AN ABSTRACT OF THE THESIS OF
Clayton S. Jeffryes for the degree of Master of Science in Chemical Engineering
presented on July 22, 2004.
Title: Silicon and Germanium Uptake and Cell Growth of the Marine Diatom
frustulum.
Nitzschia
Abstract approved:
Redacted for privacy
Gregory L. Rorrer
Diatoms are single celled algae that form cell walls made primarily of silicon
dioxide (Si02). The metabolic machinery that gives diatoms the ability to biogenically
form highly ordered solid silica from soluble silicon extracted from their external
environment provides a unique platform to create novel nanostructured materials
inexpensively and with little environmental impact.
Of particular interest are
nanocomposite materials made from silicon doped with germanium, which can display
unique characteristics, e.g. photoluminescence. Therefore, this investigation focused on a
method to introduce germanium to diatom systems in such a way that diatoms are still
able to grow, which implies the ability to continue making nanostructured materials.
Diatoms were cultivated in bubble column photobioreactors, with a doubling time of
approximately 36 hours, to a cell density of approximately 0.3 g U', at which point their
external medium was depleted of silicon. Upon reaching a state of silicon starvation
Phase Two of diatom cultivation began with the addition of varying amounts of Ge
andlor Si to the cell culture. Phase Two concentrations as high as 11.52 mg U' elemental
Ge and molar Ge/Si ratios as high as 0.83 mol Ge mol Si' were measured. It was found
that cultures which received germanium only during Phase Two initially consumed the
soluble germanium only to efflux most of the Ge back to the bulk medium within four
hours and never experienced an increase in cell mass density. All diatom cultures that
were given silicon or a combination of Si/Ge at the onset of Phase Two experienced an
increase in cell density regardless of germanium concentration, with doubling times of
approximately 100 hours. Germanium was not effluxed by cultures receiving both Si and
Ge as it was by the Ge only cultures. These results show that when diatom cultures are
grown to silicon starvation and then fed a Ge/Si combination the diatom cell is able to
process the germanium with silicon in such a way as to permanently incorporate the
germanium into the cell mass. When silicon starved diatom cultures in stined tank
photobioreactors were given one time additions of germanium the rate of uptake was
found to follow Michaelis-Menten kinetics with a maximum uptake rate of 90.5 ± 18.9
mg Ge g dry cell mass' hf' with a half saturation constant of 5.02 ± 3.17 mg Ge U'.
©Copyright by Clayton S. Jeffryes
July 22, 2004
All Rights Reserved
Silicon and Germanium Uptake and Cell Growth by the Marine Diatom
Nitzschiafrustulum
by
Clayton S. Jeffryes
A THESIS
submitted to
Oregon State University
In partial fulfillment of
the requirements for the
degree of
Master of Science
Presented July 22, 2004
Conmiencement June 2005
Master of Science thesis of Clayton S. Jeffryes presented on July 22, 2004
APPROVED:
Redacted for privacy
)
Maj or professor, representing Chemical Engineering
Redacted for privacy
Head of the Department of Chemical Engineering
Redacted for privacy
Dean of the Gka'duat'e School
I understand that my thesis will become part of the permanent collection of Oregon State
University libraries. My signature below authorizes release of my thesis to any reader
upon request.
Redacted for privacy
S. Jeffryes, Author
ACKNOWLEDGEMENTS
I would first like to thank my advisor and mentor, Dr. Gregory Rorrer, who
provided unprecedented levels of guidance and patience in helping me create this
document. The level of support and encouragement he provides to the graduate students
under his study is unparalleled.
I would also like to thank my friend and lab mate, Tavi Cruz, who also provided
me with encouragement and made our laboratory a fun place to work. I also owe thanks
to my parents Steve and Irene Jeffryes and to my fiancé, Caitlin. It would have been
difficult to achieve anything without the strong framework of family support that they
provided. I would also like to thank Dr. Goran Jovanovic, who convinced me that I was
capable of attending and succeeding in graduate school.
I would like to thank the National Science Foundation Nanoscale Exploration
Research Program for funding the research that led to the creation of this document.
Lastly, I would like to thank Dr. Chih-hung Chang, whose research partnership with Dr.
Rorrer brought such an interesting project to fruition.
