIME
MINN=
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MENEM
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mom
IINME =NM MINI" INW171
MINN 1IrT1 1111Wilsoorr
111=11
MIRE MINN mown morm, marra mom
EMT/ NEVI NINIrm
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MONNE
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MONTI NEM NNITI31111.15=
MEM
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!In 111111,1 11111111111M5V11111111,1
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1MM
MOM
MINEINI
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MITI MINE NORM ININITIM1/11
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M117/1
11111,. IIIINIF111111.7:1 MEM NWT/
ENVIE MIWT1., NIN""In mT.Irmarri
11111111 IINIrrti mourn IMMIC1IMIIF11 IN11%71
omm
111111rE
M11177111175M
MIME
MEE NM/1 MIMI !PI =111=
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moirm mom
5777111 CON= rr.r.Trru mum
Immo
tll Ift
1111171111MErn INNIMT111WM1 11111N71
11111111111111115.711MENTM IIMIC/11
1/11
N1M7131711
111.1 Mgr n .1mm
NEMO
EMM11111
NEM] MEM
820.0
MIMI Minn
Table A-1.(cont.) Laminaria saccharina gametophyte cell cultivation Run 4.
07/22/95
4-10-1
18-5:45pm
17.99
4-10-2
07/25/95
4-11-1
21-1000om_
21 17
8.60.
4-11-2
5.0
601
9.79
5...Q.
596
9,71
5.0.
671
1.0.93
5.0
664
10.82
9.75
20.0
20.0
820.0
174.97
9.76
10.87
40.0
20.0
800.0
186.47
9.15
1219
20.0
40.0
820.0
208 35
951
15.0
.
15.0.
0712895
4-12-1
24-5-350m
23.98
4-12-2
07/3095
4-13-1
26-11:45pm
26.24
4-13-2
08/01/95
4-14-1
4-14-2
28 -10'45a
27.70___8.632
0.0759
0.0761
0.0930
0.0932
789.3
788.4
788.9
0.5
5.0.
5.0
717
780
1158
5.0
5,0.
809
816
13.18
13.29
13.24
20.0
20.0
820.0
218.75
9.32
54
817
1131
13.49
13.40
500
0.0
770.0
218.75
883
5.0
20.0,
828
12.71
20.0
0.0760
0 0752
0.0996
0 0997
916.7
964.4
940.5
23.9
20.0
20.0
0 0757
0 0999
65
94993
947 7
934.8
941.2
0.0751
I
I
I
.
11. 1&
=MI [MN, =MI NEM NUM INIM1111
MIMI MIMI
11111111
II1E11 =owl
rt7=1111
Mr= MENU PFETTM MWEll
MN=
MIMI I MIMI
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mrmin worm
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MINT 71 Mir 17.1
11111111
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!WI !ME!! MUM =IrrTI
MrT",.-M tri=111
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Norrm Ear= wisrul
MUM 11111114111111=T TI
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MINILI1 Mir 1 MIT"
MINIM
WWI =NM MINIM MUM"
M11111111
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morn mr:ri
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1111M7111Intil
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=ran ff1111111rnms Nor In =En 1111111MMIr14, EMMA." 11111F741
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11711 MEM
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mown 11111111r!.. ,11111111*"..111 MEM 111111,7,
OMIT"
=WTI =KM 11111W111
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M111111
morn mrri
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111=1MINIIIITTI
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11111N
ammo
WM, =PM =NM Normi morri
mn-rm
MIME
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EM1111111
MEM] 1111F71
1111M111/ MEM
Table A-1.(cont.) Laminaria saccharina gametophyte cell cultivation Run 4.
072295
4-10-1
18-5.45om
17.99
4-10-2
0725/95
4-11-1
-21-10:00nm
21.17
8.43
4-11-2
5.0
5.0
464
454
7.56
7.40
7.48
5.0
5.0
497
507
8.10
8.26
8.1dI
15.0
15.0
072895
07/30/95
4-12-1
4-12-2
24-5:35m
4-13-1
26-11:45vm
23 98
2624
4-13-2
08/01/95
4-14-1
4-14-2
28-10:45am
27.70
8.556
0.0758
0.0762
5.0
5.0
491
5.0
5.0
523
5.0
5.0
8.00
8.18
809
8.52
8.65
858
531
540
518
8.80
8.44
8.62
502
0.0904
0.0913
623.1
638.9
20.0
20.0
820 0
936.82
9.76
40.0
20.0
800.0
998.40
9.15
20.0
40.0
820 0
1115 55
9.51
20.0
20.0
820.0
1171.27
9.32
50.0
0.0
770.0
117127
8.83
15.7
654.6
20.0
20.0
0.0753
0.0748
0.0991
0.0931
929.1
20.0
20.0
0.0750
0.0755
0.0988
0.0992
830.1
792.5
36.6
876.8
46.6
655.8
923.4
Table A-1.(cont.) Laminaria saccharina gametophyte cell cultivation Run 4.
#1 t= 2 to 10 da s
#3 t=2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.148
0.052
0.987
5.000
3.000
0.120 1/day
0.008
#2 t = 2 to 10 da s
X Coefficient(s)
Std Err of Coef.
-0.341
0.116
0.955
5.000
3.000
0.145 1/day
0.018
#4 t=2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
Regression Output:
-0.326
0.034
0.998
5.000
3.000
0.201 1/day
0.005
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.554
0.149
0.967
5.000
3.000
0.219 1/day
0.023
Table A-2. Laminaria saccharina gametophyte cell cultivation Run 5.
Run Montt Deaden
Bioteador Run 9:
Culture Loading
BS3-1
Witte:
Inoculum Rados:
Bioreactor Desaiption:
Belco Stirred Tank Bioreactor
Time Slatted:
2:30 pm
Date Started:
08/O995
Age of Inocuksn:
Inoculum Cal Density PR:
Inoculum Volume (V0:
GP2 Medan Volume (Vm):
Total Culture Volume (VT):
SParger Design:
Ak Flow Setting:
635 mgt
397 rdAnin
Gas Tempenatre:
919 rnL
809.0 nt.
900.0 nt.
94.0 mg DCWA_
50
059360
Air Row:
1186.3 mg DCWA.
NaNO3 Conc. GP2 Rectum:
NaHCO3 Conc. GP2 Medium
Nutrient Loaded
Cawing Ratio =
4,1 Siren Holes under impeller
Air Raw:tater Silt
49,50 days
Initial Cell Density (Xo):
GP2 eau per Men
Prams Parameters
1.. sacchwina female garnetcplytes
Lfr15-1,36,610(dated 06/21/95)
Ds-17-39(dated 06120'95)
Impeller Design:
two-blede paddle, H =2S cm, W =5.3 cm
taxer Setting:
6
Mar A:
200 'pm
33 uEAMSsee
krpeler Speed:
340.0 matt
Illuminator lo:
Illuminator Position:
None - Control
0 X Base Medium
0.0693 grtiker
14 cm from vessel °entwine
8.5 cm from vessel stsface
laminator Design:
Photoperioct
Wt Dried
FRI.Celis+GP2
X
0.0881
4769
0 0880
427
10 9 Walt cool white Duke( lamp, two sides
16 twOW
Average
Average
X
X14-1s
as n
0.44 vwn
12 C
13 C
11 C
Vessel Temperature:
Selpoll Temperature:
49:
187
160
4522
24.7
8 hr OFF
Table A-2.(cont.) Laminaria saccharina gametophyte cell cultivation Run 5
08/27/95
5-10-1
18-5:00pm
18.13
5.0
5.0
5-10-2
08/29/95
5-11-1
20-2:45om
24.03
5-12-1
22- 3:OOpm
22.04
5-12-2
6.53
412
6.71
482
475_
7.85
7.74
7.79
_5.0
5.0
5.0
495
462
8.06
7.53
7.79
_5.0
5-11-2
08/31/95
401
8.53
6.62
15.0
t5.0
09/03/95
5-13-1
25-3:00pm
25.04
5-13-2
09/06/95
5-14-1
5-14-2
5.0
5.0
28-230om
2E40
486
502
8.62_ 5.0446
£0
15.0
15.0
554
7.92
8.18
8.05
8.08
9.02
8.55
0.0767
0.0767
0.0934
0.0947
759.0
845.6
0.0756
0.0756
0.0973
0 0966
1097.4
1050.7
802.3
1074.1
43.3
23.3
I
0
II I
.
tIT1 trilat7171.
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Table A-2.(cont.) Laminaria saccharina gametophyte cell cultivation Run 5
08/27/95
5-10-1
18-5:00om
18.13
5-10-2
08/29095
5-11-1
20-2:45orn
20.03
5-11-2
08/31/95
5-12-1
22-3:00cm
22.04
8.70
5-12.2
5.0
5.0
562
5.0
5.0
780
5,0
5.0
9.15
9.79
9.47
20.0
0.0
640.0
635.00
0.00
12.71
12.45
12.58
764
20.0
0.0
_820 0
635.00
0.00
832
786
13.55
12.80
13.18
40.0
0.0
580.0
635.00
0.09
20.0
0.0
560.0
635.00
0.00,
n
0.0
5100
635 00
0 OD
601
15.0
15.0
09103,95
5-13-1
25-3:00om
25.04
5-13-2
09/06/95
5-14-1
5-14-2
28-230cm
28 OD
871
0.0759
0.0767
5.0
5.0
854
13.91
831
13.54
5.0
5,0
906
876
14 76.
15.0
15.0
0.0945
0,0948
889.3
852.3
870.8
18.5
13.72
14 51
14.27
0 0755
0.0754
0 1007
0.1022
1331 2
1438.3
1364 8
536
S
s
II
11
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11.1
1111Mr1 BMW Mirri
MM.
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mnior Nommi
moms
morn smirmi worm, mir nin, moms
m
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Table A-2.(cont.) Laminaria saccharina gametophyte cell cultivation Run 5
0827/95
5-10-1
5-10-2
18-5:00om
08/29/95
5-11-1
5-11-2
20-9:4,5om
2003
08/31/95
5-12-1
5-12-2
_2g-3:0Com
22.04
18.13
8.24
5.0
5.0
461
7.51
472
7.69
5.0
5.0
564
595
5.0
665
596
5.0
7.60
20.0
0.0
640.0
64 00
0.00
9.19
9.89
9.44
20.0
0.0
620.0
64,90
0.00
10.83
10.27
40.0
0.0
580.0
64 00
0.00
9.71
1E0
0.0762
0.0769
15.0
09/03/95
5-13-1
5-13-2
25-3:00om
25.04
&141
2&22013m
2800
5-14-2
5.0
5.0
5.0
150
15.0
702
572
0.0985
0.0996
1134.6
1158.1
1146.3
11.7
11.44
9.32
10.38
20.0
0.0
560.0
64.00
0.00
14 35
12.97
13.66
500
00
510 0
64 00
000
0.0759
0.0755
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01035
1416.0
1517.9
1486 9
50.9
tA9
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MIME
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11
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Table A-2.(cont.) Laminaria saccharina gametophyte cell cultivation Run 5
08127/95
5-10-1
18-5:04xn.....
18.13
5.0
5.0
5-10-2
1..: PS!
g,
..1
.1 H
41 IS
1
5-11.2
08/31/95
5-12-1
22-3:000m
22.04
8.42
5-12-2
09/03/95
09106195
5-13-1
5-13-2
25-3:00pm
5-14-1
28-2:30pm
5-14-2
25.04
28.00
8.34
8.73
.
8.54
1
5.0
601
9.79
5.0
5.0
666
672
10.85
20.0
I I
1
10.90
0.0
1 1
640.0
:,...,1
1
8.44
0.00
.:
1,0
:
40.0
0.0
580.0
8.44
0.00
10.95.,
15 Q
0.0761
09919
15.0
0.0766
0.0926
5.0
8.59
512
536
701.8
712.8
707.3
5.5
654
697
10.65
11.35
11.00
20.0
0.0
_5 0
560.0
8.44
0.00
5.0
5.0
758
896
12.32
11.83
50.0
0.0
510.0
8.44
0.00
15.0
15.0
1134
0.0758
0.0780
0.0962
0.0966
1009.8
1022.2
1016.0
6.2
Table A-2.(cont.) Laminaria saccharina gametophyte cell cultivation Run 5.