TABLE OF CONTENTS
Page
iNTRODUCTION.............................................................................................................. TI
Dry cell weight determination of samples larger than 30 mL .................................. 33
Dry cell weight determination of samples smaller than 30 mL ................................ 35
HemocytometerCell Count ...................................................................................... 37
RESULTSAND DISCUSSION ....................................................................................... 38
PHASE ONE GROWTH: SILICON CONSUMPTION .............................................................. 38
PHASE
Two GROWTh: Si AND GE CONSUMPTION IN A BUBBLE COLUMN
PHOTOBIOREACTOR....................................................................................................... 43
Growth Observations for Quadrants I-N ................................................................. 45
QuadrantI Experiments ............................................................................................ 48
QuadrantII Experiments .......................................................................................... 52
QuadrantIII Experiment ........................................................................................... 58
QuadrantIV Experiments ......................................................................................... 60
PHASE Two: SILICON AND GERMANIUM INCORPORATION INTO BI0MASS .................... 66
GERMANIUM AND SILICON SPECIFIC SURGE UPTAKE RATE COMPARISON
.................... 69
KINETIC RATES OF SURGE GERMANIUM UPTAKE .......................................................... 73
CONCLUSIONS.............................................................................................................. 76
FUTuREWORK ..............................................................................................................
BIBLIOGRAPHY ............................................................................................................. 80
APPENDIXA: SPREADSHEETS ........................................................................................ 83
APPENDIX B: EXPERIMENTAL PROCEDURES
................................................................ 137
APPENDIX C: CALCULATIONS AND CALIBRATIONS ...................................................... 157
LIST OF FIGURES
Figure
1.1
A scanning electron microscope image of the frustule (silica
based skeleton) of Nitzschia sp ..........................................................
1
...................................................................... 3
1.2
Diatom cell division
1.3
The active site of silaffin proteins ....................................................... 4
1.4
Silicic acid monomers, Si(OH)4, and a silaffin protein within
the Silicon Deposition Vesicle ...........................................................
4
1.5
Silicic acid polymerization and nanostructuring ....................................... 5
1.6
Silicon oxide nanospheres embedded with silaffin proteins
forming into diatom cell wall ...............................................................
5
1.7
Germanium incorporation into solid metal oxide ..................................... 8
2.1
Overhead view of a 2 L or 3 L bubble column photobioreactor ................... 18
2.2
Schematic of a 2 L or 3 L bubble colunm photobioreactor ........................ 19
2.3
Overhead view of a stirred tank photobioreactor .................................... 21
2.4
Schematic of a stirred tank photobioreactor ..........................................22
3.1
The dry cell mass density and soluble phase silicon concentration
versus time during Phase One for Nitzschiafrustulum .............................. 38
3.2
3.3
The dry cell mass density and soluble liquid phase silicon
concentration versus time during growth related silicon
consumption in an ideal reactor ........................................................
40
The initial phase one soluble silicon concentration versus
specific growth rate ......................................................................
41
3.4
Initial silicon and germanium concentrations for Phase Two ........................ 44
3.5
Soluble germanium concentration versus time curves for runs
BC-Ni-27 and BC-Ni-28 (Quadrant I) ..................................................
48
LIST OF FIGURES (Continued)
Figure
3.6
The soluble liquid phase germanium versus time for the
experiment BC-Ni-26 (Quadrant II) ................................................... 53
3.7
The cell mass density (XDW), liquid phase soluble germanium
concentration
(CGC), and intracellular germanium concentration
(Cyje) versus time for the experiment BC-Ni-63 (Quadrant II) .................. 55
3.8
The initial germanium uptake and germanium efflux for the culture
within the experiment BC-Ni-49 (Quadrant II) ..................................... 56
3.9
The liquid phase soluble germanium concentration (CGe), and
intracellular germanium concentrations (CGe/X) versus time for
the quadrant III experiment BC-Ni-Si ................................................ 59
3.10
The cell mass density (XDW), and liquid phase soluble germanium
concentration (CGe), versus time for experiments BC-Ni-36,
BC-Ni-37 and BC-Ni-38 (Quadrant IV) .............................................. 61
3.11
The soluble liquid phase silicon concentration
(Cs1), and the
cell mass density (XDW), versus time for experiment
BC-Ni-41 (Quadrant IV) ................................................................ 63
3.12
The cell mass density (XDW), liquid phase soluble germanium
concentration
(CGC), and intracellular germanium concentration
versus
time
for the experiment BC-Ni-62 (Quadrant IV) ................. 65
(CXJGe),
3.13
The natural log of germanium and silicon concentration versus
time for experiment BC-Ni-36 ......................................................... 72
3.14
The maximum germanium uptake rate versus initial germanium
concentration in stirred tank photobioreactors ....................................... 73
LIST OF TABLES
Table
2.1
Bubble column photobioreactor dimensions .........................................
17
3.1
Phase One specific growth rates and silicon yield coefficients for
bubble column photobioreactors growing Nitzschiafrustulum .................... 42
3.2
Initial silicon and germanium concentrations measurement during
Phase One and Phase Two ..............................................................
44
3.3
Phase Two growth rates tabulated by quadrant ...................................... 46
3.4
Initial and final Phase Two silicon and germanium measurements ............... 67
3.5
Two initial and final dry cell mass measurements and Phase Two
change in measured values ..............................................................