#1, t = 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.445
0.085
0.982
5.000
3.000
0.172 1/day
0.013
#2, t = 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
X Coefficient(s)
Std Err of Coef.
-0.141
0.090
0.976
5.000
3.000
0.157 1/day
0.014
#3, t = 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
-0.630
0.111
0.975
5.000
3.000
0.190 1/day
0.018
#4, t = 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.214
0.109
0.969
5.000
3.000
0.167 1/day
0.017
Table A-3. Laminaria saccharina gametophyte cell cultivation Run 6.
Rim idontification
Bioteactor Run #:
Culture Loading
Fromm Parameters
L sacchafina female gametophyles
Ds-19-82,7,3,5(dated 0922/95)
Ls17-7,8,4,10,6(dated 08/2695)
28, 24 clay.
796.1 mg DCWA.
Culture:
hoc:Warn Rants:
136.6-1
Bloreactor Description:
1-1_ Seam Stirred Tank Roreactor
Age of inoculum
Inoctium Cal Density (Xi):
1160 an
Date Started:
GP2 Medium Vokime (Vm):
To Culture Volume (Vi):
Initial Cell Density (Xo):
NsNO3 Corp. GP2 Mecitsn:
NaHCO3 Corp. 0P2 Merlon:
Nubian' Loaded
Loading Ratio=
GP2 salts per filter
09/19195
4,15mm Holes under impeller
50
059380
Al Flow:
397 intAnin
Gas Tenperature:
105.0 rnt
795.0 rut
Inoculum Volume (Vi):
Time Started:
Speyer Dodge:
Air Row Setting:
A k Ronmeter
Vend Temperature:
900.0 mL
Seboint Temperature:
*metier Design:
109.6 mg DCWIL
former Selling:
63.5 mgt.
340.0 mgt
Mosier Speed:
33 tzElntZsec
illuminator Position
0 X Base Moium
0.0593 gAlter
14 cm from vessel centerfsne
8.5 on tromvessel surface
Illuminator Design:
1 0 9 Watt cool-white Duka lamp, two aides
16 NON/
Pholoperiod
Sample Identification
Date
Run #
Sample #
Culture Growth
Day:Hrhim
6-1 1
PH
Time
MAO
TM*
09119196
Cultivation
Semple
Volume
null
AU 0
665 nm
mAU
Average
CH a
Wt died
NA. Dried
Chia
FP
FP+CellmOP2
/mot)
Mall
(al
lel
Average
X
X
(ma DCW# I (ma 11CW/11
Average
X-W-1s
Inn ncwit
0-11.
6-1-2
0.70
00756
00629
1974
n 07 ft1
0 0108
511
00355
0.0947
2163
0 0859
00675
00160
0 0960
378 5
407 7
um 6
976
3696
131
e
11111 IMMI
6-9-2
5.05
two-blade paddle, H e 2.5 on W
cm
6
!Aker #:
200 rpm
Imitator b:
None - Control
0.44 wm
12 C
13 C
11 C
8 hr OFF
Table A-3.(cont.) Laminaria saccharina gametophyte cell cultivation Run 6.
10/09/95
6-10-1
20- 11:OOam
20.00
6-10-2
10/11/95
6-11-1
22-9:00om
22.42
831
6-11-2
5.0
5.0
281
5.0
5.0
295
303
292
4.58
4.76
4.67
4.81
4.87
4.94
15.0
15.0
0.0880
0.0885
10/13/95
6-12-1
6-12-2
24-8:00Dm
24.38
5.0
5.0
313
286
5.10
4.66
4.88
10/17/95
6-13-1
6-13-2
28-1:000m
28.08
5.0
5.0
273
266
4.45
4.33
4.39
10/19/95
30- 11:OOam
30.00
8.38
0.1041
0.1045
666.8
657.8
662.3
4.5
,
15.0
15.0
0.0908
0.0909
0.1092
0.1108
807.2
906.7
856.9
49.8
15.0
15.0
0.0920
0.0916
0.1114
0.1116
868.3
889.2
20.9
910.1
I
,
Ai
I
to
of
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r,
rim:mm.1mm rizrmrs Nom mirrIl =WI IM11111r1 MEM =WM
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r411 immti
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mom morn
ism
....=
mom
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11111111M1
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morr,
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namirrn
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mom
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merrn morn mom morn morn mirm
mum
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000
Table A-3.(cont.) Laminaria saccharina gametophyte cell cultivation Run 6.
10/09/95
6-10-1
20-11:00am
20.00
5.0
6-10-2
10/11/95
6-11-1
22-9:00oM
2242
&61
6-11-2
10/13/95
10/17/95
6-12-1
6-12-2
24- 8:OOom
6-13-1
28-1:00pm
24.39
28.08
6-13-2
10/19/95
30-11:00am
30.00
8.58
48
85
0.78
5.0.
5.0
5.0
63
70
t.03
1.08
20.0
0.0
640.0
0.00
1.38
n/a
n/a
40.0
00
600 0
0.00
n/a
0.83
20.0
0.0
580.0
0.0Q
n/a
0.90
40.0
0.0
540.0
0.00
n/a
40.0
0.0
500.0
0.00
n/a
1.06
1.14
15.0
0.0867
15.0
0.0766_,
5.0
5.0
46
58
5.0
5.0
71
1.16
39
064
0.75
0.0989
0.0857
412.8
252.8
332.8
80.0
0.91
15.0
15.0
0.0909
0.0923
0.1019
0.1023
313.4
240.2
276.8
36.6
15.0
15 0
0.0908
0.0909
0.1022
0 1013
340.5
273 4
306.9
33.6
I
.
I
^
.
I
II
.
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.
I
.
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01
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Table A-3.(cont.) Laminaria saccharina gametophyte cell cultivation Run 6.
10/09/95
6-10-1
20-11:00arn
20.00
6-10-2
10/11/95
6-11-1
6-11-2
22-9:000m_
2292
8.42
5.0
5.0
342
5.0
5.0
385
363
331
5.57
5.39
5.48
20.0
20.0
840.0
63.50
0.00
6.27
6.09
40.0
20.0
820.0
63,50
0.00
5.91
0,0879
0.0877
15...0...
15.0
0.1117
0.1062
1180.6
828.2
1004.4
176.2
10/13/95
6-12-1
6-12-2
24-8:00om
24.38
5.0
5.0
386
325
6.29
5.29
5.79
20.0
20.0
820.0
63.50
0.00
10/17/95
6-13-1
6-13-2
28-1:401277/
25,05
50
286
323
4.66
4.96
40.0
40.0
820.0
63.50
0.00
526
40.0
0.0
780.0
63.50
0.00
5.0
15,0
15.0
10/19/95
30-11:00arn
30.00
8.61
15.0
15.0
0.0893
0.0907
0.1118
0.1139
1087.4
1127.6
1107.5
20.1
0.0911
0.1119
0.1115
965.8
974.8
970.3
4.5
0.0906
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Table A-3.(cont.) Laminaria saccharina gametophyte cell cultivation Run 6.
10!0995
6-10-1
2090
20-1190an­
6-10-2
10111/95
6-11-1
22-9:00=
22.42
8,40
6-11-2
89
591
585
9.63
9.53
9.58
5.0
20.0
0.0
640.0
222.25
0.00
59
603
563
9.82
9.17
9.50
40.0
0.0
600.0
222 25
0.00
5.0
159
0,0876
0.0879
15.0
1011395
6-12-1
24- 8:QOpm
6-12-2
10/17/95
6-1.3-1
707.9
32.6
686
603
11.17
9.82
10.50
209
0.0
580.0
222.25
0,00
28.08
5.0
5.0
586
9.55
10.47
10.01
40.0
0.0
540.0
222.25
0.00
40.0
0.0
500.0
222.25
0.00
159.
15.0
30-11:00am
675.3
740.6
5.0
5.0
6-13-2
10/1995
0.1051
24.38
.
28-190om
0.1038
30.00
8.42
129
15.0
643
0.0900
0.0925
0.1179
0.1146
1444.2
1046.0
1245.1
199.1
0.0918
0.0917
0.1181
1329.2
1283.0
1306.1
23.1
0.1173
Table A-3.(cont.) Laminaria saccharina gametophyte cell cultivation Run 6.
#1 t= 2 to 10 da s
#3 t = 2 to 10 da s
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
Regression Output:
-0.807
0.098
0.986
5.000
3.000
0.219 1/day
0.015
#2 t= 2 to 10 da
X Coefficient(s)
Std Err of Coef.
-0.344
0.213
0.908
5.000
3.000
0.180 1/day
0.033
#4 t = 2 to 10 da s
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
-0.501
0.105
0.976
5.000
3.000
0.179 1/day
0.016
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.380
0.108
0.514
5.000
3.000
0.030 1/day
0.017
Table A-4. Laminaria saccharina gametophyte cell cultivation Run 7.
Run Identification
Culture Loading
Bioreactcr Run 9:
Bioreador Description:
1-1 Belco Stirred Tank Bioreactor
Tine Started:
L. saccharine kanale gametaphytes
Ls-18-2507 and Ds-19-8
71 ,41 days
19503 mg DCW/L
75.0 tit
Inoculurn Vokane (A):
GP2 Median Vokxne (Wm):
Total Culture Vokxne (V1):
Initial Cal Density (Xo):
NaNO3 Conc. GP2 Medium:
NaHCO3 Conc. GP2 Medium:
Nutrient Loaded:
10/30,95
50
059360
397 intimin
Vessel Tencerakre:
825.0 nil
Setpoint Terrperahre:
lirpeler Dosigre
Mxer Setting
kipeker Speed:
laminator lo:
900.0 mt.
120.0 mg DCWA.
53.5 mgt
0.0 mot
None - Control
Lowing Ratio.
GP2 salts per Mir
4,1.5mm Holes under impeller
SPartier Design:
Air Row Selling:
Air Flovaneter Sit:
Air Fbw:
Gas Temperature:
Ds-204,50,7
Aged Inoculum:
Inoculum Cal Dandy (Xl):
100 pm
Data Started:
Process Parameters
Cuktes:
Inomian Resits:
BS-7-1
Ilitnanator Position:
0 X Base Medium
0.0693 Oiler
Run N
Day:HrMin
Sample if
Trifle
10.30/95
11/1195
711
7-1-2
7-2-1
0.100 am
2-1:00 cm
744
111"455
11/5/25
7.5.1
7-3-2
4.1
7-4-1
&ISO am
740
11/7/95
7-5-1
7-5-2
55,
,t1:00
7.6-1
7.6-2
.10.100 an
11/11/95
7-7-1
7-7-2
124a2mn
11/1395
754
--.1+1010.1
11/1015
Semple
Volume
(ml I
0.00
AU
Average
Wt. Dried
665 nrn
Chi a
Chia
FP
moll
fmN11
Mind 1
Mat
15.0
15.0
2.03
AM)
600
8.00
a10
74-2
pH
521
0.0973
411
5.0
53
50
0
054
075
0R4
0.86
0 99
023
81
50
72
56
1 17
0.91
IRS
50
50
104
100
189
155
5.0
133
153
217
_249
150
150
296
300
281
1FIR
12.08
5.0
5.0
201
196
3.27
3.19
523
14 00
50
50
92?
222
382
352
992
253
150
7-9-2
1607
5.0
50
242
272
Average
X
(mef1CW/I1
70.0
125.7
n mom
0,0843
0 MVO
114
0.0919
1172
00847
0 0931
0 0543
00217
169 7
237
X
Average
)(4/-la
tn. ncwil Mme fawn
97.9
27.9
2.33
157
1607
tai
1
50
50
1000
Wt. Dried
FP+Cells+GP2
0.0944
0.0916
00639
50
50
50
11/5/55
11/9/95
Cultivation
Tkne
Maid
3,94
4.43
4.19
two-blade paddle, H = 2S cm, W 5.3 on
8
Mbrer
200 ma
33 uEMY2-sec
14 cm from vessel cerdedine
8$ cm Ism VOSSel surface
1 x 9 Watt coci-whke Dart lamp, two sides
16 hr ON/
8 IT OFF
Illuminator Design:
Photoperiock
Sample Identification
Date
0.44 win
12 C
13 C
11 C
110
VI ft
9109
343
Table A-4.(cont.) Laminaria saccharina gametophyte cell cultivation Run 7.
11/18/95
11/20/95
11/23/95
11/26/95
7-10-1
7-1(0-2
19-1:00 pm
7-11-1
7-11-2
.21 -1:00 Dm
7-12-1
7-12-2
24-1:00 Dm
7-12-1
7-12-2
27-1:00 om
27.00
29-1:00 pm
29.00
11/28/95
11/30/95
7-13-1
19.00
2t00
24.00
8.51
7-14-1
7-15-1
7-15-2
4.90
3.62
426
222
5.0
5.0
363
342
5.91
5.74
5.57
5.0
5.0
462
7.53
6.53
7.03
401
5.0
5.0
392
432
6.39
7.04
6.71
15.0
15.0
31.00 ,
5.0
5.0
386
408
6.29
6.65
6.47
38-1:00 Dm
38.00
5.0
5.0
393
389
6.40
6.34
6.37
38.00
15.0
15.0
7-14-2
12/10/95
301
31-1:00 om
7-13-2
12/07/95
5.0
5.0
41-1:00 pm
41.00
8.56
5.0
5.0
15.0
15.0
515
477
8.39
7.77
0.0880
0.0874
0.0998
0.1005
469.5
0.0878
0.0879
0.1006
0.1013
0.0879
0.0877
0.1010
0.1029
424.8
44.7
447,2
4872_
467.5
19.8
467.2
608.2_
537.7
70.5
380.1
8.08
I
A
I
I
0
I
.
II
en
11
.
u
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er/i
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worm mirrn
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morrl morn mom
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IMIWT:^41
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mom
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ME111111
Table A-4.(cont.) Laminaria saccharina gametophyte cell cultivation Run 7.
11/1895
11/29,95
11/23/95
11/26/95
7-10-1
7-10-2
19-1:00om
7 11 1
7-11-2
21 1.03om
7-12-1
7-12-2
24-1:00cm
7-12-1
27-1:00om
19.00
5.0
21.00
5.0
5.0
24.00
27,00
7-12-2
11/28/95
11/00/95
7 -13-1
29-1:00om
29.00
31-1:00om
31.00
5,63
7-13-2
1 2/07195
7 -14.1
38-1:00om
38,00
7-14-2
38.00
355
386
5.78
6.29
6.04
20.0
20.0
820.0
174.69
9.22
421
686
6.613
399
6.50
20.0
40.0
840.0
196.61
9.98
7.67
20.0
40.0
860.0
217.00
10.07
7.84
40.0
20.0
840.0
226.95
9.53
7/2
20.0
40.0
860.0
245.93
927
8.34
40.0
80.0
900.0
280.52
8.9
40
0.0
860.0
280.52
8.34
510
8.31
5.0
432
7.04
5.Q
5.0
501
461
8.16
7.51
15.9
15.0
0.0876
0.9870
5,0
5.0
456
492
7.43
5,0
5.0
521
8.49
8.19
503
7-15-1
7-15-2
41-1;00cm
41,00
15.Q.
8.53
5.0
5.0
0.0876
0.0872
15.Q.
15.0
514
478
702.0
751.4
726.7
24.7
8.01
15.0
12/10/95
0,1042
0.1043
8.37
7.79
0.1059
0.1087
815.3
856.2
40.9
897.1
8.08
0.0888
0.0885
0.1077
0.1087
849,7
937.8
893.8
44.0
I
1
. I.
ta,
I
Lk71717-10TTIN
111
mnrrm nom= norm mown
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morn mono mirrn11111111Tril 11111.ran
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MEN=
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morn
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mmrri =pm
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Table A-4.(cont.) Laminaria saccharina gametophyte cell cultivation Run 7.
11/18/95
7-10-1
19-1:00 om
19.00
7-10-2
5.0
5.0
399
363
690
6.21
20.0
20,0
820.0
17.61
9.22
5.91
112045
7-11-1
7-11-2
21-1:00 om
21.00
5.0
5.0
433
412
7.45
6.71
6.88
200
40.0
840.0
19,82
9.98
1123195
7-12-1
7-12-2
24-1:00 om
24.00
5.0
5.0
573
503
9.33
8.19
8.76
20.0
40.0
860.0
21.87
10.07
1126195
7-12-1
27-100 om
27.00
5.0
5.0
601
9.79
9.15
9.47
40.0
20.0
840.0
22.87
9.53
7-12-2
1128/95
29-1:00 om
29.00
8.51
562
15.0
15.0
0.0877
0.0880
0.1103
0.1123
1101.5
1213.4
1157.5
56.0
11/30/95
7-13-1
7-13-2
31-1:00 om
31.00
5.0
5.0
881
603
11.09
9.82
10.46
20.0
40.0
860.0
24.79
9.27
12/07/95
7-14-1
7-14-2
38-1:00 om
38.00
5.0
5.0
682
650
11.11
10.85
40.0
80.0
900.0
28.27
8.99
40
0.0
880.0
28.27
8.34
38.00
12/10/95
7-15-1
7-15-2
41 -1:00 pm
41.00
10.59
15.0
15.0
8.56
5.0
5.0
15.0
15.0
0.0881
0.0851
890
654
11.24
10.65
0.1096
0.1071
1028.3
1073.5
1049.9
23.6
10.95
0.0879
0.0883
0.1086
0.1075
973.9
872.1
923.0
50.%
I
A
.
11
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,
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771
Table A-4.(cont.) Laminaria saccharina gametophyte cell cultivation Run 7.
11/1895
7-10-1
19-1:00 pm
19.00
7-10-2
112095
7-11-1
7-12-1
7-12-1
7-12-2
112895
11/3005
7-13-1
7-14-1
20.0
20.0
820.0
2.32
9.22
5.0
5.0
485
453
7.90
7.38
7.64
20.0
40.0
840.0
2.61
9.98
24-1:00 pm
24.00
5.0
5.0.
510
8.31
8.89
M.,
20.0
40.0
860.0
2,88
9.46
10.07
5.0
5.0
603
582
9.82
9.48
9.65
40.0
20,0
840 0
3.02
9.53
27-1:CO DM
27.00
29-1:00 pm
29.00
31-11Y) pm
31 00
5.0
5.0
688
642
11.17
10.46
10.82
20.0
40.0
860 0
327
927
_38-1:00 pm
38.00
5.0
5.0.
612
700
9.97
10.69
40.0
80.0
900.0
11 40
3.73
8.99
40
0.0
860.0
3.73
8.34
15.0
15.0
8.51
7-13-2
12/07/95
6.60
6.68
6.64
410
21.00
7-12-2
1126/95
405
21-1:00 cm
7-11-2
112395
5.0
5.0.
7-14-2
0.0877
0 0878
150
38 00
0 0879
0.0877
15.0
12/1095
7-15-1
41-1:00 pm
41.00
8.56
5.0
50
7-15-2
766.
838
12.48
10.39
0.1075
0.1079
0 1079
0.1071
914.8
934 4
927 2
888.2
924.6
907 7
9.8
195
11.44
.­
15 0
15.Q..
0 0891
0.0879
0 1084
0.1080
875 n
933.9
904 5
PA 4
29 4
Table A-4.(cont.) Laminaria saccharina gametophyte cell cultivation Run 7.
RUN # 7 Specific Growth Rate Calculations
= 2 to
da s
Regression Output:
-0.497
0.092
0.973
5.000
3.000
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
Growth Rate =
Growth Rate +/- 1s =
0.167 1/day
0.016
Growth Rate =
Growth Rate +/- 1s =
-0.313
0.076
0.982
5.000
3.000
0.172 1/day
0.013
#4 t = 2 to 9 da s
#2 t = 2 to 9 da s
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
Growth Rate =
Growth Rate +/- 1s =
#3 t = 2 to 9 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
-0.699
0.069
0.990
5.000
3.000
0.207 1/day
0.012
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
Growth Rate =
Growth Rate +/- 1s =
-0.544
0.069
0.990
5.000
3.000
0.209 1/day
0.012
Table A-5. Laminaria saccharina gametophyte cell cultivation Run 8.
Run Identification
Etioreactor Runt.
Culture Loading
BS-8 -1
Process Parameters
Culture:
L. saccharina female gametcphytes
Inoculurn Resta:
Ls-D2-22(A)-1,2,3,45,9,10
Dated 03,07198
Bioreacbr Desaipt3n:
Age at Inoakm:
1-1. Beam Stirred Tank Bioreador
The Staled:
700 pm
036408
Date Started:
Date
Rim
Day:Hrlan
100.0 mL
Vessel Tempera*:
GP2 Medium Volume (Vm):
Total Cukuie Volume (VT):
Initial Cell Density Q(o):
NeNO3 Conc. GP2 Medium:
NaHCO3 Conc. GP2 Medium:
Kor) rd.
Sapoint Temperature:
timelier Design:
900.0 rei.
124.1 mg DMA.
340.0 mgt.
Vokime
AU 0
885 we
Chi a
O
1,4
Average
CH a
15,0
1_1
150
545-an
2-700 cal .
0328913
30
2.00
8.2-2
011'MVAR
ontravas
0.3.1
8-3-2
4-3911
a-4-1
6-930 sm
5-9.810
5.0
5.0_
54-0
0401/98
046398
8-5-1
861
8-700=
10.890ont
8.00
6.32
1004
04/0596
6-7-1
12-19.0Cbm
12.13
843-1
14410Elom
14 08
8-8-2
04/0950
8-9-1
8-9-2
1139-00om
1666
838
39
42.
0.64
0.66
085
79
71
117
133
159
217
745
739
146
238
483
3.50
14 cm tan vessel contains
I c 9 Watt cool-Mite Duka lamp, two sides
Numinedor Design:
Photoperiock
W1. Dried
1M. Dried
Average
FP
FP+CellsGP2
X
...IA
II
16 MOW
Average
X-11-1s
I
II
180.8
87.4
124.1
387,
919 5
240.4
9100
'in
00622,
QMYi1
4795
4505
219
0 0881
471 9
0.0883
0 0893
0.0979
00797
00735
(1118,1
00744
00736
00968
11R
441
6.0_
332
5.41
4.90
5.16
301
511
397
66_
541
504
410
6.68
418
394
159
1568.58
6.98
6,77
Maur it
33 tent2-sec
1.10
271
949
J1-7-2
104417/95
059
067
0 AO
aft
SQL
5.11_
R.R.9
6
200 ipm
85 cm from vessel surface
II
8-1-
two-blade paddle, H = 2.5 cm, W = 5.3 an
Iluminator b:
Iluminstor Poskiore
0 X Base Mahan
0.0693 renter
0.44 wm
12 C
13 C
11 C
Mixer Setting:
In-peller Speed:
63.5 mgt
None - Control
Culture Growth
Sample
Cultivation
pH
Tab
Sample It
mg DCWA.
Inoaiun Volume (V):
Ruined Loaded:
Loading Ratio.
GP2 salts per filer
Sample klentilloation
Air Ammeter S14:
Air Fbw.
Gas Terrperature:
18 days
-
109CLIPUM Cell Density Q0):
4, 1.5rnn tales under inpeller
50
059380
397 mUmin
SPaKter Design:
Air Flow Sating:
8 hr OFF
Table A-5.(cont.) Laminaria saccharina gametophyte cell cultivation Run 8.
04/12/96
04/14/96
04/17/96
8-10-1
8-10-2
19-10:00Dm
8-11-1
8-11-2
21-7:00 Dm
8-12-1
8-12-2
24-8:30Dm
19.13
5.0
5.0
21.00 ,
24.06
5.0
5.0
8.51
5.0
5.0
396
431
404
401
414
421
6.45
7.02
6.74
6.58
6.53
6.56
6.74
6.86
6.80
15.0 ,
15.0
04/20/96
8-13-1
8-13-2
27-6:45Dm
26.99
04/26/96
8-14-1
8-14-2
33-7:45Dm
33.03
05/01/96
J -15-1
8-15-2
38- 1 :OOam
37.70
8.421
8.34
5.0
5.0
388
400
6.32
6.52
6.42
5.0
5.0
366
402.
5.96
6.55
6.26
15.0
15.0
,
5.0
5.0
453 ,
406
15.0
15.0
0.0959
0 0966
775.9
765.6
770.8
52
0.0799
0.0796
0.0966
0.0953
744.2
678.9
711.6
32.6
0.0787
0.0769
0.0956
0.0933
763.1
738.1
750.6
12.5
0.0788
0.0796
7.38
,
7.00
6.61
.1
1
.4 I_ ,
.
C
B.
.
_
I
.
-
IA,
.
.
N.
11.
trrirrrr,',.
Ii 14
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toic.K12311 MUNN laiLi1T21
INNEN rlIFIIIII
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.
A
9
1.0971 MIMI
MEM NEM= 11111-rin
11111=n51
MINIM
0.003 mum
I
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mom =two Nom
mirira Noma =lora
rripas
WM11 MIME ?MM., Mr=
[MEM
OM:11/96
mum
1111111111
IMIF-71111111 I 11=771111W7111MENErq
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Illir1111111711111M71
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MMIMI
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np-sim RUM"
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ta_c..e_iAllIEMIIMM MOM INE771
[19111111 MINN
MEM
mrriii
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rira
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Mt=
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MEN
va
IWTO
MIMI 111=MM
MINIM
11.1111FlO IIMMILMI I
MEM
11111111
mirira
A. 1 silt! MN=
mom
rm,mn. =MN IMMI worn =Inn nra mirn.
rr .
MIMI =OM Mir
IMPW111111MEA1
imm mom mom
mow
Immi
51777111%. rT311111 ...1777111111111571
=mu
mum
mom mims
=WEI 1111 EINEM
mirm mom morn
NEI=
INIIMI1111117.
=lira MIIMITA
mom Numa =win =17:11 Elf=
Ir71
orrnri. ffra
0.0941
0.0942
0.1102
MENEM
mmni mirmin =Err) MIME] IMMO IMERTII 1373
Table A-5 (cont.) Laminaria saccharina gametophyte cell cultivation Run 8.
04/12/96
8-10-1
19-10:00om
19.13
8-10-2
04/14/96
8-11-1
21-7:00 om
21 00
8-11-2
04/17/96
8-12-1
24-8:30pm
24.06
8.40
8-12-2
5.0
5.0
799
5.0
5.0
861
5.0
5.0
888
891
803
871
13.02
14.51
13.77
20
20.0
820 0
112,92
4 07
14.03
13.08
13,55
20
200
820 0
117 Al
408
14.47
14.19
14.33
40
40.0
820.0
127.64
4.20
14.04
20
40.0
840.0
136.69
427
13 50
40
60n
AR0
14asn
409
40
0.0
820.0
149.30
3.58
15.0
15.0
04/20/96
8-13-1
27-6:45om
28.99
8-132
0/40A/A8
A-14-1
33-745nm
X 03
41
8-14-2
0.0790
0 0789
5.0
861
1403
50
883
14.06
50
555
803
13 91
13.08
5.0
15.0
15.0
05/01/96
8-15-1
8-15-2
38- 1:OOam
37.70
8.42
5.0
5.0
15.0
150
0.0777
0.0781
902
812
14.69
13.23
0.1083
0.1019
0.1013
0.1046
1588.4
1168 8
1214.4
1405.8
1378.6
1310.1
13.96
0.0768
n0791
0.1023
1064
13452
1454
1399.9
.
/
/
i
.
D
ID
II
tk-n 1,7a.dri71,1
J II
rim.
I
leg
1
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tr,',7711173111111111=1"Ell 111111111M
IP:M =Fri MIMI Mimi mown omml iiimiumm morn mrtri
ism. IMIEE
ME=7"71111111711 =WE
IIIIIIIMM
EMMMI
MEM Emir, ww:ri, iwr,
mmirEIIMIIIII
as
MEE MIMI MOM
M111
MEIN.
mown morn cri
MINIMIIMIIIVITI mem
MEM
ismorn morn imortm
rrnmi mrnim
E61111111MTIIIIIIWITI
ITEMIMI
knrfrall MIME CRIIITIE
L9RIYIIM
MIEN
ITEM
1111 MIEN
RM7111111111!MM IMMO MEM,
fr7"111 /11=E .rrArtE
rm.. sm.
=Mtn
11111111
WI­
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MEV,
111111111011=r1I1 MEM 1111111rm morn
Rtri, MIMI MIMI
EEM1774111117771
!VI MEM
11I
=Irti
mem
MINIM
111M111111 MEM
MEM
I111111
MINIM 17:7111=EUI 111111MT1
11111E!TI MEM
MEM INEETE
MINIM
IENIM
NIIIME MEM MEM
mum imirm Nwri. morn
MIMI MINIM 111M MEM
MEM
MEIN
..1,111111111E"E MrR7111MIM
11111=1111
1111711111MTI
MOM MENEM
rrrn Nur 771
MIMI
11-111, imarini =awl
MIME
MEE= IMP, E111111r1 mom Nom
MEM =MUM
pm:morns= IttrrE
immom... 1".71
11111111 MIMI IIMMTI EllMra
Mir= EINIIErq
MErfirn mirn
I11 EINEMMINIM
MOM Norm Norm MEM,
MIMI MIME
ENE= EEIr1711711.srri
ITIrMI MEM Ear
11111111E
Ill="Arl MEM, isrien
MEM
MIEN
Table A-5.(cont.) Laminaria saccharina gametophyte cell cultivation Run 8.
04/12/96
8-10-1
19-10.03otri
19.13
8-10-2
0414/96
04/17/96
8-11-1
8-11-2
21-7:00 om
8-12-1
24-8:30orn
21.00
24.06
8.38
8-12-2
834_
5.0
5.0
512
546
5.0
5.0
508
824
531
8.66
521
8.49
8.49
5.0
5.0
521
8.62
20
20.0
820.0
236.46
14.24
8.45
20
20.0
820.0
253.93
14.28
8.49
40
40.0
820.0
288.01
14,69
8.85
20
40.0
840.0
319.65
14.95
9.16
40
60.0
860.0
363.80
14.32
40
0.0
820.0
363.80
12.54
8.89
15.0
15.0
04/20/96
8-13-1
27-6:45om
26.99
8-13-2
0426196
8-14-1
33-7:45om
33.03
8.47
8-14-2
0.0790
0.0793
5.0
5.0
546
8.89
541
8.81
5.0
5.0
602
523
9.81
8-15-1
8-15-2
38-1:00am
37.70
8.52
5.0
5.0
15.0
15.0
555
631
1241.7
1080.3
1161.0
80.7
8.52
15.0
15.0
05501/96
0.1031
0.1010
9,04
10.28
0.0786
WOOL
10702
0.0791
0.1013
1114.6
1092.4
22.2
9.66
0.0793
0.0791
0.1010
0.1023
1080.3
11812
1130.8
50.5
LIJ
oo,
Ai
(*Tr AT71.=i1
Iy,i
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Prittr=
rrir
Frrerill
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mirm,
morra MIIICIT111111Mr1
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ntra."11M 1 ?MIMI" 111111WIVI
MELT, 101111171I1
Pt:Tim rm.. rnmrie
sin sir IMM[171
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mr-n, Imirrrilmrm
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mum=
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MINOTIIIIIIIMMIIIIIM7M11111147V1
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wsirm2 Ism Ewen
mom-fa own =EMI
....
mwri, =Km NINTTIMIMMIMIrTAI
1111111M11
11Mr17111111MTII
MIMMTTI =Ern mom lssss sorr-mwrgrnomrr IMMTTIIMMIM Ilinsmrnmr.: ,mnisimnr.n
MEM
1111111111
M111=73 MMIIIMI MIK=
'1 II
,IWI =VIII
MEM
Table A-5.(cont.) Laminaria saccharina gametophyte cell cultivation Run 8.
04/12/96
8-10-1
19-10:03pn
19.13
8-10-2
04/14/96
8-11-1
21-7:00 Dm
21.00
24-8:30pm
24.06
8-11-2
04/17/96
8-12-1
8-12-2
8.38
5.0
5.0
623
653
10.15
5.0
5.0
707
668
5.0
5.0
10.39
20
20.0
820.0
360.01
24.42
11.52
10.88
11.20
20
20,0
820.0
389.95
24.48
717
11.68
11.43
40
40.0
820.0
448.37
25.19
686.
11.17
10.64
15.0
15.0
0420/96
8-13-1
27-6:45pm
26.99
1E132
0406/96
8-14-1
33-7:45nm
33 03
843
8-14-2
0.0793
0.0792
5.0
50
691
_5 0
655
5.0
701
681
8-15-1
8-15-2
38-1:002m
37 70
848
50
5.0
15.0
15 0
1597.2
631
1534.1
11.17
20
40.0
840.0
502.61
25.62
10.67
11.42
11 04
40
R0.0
860.0
578.30
24 54
40
no
KO 0
578.30
21 50
0.0793
0.0792
703
713
1660.3
11.26
11 09
15.0
15.0
05/01/96
0.1097
0.1077
11 45
0.1099
0.1074
1673.6
1593.9
79.8
1514.1
1153
11.61
0.0794
0 0797
0.1101
1679.8
0 1099
16451
1662.5
17.4
Table A-5.(cont.) Laminaria saccharina gametophyte cell cultivation Run 8.
#1 t= 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.772
0.173
0.962
5.000
3.000
0.237 1/day
0.027
#2 t
2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.179
0.226
0.936
5.000
3.000
0.233 1/day
0.035
#3 t = 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
0.163 1/day
0.025
#4 t = 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.132
0.162
0.933
5.000
3.000
-0.072
0.229
0.910
5.000
3.000
0.197 1/day
0.036
Laminaria saccharina gametophyte cell cultivation Run 9
Table A-6.
Run Identification
Bioreactor Run It
Culture Loading
66-9-1
Process Parameters
Culture:
Mom lum Rasta:
L. saccharine female gametcphytes
Srodaer Design:
Ds- 25(8)-1,5,6,9,10,11,12 (0401/98)
Al Row Setting:
4 x 1.5 min holes tinder knpeller
50
Blass:tor Descrialon:
Age of Inioulunx
38.0 days
Al Fbvanoter SRN:
1-L Bolos Stimsd Tank Bioreactor
boat= Cell Density (XI):
977.9 mg DCW/L
Air Row
inocukan Volume (VI):
110.0 int
GP2 Medium Volume (Vm):
790.0 mL
903.0 mt.
Gas Tenperature:
Vessel Temperature:
Setpolnt Ten -pasture:
frivoler Design:
Mixer Setting:
Invoker Speed:
Illuminator lc
Illuranator Position:
Tine Marled:
300 pm
Date Started:
05/1056
Total CuRuns Volume (VT):
Initial Cell Density (Xo):
127.3 mg DCW/t.
63.5 mgt.
306.0 mgt
NaNO3 Cone. GP2 Medan:
NaHCO3 Conc. GP2 Makes
Nutrient Loaded:
Loading Ratio=
GP2 sags per filter.
None Conant
0 X Base Medium
059350
397 mUmin
0.0693 glitter
Muminator Design:
05/1003
16 MOW
Culture amain
Day:HrMin
Cultivation
AU 0
Sample N
Tine
Sample
Volume
665 urn
Cite
trIev1
fell
Cite
Tad I
mALI
(meat 1
(mail 1
9-1-1
0320 um
0.02
PH
8.26
9-1-2
2-390 an
202
05/14/9A
05/16/98
5-3-1
9-3-2
4.4.00 pm
9-44
6-7110 an
eta
617
5-41-2
06/15/96
9-5-1
8-1220 an
7.90
830
9-5-2
06/22/98
945-1
A-5-2
10603 orn
9-71
12-3:00 cm
115AVAIR
05/2606
9-94
9-02
14-34X1 am
9-9-1_
18-100 art
14 011
15 99
833
Average
Average
X
X4 -ls
tmn7X1W/1 I Imo nr:wn
15.0
0.0748
0.0842
2811
262.9
182
150
0.0740
3179
460
43
52
070
nys
077
71
1.16
090
39
064
FO
5,0
77
1 All
125
50
5o
140
104
228
116
1'0
199
125
12
1.89
9191
752
235
249
2 41
50
5.0
212
196
3.45
3.32
50
319
0
272
413
5.0
231
3.76
144,
12.00
X
Inn ncum 1
1273
BLOB
9-7-2
kti
133.7
120 8
411
5.0
MOMS
Wt. Dried
FP+Ceilsr43P2
0.0837
0.0834
50
50
94-2
WI. Dried
FP
0.0764
0.0763
0
9-2-1
Average
150
150
5.0
05/12/96
Iwo-blade paddle. H 2.5 an, W = 5.3 an
6
Mixer 0:
200 rpm
33 uEMVI-sec
14 an from vessel contain.
8.5 as from vessel surface
1 x 9 Watt cool-white Duke( lamp, two sides
Photopedod:
Sample Iden80callon
Date
Run I
150
0 0771
00768
341
5.65
50
340
554
244 8
410
9-92
5.0
008
5.55
00879
0 new
3638
971 9
0.44 Arm
12 C
13 C
11 C
8 hr OFF
Table A-6.(cont.) Laminaria saccharina gametophyte cell cultivation Run 9.
05/29/96
9-10-1
9-10-2
19-6:00 pm
18.13
5.0
5.0
389
456
6.34
7.43
6.88
05/31/96
9-11-1
21-7:45 DM
20.20
5.0
5.0
403
410
6.56
6.68
6.62
24-11:00arr
23.83
9-11-2
06/03/96
9-12-1
8.41
9-12-2
06/06/96
06/12/96
2721
9-13-1
9-13-2
27-8:00 pm
9-14-1
33-2:00 orn_
32.96
35-7:00 DM
35.17
9-14-2
06/14/96
06/17/96
9-15-1
9-15-2
38-1:00 om
37.92
8.51
8.55
15.0
15.0
0.0744
0.0741
602.9
524.3
563.6
39.3
0.0871
0.0755
0.0754
0.0903
0.0906
637.9
665.0
651.4
13.6
15.0
15.0
15.0
0.0788
0.0849
0.0974
0.1026
875.9
787.8
831.9
44.1
5.0
5.0
388
415
6.32
6.76
6.54
5.0
5.0
411
6.70
6.68
6.69
410
5.0
5.0
386
372
6.29
6.06
6.17
15.0,
5.0
5.0
481
403
7.84
6.56
720
0.0886
I
.
II
I
.
II
n
.
.
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7.7.1;77 MUM Irrirri
mom mos
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n
moms mrrn smwr-zi
mem
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Nm
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simerl
1111111111MI
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mu..
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morn MINIM INIIETTI norm
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111M111
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III1,717M1f9f1111M EMIT,
ETIMI
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MEM
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MINN
Ini
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MTIT'l NMI?,
NMI mom mrr,"arm Immo
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MEM
mow
airri
Table A-6.(cont.) Laminaria saccharina gametophyte cell cultivation Run 9.
05/29/96
05/31/96
9-10-1
9-10-2
19-6:00 pm
9.11 -1
21 7.45 om
18.13
20.20
9-11-2
06/03196
9.12 -1
24-11:00am.
23.83
8.45
9-12-2
030656
9-13-1
27-8.03 cm
2721
9-13-2
06/1256
9-14-1
33-2:00 Pm
32.96
9-14-2
06114/96
06/17/96
9-15-1
9-15-2
5.0
5.0
551
8.98
602
9.81
5.0
5.0
591
9.63
602
9.81
20.0
40.0
820.0
177.41
9.90
9.72
20.0
20.0
820.0
188.57
9.75
40.0
40.0
820.0
210.35
9.70
10.0
15.0
0.0741
0.0741
0.0862
0.0888
896.5
637.7
667.1
29.4
5.0
5.0
615
666
10.02_
5.0
645
671
10.51
10.93
10,72
5.0
20.0
40.0
840,0
230.57
9.67
5.0
5.0
642
10.46
10.93
10.69
20.0
60.Q.,
880.0
258.15
9.30
671
10.43
10.85
35-7:00 pm
35 17
8.50
15.0
15.0
0.0789
0.0764
0,0959
0.0945
768.8
853.7
811.3
42,4
30.0
20,0
870 0
266.81
9.10
38-1.00 DM
37.92
8.59
15.0
15.0
0.07(19
0.102a
23.1
40.0
0.0
830.0
26681
8.44
0.1031
1195 5
1241.7
1218.6
0.0790
430.0
360 0
5,0
5.0
Total
9.39
711
702
11.58
11.44
11.51
.
le
I
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St
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L
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e
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4
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rmrrimirrzm. crrnrim lowri
Imrri
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rylr film MIMI !TIMM MWTM
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II
FITIMIIII MINE
11011
MERIN 111111
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MIME
rsno.
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MIMM
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rm
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MIN=
MINIM
MIMI =MIMI 111111W13
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1111177A
MIMI=
EMMINIWIl.kI 11.11rrn, 111111.1=IIMErIrli
milwrrn mom mrrrinissrn
FITI IMWEV1 MINIM INEMIll
Vr771111111 rramr. inzzln Esmi
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111111Mr7/1 IMMITMI NWT!
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IIMMTI IIII=Irl - I MI
MEM
MEIN
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MIMI
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MEM"
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rimmmt
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MM
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moo. mum
et.
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1111111M1 IMIMS7r1 NMI=
=NM
Table A-6.(cont.) Laminaria saccharina gametophyte cell cultivation Run 9.
05/29/96
9-10-1
19-6:00 om
1813
9-10-2
05031/96
9.11 -1
21 -7:45 cm
20.20
9-11-2
0603196
9-12 -1
24-11:93an
2a83
8.17
9-12-2
0606496
9-13-1
27 8.00 cm
2721
9-13-2
06/12/96
9-14-1
33-2.00 om
32.96
9-14-2
35-700 om
06/14/96
06/1798
9-15-1
9-15-2
38-1.00 om
35.17
37.92
8,21
8.09
5&
549
5.0
497
8.10
5.9
5.0
566
9.22
9.30
571
8.52
20.0
20.0
800.04
308.29
2127
9.26
20.0
0.0
780.0
30829
19.09
40.0
0.0
740.0
30829
16.17
15.0
15.0
0.0742
0.0739
0.0909
0.0909
770.5
791.9
781.2
10.7
59,
641
666
10.44
10.85
10.65
5.0
5.9
5.0,
620
1910
10.43
20.0
10.77
00
720.0
308.29
14.17
661
5.0
5.0,
707
655
1t52
11 09
200
00
700.0
308.29
11.70
10.67
159
0.0788
0.0765
0.1001
0.0969
1055.9
1006.6
1031.3
24.7
30.0
0.0
670 0
308 29
10.96
15.0
15.0
15.0
0.0848
0.0844
0.1109
0.1119
13482
1395,8
47.8
40.0
0.0
630.0
30829
10.17
5.0
5.0
801
742
13.05
12.09
12.57
1443.4
I
1
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.
.
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as sin sr,
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PM:7MM
PMM'IMI t
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IAI
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Table A-6.(cont.) Laminaria saccharina gametophyte cell cultivation Run 9.
05129/96
05/31/96
9-10-1
9-10-2
19-6:00 pm
18.13
9-11-1
21.7:45 om
20.20
24-11:008m
23.83
5.0
9-11-2
06/03/96
9-12-1
8.34
__V 2-2
06/06/96
09112/96
8.37
9.56
20.0
0.0
640.0
228.60
5.0
5.0
612
603
9.97
9.82
9.90
20.0
0.0
620.0
228.80
-­
40.0
0.0
580.0
228.60
-
15.0
15.0
0.0741
0.0739
0.0915
0.0945
817.7
1031.9
924.8
107.1
5.0
5.0
602
632
9.81
10.30
10.05
5.0
617
588
10.05
9.58
9.81
50
20.0
0.0
560.0
228.60
-
5.0
5.0
_595
632
9.69
10.30
9.99
20.0
0.0
540 0
228.60
-
9-13-1
9-13 -2
27-8:00 pm
9-14-1
9-14-2
112:00 pm
32.98
35-7:00 pm
35.17
8.50
15.0
15.0
0.0736
0.0958
0 0756 ___0.0936
1140.0
850.7
995.3
144.6
30.0
0.0
510.0
228.60
-
38-1.00 pm
37 92
8 51
15 0
15.0
0.0787
0.0789
0 1041
13297
1349 3
19 5
40 0
00
470a.
228 60
0.1049
-
1368.8
06/14/96
08117/98
514
587
8.97
59
9-15.1
9-15.2
2721
5.0
50
698
742
11.37
12 09
11.73
Table A-6.(cont.) Laminaria saccharina gametophyte cell cultivation Run 9.
#1 t= 2 to 10 da s
#3 t = 2 to 10 da s
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
0.120 1/day
0.022
#2 t = 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
Regression Output:
-0.235
0.138
0.910
5.000
3.000
X Coefficient(s)
Std Err of Coef.
-0.203
0.127
0.934
5.000
3.000
0.131
1/day
0.020
#4 t= 2 to 10 days
-0.381
0.176
0.931
5.000
3.000
0.177 1/day
0.028
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.077
0.134
0.943
5.000
3.000
0.150 1/day
0.021
Table A-7.
Laminaria saccharina gametophyte cell cultivation Run 10.
Run Identification
Culture Loading
BS-101
Bicreactcv Run A:
Bioreactor Descriplon:
14. Seiko Stirred Tank Bioreactor
Time Started:
Age of inoculum
inocukin Cal Dandy AG):
072993
L. saccharine female gametophytes
SPASM Design:
Ls- 24(8)-5,7,8,910.11,12
Kr Flow Setting
Ak Romneter S44:
-
50.0 days
mg DCW4.
50
059360
397 int/min
Gas Tenvetakire:
Vessel Tampon:tire:
nao mt.
891.0 rriL
Selpoint Temperature:
knpeler Design:
107.9 mg CCM.
63.5 ing1.
333.0 m94.
Wee Selling:
Illuminator im
likrninator Position:
0 X Base Medium
Runty
Sample A
Day:Hrkin
Guth/al/0n
10-1-1
10-1-2
pH
Tine
(kw/
Trials
07/29/96
002
0401517am
801
Sample
AU 0
Varna
fntl
665 men
2-7:00
.
40
pitto/94
.44111rrn
034
33/04/96
000096
104-1
104-1
8-21111 men
8-9:45 an
641
8.01
813
1042
106-1
10-10114009
IL.;
1041
(ma nrwu Inn nnws
104 1
0 0755
0 0765
0 0833
5.6646
1712
0.088
01013
00897
493 1
38.
17
069
42
33
068
059
0.64
5O
5.0
41
O0./
0.84
073
39
5.0
5.0.
111
181
109
74.
Average
X4 -la
kw 111-MA 1
107.9
3.8
178.9
7.7
433 3
73
059
n fin
1 14
15.0
15.0
50
14-700
14 40
SD
5.0
104-2
08714/98
X
909
152
130
2.12
181
295
199
AA
1668
VI
971
..111
10-7-2
08/1298
16 MOW
Average
0.0824
0
50
1044
Wt. Dried
FP+Cells+GP2
fat
00756
on
MOWN
WI. Died
FP
lot
15
5..QJ
103-2
fm0,11
0.082311.1.1
5A
104-9
Average
Col a
0.0754
D
10-Z1
Chi a
fnr4 1
15'0
50.
07/31/96
1 x 9 Wag cool-Mlle Dukoc lamp, two sides
Culture Growth
381111281 Identification
104-1
10-9-9
184110 am
16 15
8.20
216
21711
20Q
PA
393
3.29
15.0
15.0
5.0
5.0
0 0871
277
451
251
4.09
Miter A:
8.5 an from vessel surface
Illuminator Design:
Phcloperiod:
Data
two-blade paddle, H =2.5 cm, W = 5.3 an
14 an ham vessel contain.
0.0693 griller
GP2 salts per filter:
0.45 vvrn
12 C
13 C
11 C
6
200 rpm
33 uE/M42-sec
impeder Speed:
None - Control
Lowing Ratio=
4 x 1.5 we holes umler impeller
Ak Flow.
105.0 01.
Inoculum Vulpine (A):
GP2 !blacken Volume (Vm):
Total Culture Volume (V7):
Initial Celt Densly (Xo):
NaNO3 Cont. GP2 Medium:
NaHCO3 Conc. GP2 Medum:
NtArient Loaded:
9:30 am
Date Started:
Process Parameters
Culture:
inoculum Resits:
4.30
4.37
8 hr OFF
Table A-7.(cont.) Laminaria saccharina gametophyte cell cultivation Run 10.
-------------08/17/96
10-10-1
10-10-
19 -4:00 .m
1927
5.0
InTnEll
10-12-1
IrEgriM
24-10:45
24.55
4.53
5.08
4.81
MIMI IIIIIIMMI =ETTI 1111=E1
MNIIIIMI MEM IMIMI
157STMIIMITMMEIMIIII IIIIIIM
08/22/96
278
312
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8.33
15.0
15.0
0.0771
0.0903
0.0770
523.8
560.7
36.9
97.
1111W111111mirmiti
um"
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111111=71
08/25/96
10-13-1
27-12:20
27.12
5.0
5.0
10-13-2
momMIMIMIMI
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35-4:00 m
some
MM.
10-15-1
3527
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47-3:00
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7.59
7.80
8.01
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7.23
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10-18-1
5.86
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9/14/96
6.68
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09/02/96
410
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Table A-7.(cont.) Laminaria saccharina gametophyte cell cultivation Run 10.
08/17/96
10-10-1
19-4:00 pm
5.0
19.27
08/20/96
10.11 -1
22 9:00 om
22.48
24-10:45
24.55
5.0
5.0
10-11-2
08/22/96
08/25/96
10-12-1
10-13-1
246
59
10-10-2
27-12:20 p
8.51
27.12
10-13-2
4.01
4.47
20.0
0.0
640.0
577.85
0.00
5.77
20.0
0.0
620.0
577.85
0.00
40.0
0.0
580.0
577.85
0.00
4.94
396
312
6.45
5.08
15.0
0.0755
0.0901
624.5
15.0
0.0761
0 0906
6151
5.0
5.0
402
5.0
512
50
619.8
4.7
7.23
6.55
6.89
8.34
7.35
7.84
20.0
0.0
560.0
577.85
0.00
451
08/3056
10-14-1
10-14-2
3241.30 om
32.46
5.0
5.0
606
612
9.87
9.97
9.92
200
0.0
540.0
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0.00
09/02/96
10-15-1
35-4:00 om
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9.82
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10.30
20.0
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577.85
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10-15-2
09105/96
10-16-1
10-18-2
38-8A5 om
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0 7790
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876.5
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15.0
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0.0949
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15.0
15.0
5.0
709
701
04,01/96
9/14/96
10-17-1
10-17-2
41 9.00 om
10-18-1
10-18-2
47-3:00om
41.50
47.25
8.51
50
771
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24208 9
249301
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400.0
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000
Table A-7.(cont.) Laminaria saccharina gametophyte cell cultivation Run 10.
#1 t = 10 to 20 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
0.426
0.054
0.957
5.000
3.000
0.062 1/day
0.008
#2. t =10 to 20 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std. Err of Coef.
#3. t= 10 to 20 a
X Coefficient(s)
Std Err of Coef.
0.158
0.089
0.937
5.000
3.000
0.084 1/day
0.013
#4. t= 10 to 20 da s
Regression Output:
0.443
0.076
0.885
5.000
3.000
0.051 1/day
0.011
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-1.998
0.350
0.842
5.000
3.000
0.196 1/day
0.049
Laminaria saccharina gametophyte cell cultivation Run 11.
Table A-8.
Bioreedor Run 9:
BS-11-1
L. saccharine female gametophytes
DS-28(A)-52,3,6,4
4, 15rnm hobs under impeller
50
059393
Sparger Design:
Air Flow Sating:
420 days
Age c4 Inocuknt
Inoculurn Cal Densky Q(:
nocukan Volume (Vi):
0P2 Pertain Volume (Vm):
Total Culture Whim. 0/1):
Initial Cell Dandy (Xo):
NeNO3 Conc. GP2 Medium:
NerIC03 Cone. GP2 Machin:
Nutrient Loaded:
Lowing Ratio w
GP2 salts per filter
11:45 pn
09/26/96
Date Started:
Culture:
inocuken Reeks:
Air Rwanda:SIN:
Bioreactor Desorption:
1-1. Belco Stirred Tank Bbreactor
Time Stetted:
Process Parameters
Culture Loading
Run Identification
397 milmin
Ak AM
mg DCW/L
830.0 mL
930.0 at
too-blade paddle, H w 2.5 an,W .53 cm
Impeller Design:
Mbcer Settng:
Impeller Speed:
Illuminator in
Illuminator Position:
182.5 mg DCW/L
635 me.
340.0 rngt
None - Control
6
Date
Rim 9
Sample It
14 cm from vessel centerline
85 an from vessel surface
0 X Base Medium
0.0693 gliller
10 9 Watt cool-while Duka lamp, Mo sides
Illuminator Design:
16 MOW
Culture Growth
Day:Hrkan
Trial ft
11-1-1
11-1-2
0-11:45 om
AD 0
Tens
Semple
Vokrne
1365 nm
Chl a
Average
CH a
Wt. Died
FP
kW
9811
mALI
Owen
(mot I
101
Cultivation
0.950
pH
8.11
150
50
11-2-1
2.1290 om
151
11-22
pit maw
113.1
4.020 rm
SO
11-4-1
5490 om
568
1142
1013895
114-1
10-1000on;
9.93
11-5-2
106568
11-64
11-7-1
11-8-1
0439,
5.0
51
083
50
53
0.66
n
77
79
1 25
50
50
122
199
5.0
5.0
136
142
118-1
11-9-2
2.22
226
231
15n
00757
min
inn
0.0797
0.0693
251 5
271.8
251
15.0
0 0795
0 0796
0 0905
0 0915
3519
395
298
425 6
307
14-1100om
13.97
5.0
5.0
231
3.76
4.10
272
443
50
465
855
5.0
331
5.39
19-600oM
18.76
20.0
188
507
150
15/96
182.5
2025
78
150
1
rmm nnwit
127
327
297
1512,
Inn nr.vo 1 Min nnnm
162.5
Average
X+/-1s
X
12B.
201
175
16511Cren
0.0790
0.0796
Average
X
0 65
50
50
11-84
Wt. Died
FP+Cells+GP2
rot
069
11 43
11.7-2
101286
023
36
12,1000em
11-6-2
10'1098
8.07
95-
5.11,
5.0
11-3-2
10112/96
0.0716
0.0716
15.0
50
50
444
475
723
7 74
7.49
Mbar.:
200 rpm
33 uFJm^2-sec
Pholoperioct
Sample IdertfficatIon
0.44 wm
12 C
13 C
11 C
Gas Temperature:
Vessel Temperature:
Sepoirt Terrpeniture:
70.0 04.
8 hr OFF
Table A-8.(cont.) Laminaria saccharina gametophyte cell cultivation Run 11.
MIIMIIIMMEI
---------------------10/17
rtrilli7111
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10/24/96
11-12-1
11-12-2
28-8: m
27.88
5.0
5.0
525
512
8.55
8.34
8.45
15.0
15.0
10i28/96
11-13-1
11-13-2
32-7: m
31.83
0.0711
0.0721
0.0856
0.0876
638.2
700.2
669.2
31.0
0.0796
0.0796
0.0971
0.0951
798.9
665.6
732.2
66.7
0.0799
0.0796
0.0976
0.0955
810.9
692.2
751.6
59.3
15.0
15.0
0.0798
0.0796
0.0969
0.0979
771.3
852.2
811.8
40.5
0.0943
0.0934
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730.5
724.8
5.6
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716.6
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0.0999
0.0913
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747.0
28.8
775.8
5.0
5.0
466
498
7.59
7.85
8.11
15.0
15.0
11/01/96
11-14-1
36-9:
35.89
5.0
5.0
11-14-2
491
462
8.00
7.53
7.76
15.0
15.0
11193/96
1101/96
11-15-1
11-15-2
38-10:
37.96
8.21
11/12/96
446
727
471
7.67
7.47
42.97
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15.0
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0.0771
11-17-1
11-17-2
46- 10.11.
45.93
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15.0
0.0801
0.0802
11.18-1
11-18-2
47- 10:11
15.0
15.0
0.0740
0.0745
11-16-2
11/11/96
5.0
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Table A-8.(cont.) Laminaria saccharina gametophyte cell cultivation Run 11.
10(17196
11-101
21-7:00cm
20.80
11-10-2
100'21,86
1 1-1 1-1
25-9:1M cm
24 89
8.17
11-11-2
612
602
5.0
5.0
576
5.0
5.0
591
9.89
9.97
28-8:459m
11-12-1
11-12-2
102496
27.88
5.0
5.0
31 83
11.13-2
11-14-1
36-9:00cm
35.89
9.5
9.63
9.38
0.0761
0,0756
666
671
10.85
10 93
43.11:00cm
11.16-1
11-16-2
11A38,06
38-1045om,
11-15-1
11 -15-2
110398
37 96
8 19
42.97
45.93
963
0.0954
0 0955
1113.3
10.0
20,0
40 0
910.0
910.0
9.87
10.46
11.45
11.09
40.0
0.1055
0.1059
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910.0
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132.50
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3.91
383
as()
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1358.0
10.16
0.1089
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1127
40.0
1572.9
1570.5
0.1029
n
0.0742
0 0804
150
1537.7
1494.8
1580.6
0.1054
0.1069
0.0776
0.0778
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0.1080
1526.0
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0.0795
0.0800
15.0
15.0
150
15.0
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995.0
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10.85
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0.0796
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0.0798
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666
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5.0
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11-17-1
11-17-2
11/11,96
46 93
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32-7.45cm
11-13-1
1028/96
15.0
15.0
11/131/66
15.0
15
47-1000am
11-18-1
11-18-2
11/12/96
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Table A-8.(cont.) Laminaria saccharina gametophyte cell cultivation Run 11.
#1 t= 2 to 10 da s
#3 t= 2 to 10da s
Regression Output:
Constant
Std Err of Y Est
-0.232
0.147
0.936
5.000
3.000
R quared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
0.117 1/day
0.018
#2 t = 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
0.042
0.099
0.961
5.000
3.000
0.102 1/day
0.012
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
0.131
0.940
5.000
3.000
0.109 1/day
0.016
#4 t = 2 to 10 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
-0.099
0.131
0.021
-0.032
0.174
0.928
5.000
3.000
1/day
155
Appendix B
Nitrate Analysis.
156
Nitrate Concentration Measurement
Chemetrics Nitrate-Nitrogen Analysis Kit
(Forestry Supplies, model # 78054 )
1.
A-6900 cadmium foil pack.
2.
Prescored ampoules filled with reagents sealed under vacuum.
3.
Snap cup.
4.
Color comparator.
Sample Preparation Procedure
1.
Prepare GP2 medium standards containing sodium nitrate (NaNO3) concentrations
between 0 and 600 mg/L.
2.
Rinse the vial with medium to be sampled.
3.
Fill the dilutor snapper cup with 25 mL of distilled/deionized water.
4.
Fill the sample cup with 5 mL of stock solution of known nitrate-nitrogen
concentration.
5.
Empty the contents of one A-6900 cadmium foil pack into the sample cup.
6.
Cap the sample cup and shake vigorously by hand for exactly 3 minutes.
7.
Hold the vacuette almost horizontally and touch the tip to the contents of the
sample cup. The tip will fill completely by capillary action.
8.
From the horizontal position in the sampling step above, immediately pull the
vacuette into a vertical position. This should cause a small portion of the collected
sample to fall inside the sleeve of the vacuette tip.
Note: If all of the collected sample still remains in the glass capillary, a light tap
near the shoulder of the ampoule will allow the sample to fall inside the sleeve of
the vacuette tip.
9.
Completely immerse the vacuette tip into the contents of the dilutor
snapper cup. Snap the tip of the ampoule and wait till the vacuette fills up. The
sample will fill the ampoule and begin to mix with the reagent.
Note: A small bubble will remain in the ampoule to facilitate mixing
157
Spectrophotometer Procedure
1.
Remove the vacuette from the dilutor snapper cup. Mix the contents of the
ampoule by inverting it several times.
2.
Wait 10 minutes, then carefully transfer 1.5 mL of the pink colored solution into
the cuvette immediately.
3.
Make up the total volume in the cuvette to 3.5 mL with nano-pure HPLC-grade
water.
4.
Measure the absorbance at 560 nm. Repeat the steps 1 to 3 for the duplicate
sample.
5.
Repeat steps 1 to 4 for NaNO3 stock solution of various concentrations.
6.
Plot absorbance vs. concentration of NaNO3 (mg NaNO3/L), to obtain calibration
curve and perform regression to obtain the slope and intercept.
7.
Repeat steps 1 through 4 with a sample of unknown nitrate concentration.
8.
The nitrate concentration (CN) of the sample is obtained by
CN
56°
AA 560,ref
m
where A560 is the average absorbance of the sample at 560 nm, A56J,ref is the y-
intercept of the calibration curve, and m is the least-squares slope of the
calibration curve (mAU560/(mg NaNO3/L)). Values for "A560,ref"and "m" from
calibration data are given in Table B-1.
I
I
0
01
I
I
I
.
4
II II
cuF1
_.
.
I
Absorbance (mAU at 560 nm)
I
I
O
Iv
159
Table B-1. Calibration data of nitrate standard versus absorbanct, at Joy nm.
Nitrate Analysis Calibration Data
Date
12/06/95
Standard
NO3-N
(mq/L)
Standard Absorbance at 560 nm (mAU)
NaNO3
(mq/L)
Trial #1
Trial #2
0
0
10
60.7
151.8
303.6
25
50
75
100
4554
607.1
22
36
88
117
164
Average
19
42
86
113
161
Calibration Parameters
Regression Output
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
3.265
5271
0.994
5.000
3.000
0.258 mAU/(mg NaNO3/I-1
0.012
0
20.5
39.0
87.0
115.0
162.5
Predicted
3.3
18.9
42.5
81.7
120.9
160.0
160
Table B-2. Estimation of the yield coefficent for nirate (Yx/N) in a stirred-tank bioreactor.
_
Run
No.
Bioreactor
No.
4
5
6
7
8
9
Xo
Xf
mg DCW/L mg DCW/L
1
118.3
94.0
109.6
97.9
1
124.1
1
127.3
1
1
1
Cn,o
mg/L
Cn,f
mg&
Y X/N
mg DCW/
mmol NO3
822.5
84.1
26.1
1032.0
1074.1
91.9
80.3
80.3
80.3
37.7
33.8
41.6
22.2
37.7
Average
+/- 1s
1537.1
1425.1
889.2
537.7
750.6
831.9
84.1
966.0
917.0
1290.8
1194.7
236.4
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162
Table B-3.
contd.
Sample Identification
Runk Date
Reactor #
Nitrate An lysis
Cultivati
I.
I
Analysis Absorbance at 560 nm (mAU)
Date
Trial #1
Trial #2
. f ,A: .
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2.
Time
(days)
II
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12/06/95
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163
Appendix C
15-HETE Measurement by EIA
164
Analysis of 15-HETE from the Female Gametophytes of L saccharina
Sample Extraction
1.
Measure the wet weight of the biomass sample (typical weight, 0.15 g).
2.
Cool the sample in a -80 °C freezer for 2 hours.
3.
Freeze-dry the biomass sample.
4.
Weigh the freeze dried sample (± 0.1 mg), then transfer to a 55 mL "Potter-
Elvehjem" (PE) style tissue homogenizer. The typical weight of freeze dried
biomass sample used for analysis is 15.0 mg.
4.
Add 10 mL of DCM/Me0H (2:1 v/v) mixture to the PE tissue homogenizer
containing the freeze dried biomass tissue sample.
5.
Macerate the tissue completely. Specifically, attach the pestle to an overhead
mixer. Set the mixing speed at 150 rpm, and macerate until the pulp is completely
solubilized. Make sure homogenizer is immerzed in an icebath.
6.
Filter pulp one more time and re-extract. Pool all filtrates (total volume 20 mL)
7.
Store supernatant extract samples cooled at around 4 °C. Retain the supernatant
for testing.
Preparation of ODS Silica Cartridge and Oxylipin Isolation
1.
Wash the ODS-Silica cartridge (Varian Sample Preparation Products, 1211­
3027) with 10 mL of HPLC-grade water followed by 10 mL of ethanol (95%) and
another 5 mL of HPLC-grade water.
2.
Blow down extract sample to dryness over flowing nitrogen at room temperature.
3.
Reconstitute the dried residue in 0.1 M sodium phosphate buffer.
Note: Prepare 0.1 M sodium phosphate solution and adjust the pH to 7.0 by
adding 3 % (v/v) formic acid (typical volume, 3 mL for 1L of phosphate stock
solution)
165
4.
Acidify the sample to pH 3.0 with 3 vol % formic acid and apply to a ODS-silica
cartridge. All the non-polar components in the buffer get adsorbed onto the
cartridge. Discard the liquid fraction.
5.
Wash the cartridge with 5 mL of HPLC-grade water followed by 5 mL 15 vol %
ethanol followed by 5 -mL of HPLC grade petroleum ether.
6.
Elute the non-polar components from the cartridge with 10 mL of HPLC grade
ethyl acetate.
7.
Evaporate the eluted solvent to dryness under flowing nitrogen at room
temperature.
8.
Reconstitute the dried residue in a 0.2 mL EIA kit buffer.
166
EIA Assay
1.
Bring all reagents to room temperature.
2.
Mix all reagents thoroughly before use.
3.
Determine the number of strips required to test the desired number of samples
plus the number of wells needed for running substrate blanks, buffer blanks and
standards.
4.
Remove the extra strips from holder and store at 2-8°C in the storage bag
provided.
5.
If more than one assay will be performed, the standards must be stored at -20°C.
Note: Do not freeze thaw the standards more than three times.
6.
Leaving the substrate blank well empty, pipette 100 !IL of standard, buffer or
sample ( in duplicate) into the antibody precoated wells according to the following
scheme:
Wells
1A, 1B
Substrate Blank
1C, 1D
15 HETE Standard (0 pg/mL)
1E, 1F
15 HETE Standard (50 pg /mL)
1G, 1H
15 HETE Standard (100 pg/mL)
2A, 2B
15 HETE Standard (250 pg/mL)
2C, 2D
15 HETE Standard (1000 pg/mL)
2E, 2F
15 HETE Standard (2500 pg/mL)
2G, 2H
15 HETE Standard (5000 pg/mL
3A -
7.
Samples
Other Samples
Gently agitate the plate by tapping the edge of the holder for at least 15 seconds to
thoroughly mix contents of each well.
167
8.
Leaving the substrate blank well empty (1A, 1B), pipette 100 !IL of 15-HETE
antibody to all other wells and tap gently to mix.
9.
Cover the wells with a plate sealer and incubate overnight at 2-8°C. (Optional:
incubate for 2 hours at room temperature on a horizontal orbital rotator set at
150+1- 50 rpm)
10.
Leaving the substrate blank wells empty (1A, 1B), add 100 !IL of 15-HETE
Alkaline Phosphate conjugate to all other wells and tap gently to mix.
11.
Cover the wells with a plate sealer and incubate at 2-8°C for 3 hours. (Optional:
incubate for 2 hours at room temperature on a horizontal orbital rotator set at
150+/-rpm).
12.
Remove sealer. Aspirate solution from all wells. Wash wells 3 times with
approximately 400 p,1 of wash buffer per well with thorough aspiration between
washes. Invert and blot on paper towel after final wash.
13.
Pipette 300 p,1 of pNPP substrate solution into all wells. Incubate covered for 2
hours at 37+/-2°C. (Optional: incubate covered for 2 hours at 37+/-2°C, no
shaking)
14.
Pipette 50 pL stop solution into all wells, including blank wells. Tap plate
gently to mix the contents.
15.
Read absorbance of wells at 405 nm versus substrate blank using a 96 well
microplate reader (Biotek Instruments, Inc., Model No. EL311 SX). The
absorbance should be read as soon as possible after the completion of the assay,
but may read upto 2 hours after addition of stop solution when wells are kept
protected from light at room temperature.
16.
For standards, plot the abosorbance, A405 (vertical axis) versus the 15-HETE
concentration, C15_HETE in pg/mL (horizontal axis) for the standards using a linear
scale for absorbance and a log scale for concentration. Fit calibration data to a
three parameter logistic equation, given by
A405 = a3 + a2 1n C15-HE'TE
al On C15-HETE)2
168
where al, a2 and a3 are adjustable parameters estimated by regression analysis of
calibration data (Table C-1). Typical values for al, a2 and a3 are -0.44, 0.02, 2.76
respectively.
17.
The 15-HETE yield in the biomass is estimated by
C15-HErE Vbuffer
Y15- HETE(X
where Ci5_HETE is the 15-HETE concentration from the EIA assay, MDcw is the dry
cell mass of the tissue used for extraction, and Vbuffer is the buffer volume
containing the extract obtained from m Dcw.
18.
For each sample record the absorbance at 405 nm in each sample well.
Average duplicate samples.
19.
Locate the average absorbance value which corresponds to each sample on the
vertical axis and follow a horizontal line intersecting the standard curve. At the
point of intersection, read the 15-HETE concentration (pg/mL) from the
horizontal axis.
r-
3
rl
O
O
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Absorbance (mAU at 405nm)
170
Table C-1. EIA calibration of 15-HETE standards versus absorbance at 405 nm by
semi-log plot.
C-(15-HETE)
In C-(15-HETE)
A405
POI/MI
0.00
50.00
100.00
250.00
1000.00
2500.00
5000.00
-­
3.91
4.61
5.52
6.91
7.82
8.52
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
1.80
1.30
1.14
0.83
0.58
0.43
0.30
A405
Predicted
-­
1.26
1.11
0.91
0.61
0.41
0.26
2.108
0.054
0.986
6.000
4.000
-0.216 mAU /(pq/L)
0.013
171
2.0_
1.8_
E
O
1­
<
1.6
1.4_
1.2_
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0(1)
0.8:
.2 0.6_
0
Cl)
<
=
0.4_
0.2_
0.0
10
1
I
I
I
FI ill
I
100
1000
I
I
10000
Standard Concentration (pg 15-HETE/mL)
Figure C-2. EIA calibration of 15-HETE standards versus abosorbance at 405 nm
by logistic equation.
.1
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173
Appendix - D
Nutrient Addition in a Stirred-Tank Reactor
174
Fed-batch Cultivation of L. saccharina Female Gametophyte Suspensions in a
Stirred-Tank Bioreactor
1.
Autoclave (250 °F, 15 prig, 20 min) the bioreactors, GP2 base medium, GP2
nutrient medium stock solution and essential glassware.
2.
Pool four flasks of L. saccharina gametophyte culture and gently blend in a 500
mL blending cup on an "Osterizer" blender at "liquefy" for 5 seconds. Determine
the dry cell density of the inoculum (Xi).
3.
Set the initial cell density of each culture at 120 mg DCW/L using the equation
X°= X.
V.
'
(V; +Vm)
Specifically, determine the inoculum volume Vi and medium volume Vn,
necessay to set X0 and total volume VT (Vi+Vm) to the desired levels.
4.
Inoculate the reactors with the freshly blended cultures and GP2 medium with
nutrients at "lx" level as listed in Table 1.
5
Position the reactors on the stir plate in the incubator, and hook up air inlet tube
and thermocouple to the reactor.
6.
Add 20 mL of the GP2 nutrient medium to the bioreactor culture at two day
intervals.
7.
Do not add nutrient stock to the control during the course of the experiment.
8.
Measure the chl a concentration in the culture, in duplicate,every two days. For
each measurement, use 5 mL of culture.
9.
Measure the dry cell density in the culture, in duplicate, every eight days. For
each measurement, use 15 mL of culture.
10.
Measure the nitrate concentration in the culture, in duplicate, every eight days.
For each measurement use 5 mL of culture.
11.
The nutrient concentration in the reactor, assuming no consumption of nutrients
by the culture is given by
CN
=
(CN,i)(VT,i)(Cmi)(Vs)+(Cmo)(V0)
(VT
Vs + Vo
(1)
175
where
CNi+i = concentration of nutrient at any time in the reactor, g/L
CNJ
= concentration of nutrient in the reactor just before the addition of
feed stock, g/L
12.
CN,0
= nutrient concentration of nutreints in the feed stock, g/L
VT,j
= volume of reactor just before the addition of nutrient feed stock, L
Vs
= volume removed due to sampling, L
Vo
= volume of nutrient stock added to the reactor, L
The cummulative rate of nutrient addition is given by
RN =
where RN
[CN -CN,i ][1]
CN,i
t
(t > 0)
= rate of nutrient addition, % dayl
= initial concentration of nutrients at the start of the experiment, g/L
t
= time, days
176
Appendix E
Laminaria saccharina Decontamination Protocol
177
Laminaria saccharin Gametophyte Culture Decontamination
1.
Decant GP2 medium from flasks containing gametophyte cultures suspected to
contain microscopic contamination.
2.
Filter 100 mL of the culture on an autoclaved 20 gm nylon mesh filter.
3.
Immerse the nylon mesh containing filtered cell biomass in a beaker-filled with
100 mL sterilized deionized/distilled water. Let water drain from the mesh.
4.
Repeat step 3 five times.
5.
Rinse the cultures with sterilized GP2 base medium.
6.
Resuspend the gametophyte cultures in 100 mL GP2 medium (1 x nutrients)
containing 100 units/mL of penicillin G.
7.
At day 3 add 0.2 mL of Tween to each flask containing 100 mL culture.
8.
At day 6 decant the medium and wash the cultures with nutrient free GP2
medium for 3 times. Filter cultures on an autoclaved 20 gm nylon mesh filter.
9.
Resuspend the filtered culture in 100 mL GP2 medium (lx nutrients).
178
Appendix F
Nutrient Removal Studies
Run Identification
Cutture Loading
Bioreactor Run e:
BJ-1
Bioreactor Description:
21 jacketed Banco
Stirred Tank Bioreactor
Time Started:
12:30prn
Date Started
0325/95
Date
Run it
Day:Hr.
Sample N
Cultiva
Time
Trial#
icAE
1-1-2
. et
i
1-2-2
ormi =Pm IIIIIII771 MEM
MINIM
1-4-2
1
1-6-2
1- -1
1.7-2
Illrfl
...MFTIIM IIM:XF/
1-5-2
I
AU @
665 nm
mAU
L saccharine female gametophyte
Sparger Design:
Air Row Setting:
CO2 Flow Setting:
Total Row.
Mole% CO2
Setpoint Temperature:
Impeller Design:
Impeller Speed
Illuminator lo:
Illuminator Position:
LS-D-11-2,4,8,5,12 (2/21/95)
31.0 days
745.0 mg DCW/I_
280.0 mL
1500.0 mi.
1780.0 mL
1172 mg DCW/1_
63.5 mg'L
0.0 mglL
0.0054 g/filter
69.30 mg/g-filter
WI. Dried
FP
Wt. Dried FP
+Cells+GP2
MEM/
Illuminator Design:
Photoperiod:
X
a DCW)L
Average
Average
x
x+i-is
DCW/L
gam=
gi,
DCW/L
=Ea
Illow,: MI 11M111.11= MI .rmlIIIIMEM
0.0784
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MEM
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on
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;.rrirTE
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Sample
Volume
mL
1-3-2
,0
pH
13M573 worm mom =11117
e
Process Parameters
Culture:
Inoculum Flasks:
Age of Inoculum:
Inoculum Cel Density (X):
Inoculum Volume (Vi):
GP2 Medium Volume (Vm):
Total Culture Volume (111):
Initial Cell Density (Xo):
NaNO3 Cane. GP2 Medium:
NaHCO3 Conc. GP2 Medium:
GP2 loading per Men
MEM
NMI
mum
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Nom Nona
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0.0791
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MEM
MIME
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five 1 mm holes under impeller
1.5 =
0=
1500 mL/min =
1500.0 mUmin at
0.0 mlimin at
0.843 win 4)
0%
12 C
Vessel Temperature:
Magnetic Stir Bar (7.6 cm diameter, 1.2 an width)
21 C
28 C
21 C
12 C
150.0 rpm
63.5 uE/M2-sec inner surface, 81.4 uE/InA2-sec jacket outer surface
14.75 an from vessel centerline
6.75 an from vessel surface
2 x 9 Watt Cool White Dulux per side, two sides
16 hr ON/
8 hr OFF
:
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6.4
Specific Growth Rate
Specific Death Rate
X vs. t, 0 to 14 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X vs. t, 21 to 33 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
4.808
0.063
0.991
7.000
5.000
0.115 1/day
0.005
X Coefficient(s)
Std Err of Coef.
-0.037 1/day
0.007
7.184
0.070
0.873
6.000
4.000
Run IdentakatIon
Culture Loading
Bioreador Run a
Process Parameters
L saccharina female gams4ophyte
DS-1543,343 (4/09/95)
Culture:
Inoculurn Flasks:
BJ -2
Bbreactor Description:
2 L jadcated Belco
Stirred Tank Noreactor
5123 mg DC1AVL
4095 mt.
GP2 Mackin Volume (Vm):
Total Cutts* Volume (VT):
Time Started:
8:30 pm
Date Started:
05/1435
1750.0 nil
Inkier Cell Density (Xo):
MINOS Conc. GP2 Medium:
0P2 lowing pallier.
............
mmi....iii..=1.
Darl*Mh
Run it
Cultivation
Time
pH
Sample
AU O
Volume
65 Fin
MUM um Nom ow Elm
Pimill11111MINIIMIFIR.. 1NM
is ra
IIIIIIIINIIIMINIIIINPI
MR
mn.I.6=...=1.iimmrnii
Mii M.
PM WM WIMP!
mirm.. -=1....Isw;
r
120.0 mg DCW/L
Impeler Design:
Impeller Speed:
63.5 mgt.
laminator to
0.0 mgt
Illuminator Position:
0.0 glitter
0.0054 glitter
69.3 mgigfiker
Illuminator Design:
M. Dried
FP
IMMINIM MINIM
CLLR
Mil MR OM
11111i1
101111111t7:11
Wt. Doled FP
*Cells+GP2
X
Average
Average
X
X+/-1s
,
IIMIIIIIIIIIIPPINININIII
Mg
MUIR NIIMININIIP
miTIII=ff:.iiiiimeiir
NRIFINIRIMPIA.,
.1. _, 1mer-ii_ =Nom
NPR migmalm Noi
m71!:
0 0749
five 1 mm holes under inpeler
1S s
0
1500 intimin
0%
12 C
1500
1101111.11111111111011
"a6: 11111,11111
at
00 mUmb at
0.857 wm 0
Vessel Temperature:
Magnetic Stir Bar (7.6 an diameter, 1.2 cm width)
21 C
28 C
21 C
12 C
150.0 rpin
635 u/m^2-sec inner. surface, 81.4 riE4m^24ec jadest outer surface
14.75 cm from vessel centerline
8.75 an from vessel suface
Photoperbd:
mil mum imor. mg
11111111.1111M MIR =IN
11111111.11111111111.11
Mob% 002:
Seboint Temperature:
1340.5 ml_
NaHCO3 Cons. GP2 PAscarn:
Sample l
Sparger Design:
Air Row Setting:
CO2 Flow Selling.
Total Row
35.0 days
Age ch Mood=
hoed= Cat Density (4):
Inoculum Vamp M:
2 x 9 Wall Cool White Duke( per side, too sides
18 WON'
hr OFF
05198
2-8-1
14-6 fpm
82Q
05130
2-9-1
16-6:30Drn
16.00
06/06
06/09
06/10
06/12
5 :4
0.0895_
556.0_
15.0
0.0755
0
1
0.0897
0.0883_
597.9
0.0754
0 175
0.0883
0 1758
0.0761
0.0901
15.0
15-0
32.4
511.7
557 9
534.8
23.1
60a1_,
501.8
652.4
50.7
0.0889_
0 0754
0756
0.0875,
0.0912
45: 3
574 5
116.2
690.7
2511
0.0886
494.9
553.0
524.0
0 1751
5.040.
15 000,
43 .7
443.0
11.4
0 0748
0.0868
15.000
0.0751
0.0862
11.6
0.0841
393.0
4162_
404.6
0 1732
0.0762
0 6762
0.0867
0 0872
348.0
364.6
16.7
15000
7.39
15.000
15.000
0 1762
0.0759
0.0875
0.0866
401.3
362.7
382.0
19.3
7-7L.
15.000,
15.000
0.0757
0.0759
0 0869
396.9
342.7
369.8
27.1
0.0863_
15.400
15.000
0 0756
0.0769
0.0864
0.0873
3701
354.4
16.3
15.000
15.000
15.000
0 1765
0 1774
0.0765
1.7
365.4
12
I$770
359 9
356.4
366.6
364 3
3582
1 AM
0.0872
0.0881
0.0873
0.0878
5.11_
2-10-1
2-10-2
18-630orn
2-11-1
2-11-2
23-6:30nrn
2-12-1
2-12-2
26-5:1 cwn
2-13-1
2-13-2
21-6:00nm
2-14-1
29-61X19111
2-15-1
0.0893
0
565.4
8.19
8
t5-0
15.0
50
23 00
25.54
:3
26.98
31-5:45pm
7.32.
30.97
7.01
2-15-2
06116
0620
2-16-1
2-1&-2
33-5:1 cm
2-17-1
37-4' 5orn
32.95
91
7.36
2-17-2
06123
06/26
06/29
2-18-1
2-18-2
40-6"15gna
2-19-1
2-19-2
43-61022rn
2-20-1
46n
2 -20-2
after blending
ft r blending_
3.
,2 :
45.92_
9
15.0
150
2-14-2
06/14
017.1
138
2-9-2
06/01
15.0
1511
54 2
2-8-2
7.41
7 48_
15.000
0 08911
s 08
a,
08 1
,
5330
3813
338.1
00
tr.)
Specific Growth Rate
Specific Death Rate
X vs. t, 2 to 14 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X vs. t, 26 to 33 days
Regression Output:
Constant
Std Err of Y Est
R Squared
No. of Observations
Degrees of Freedom
X Coefficient(s)
Std Err of Coef.
4.747
0.078
0.980
7.000
5.000
0.116 1 /day
0.007
X Coefficient(s)
Std Err of Coef.
7.997
0.030
0.981
5.000
3.000
-0.064 1/day
0.005
185
Appendix G
Temperature in Four-Reactor Incubator System
186
Table G-1. Temperatures in the four-reactor incubator system.
Reactor
No.
1
2
3
4
Reactor Location
Lights ON
TR ( °C)
Top Shelf, Left
Top shelf, Right
Bottom shelf, Left
Bottom shelf, Right
TR ( °C)
vessel temperature
TG ( °C)
gas temperature
13.2
13.2
15.1
15.1
TG ( °C)
13.0
13.0
14.5
14.5
Lights OFF
TR (°C)
TG ( °C)
12.5
12.5
13.0
13.0
12.2
12.2
13.5
13.5
187
Appendix H
Control Run Repeatability
188
Table H-1. Control run repeatability.
Run No.
11
day-1
Average Chl a
mg chl a/L
Xf (a)
mg DCW/L
4
0.12 ± 0.008 (n = 5)
9.51 ± 0.12 (1s, n = 5)
(b)
5
0.17 ± 0.013 (n = 5)
8.05 ± 0.36 (1s, n = 5
(b)
6
0.22 ± 0.015 (n = 5)
4.70 ± 0.23 (1s, n = 5)
(b)
7
0.17 ± 0.016 (n = 5)
6.91 ± 0.79 (1s, n = 5
502.6 ± 49.6 (1s, n = 2)
8
0.24 ± 0.027 (n = 5)
6.62 ± 0.34 (1s, n = 5)
731.1 ± 27.6 (1s, n = 2)
9
0.12 ± 0.012 (n = 5)
6.65 ± 0.43 (1s, n = 5)
741.7 ± 127.6 (1s, n = 2)
10
0.06 ± 0.008 (n = 5)
7.62 ± 0.15 (1s, n = 5)
672.0 ± 30.2 (1s, n = 2)
11
0.12 ± 0.018 (n = 5)
7.89 ± 0.41 (1s, n = 5)
731.8 ± 21.5 (1s, n = 2)
Overall
0.15 ± 0.06
-­
(ls, n=8)
(a)
stationary phase average.
(b)
multiple stationary phase points not obtained.