67
3.6
Two maximum specific silicon and germanium uptake rates ...................... 72
3.7
Germanium concentrations and initial germanium uptake rates ................... 74
LIST OF APPENDIX TABLES
Table
Pag
Bi
Diatom Nitrate LDM Medium ........................................................
141
B2
Bubble colunm vessel geometry ......................................................
142
B3
Reactor operating parameters .........................................................
143
Nomenclature
as1,i
Quadratic silicon assay coefficient (mg Si U' mAU2)
Linear silicon assay coefficient (mg Si U' mAU')
a53
Constant silicon assay coefficient (mg Si U')
aGe,,
Quadratic germanium assay coefficient (mg Ge U' mAU2)
aGe,2
Linear germanium assay coefficient (mg Ge U' mAU')
aGe,3
Constant germanium assay coefficient (mg Ge U1)
a,1
Quadratic spectrophotometric cell number density assay coefficient (cells
mU' mAU2)
a,2
Linear spectrophotometric cell number density assay coefficient
(cells
mU' mAU')
a,3
Constant spectrophotonietric cell number density assay coefficient (cells
niU')
CGe,2
Phase Two liquid phase germanium concentration (mg Ge U')
CGe,2,O
Initial Phase Two liquid phase germanium concentration (mg Ge U')
Cs1,,
Phase One liquid phase Silicon concentration (mg Si U')
Initial Phase One liquid phase Silicon concentration (mg Si L')
Cs,,,f
Final Phase One liquid phase Silicon concentration (mg Si U')
Phase Two liquid phase silicon concentration (mg Si
U5
C1,2,0
Initial Phase Two liquid phase silicon concentration (mg Si U'
CGe/X
Solid phase mass ratio of germanium to biomass (mg Ge g DCW')
Sample dilution (ml final volume ml sample volume')
Hemocytometer chamber volume (mm3 sample volume hemocytometer
squar&1)
Kije
Michaelis-Menten half saturation constant for germanium (mg Ge U1)
Michaelis-Menten half saturation constant for silicon (mg Si U')
Monod half saturation constant for silicon during silicon limited growth,
(mg Si U')
k'G
k's1
Specific first order Ge uptake rate constant (L g DCW' hr)
Specific first order Si uptake rate constant (L g DCW1 hf1)
MDW
Dry cell mass (g DCW)
MDW+f+s
Measured mass of cells, filter and salts (g)
Mf
Filter Mass (g filter)
Ms
Salt Mass (g salt)
N
Number of cells in hemocytometer cell sample (number cells)
Number of hemocytometer squares in cell count (number squares)
RGe
Apparent Ge uptake rate (mg Ge L hr')
R'Ge,O
Initial specific Ge uptake rate (mg Ge g DCW' hr')
Rs1
Apparent Si uptake rate (mg Si g DCW1 hf1)
Sc
Salt retained by filter during dry cell mass density measurements (g salt
gfilter')
td
Cell culture doubling time (hr)
V
Cell culture volume in reactor vessel (L)
V
Volume of culture sample (L)
XDW
Dry cell mass density (g DCW U1)
XDW,O
Initial dry cell mass density (g DCW U')
XDW,f
Final dry cell mass density (g DCW U')
XDW,2,O
Initial Phase Two dry cell mass density(g DCW U')
XDW,2,f
Final Phase Two dry cell mass density (g DCW U')
XN
Cell number density from cell count measurements (cells/mL)
YXJGe,2
Biomass yield per Ge consumed during Phase Two (mg cell mass
mg G&1)
Yxis
Biomass yield per Si consumed during Phase One (mg cell mass mgSi')
YXJSI,2
Biomass yield per Si consumed during Phase Two (mg cell mass
mg Si1)
Greek Letters
K
Cell mass to liquid phase germanium partition coefficient (L culture L
fresh cell volume')
Specific growth rate (hf')
Phase One specific growth rate (hr')
Phase Two specific growth rate (hr')
1L,max
Maximum specific growth rate (hf')
p
Fresh cell mass density (g fresh cell mass L fresh cells')
Abbreviations
DCW
dry cell weight
Introduction
Diatoms
Diatoms are ubiquitous single-celled algae of the class Bacillariophyceae. They
are easily identified by their cell walls made of highly ordered silicon. While diatoms are
not the only organisms to utilize silicon to build biological structures (Simpson, Volcani,
1981) they are the world's largest contributor to biosilicification (Martin-Jezequel et a!,
2000). Each diatom is easily identified by the ornate patterns of silicon that make up its
cell wall.
Figure 1.1. A scanning electron
microscope image of the frustule (silica
based skeleton) of Nitzschia sp.
In Vivo Biosilicification
Biosilicification is the process by which an organism takes up soluble silicon from
the environment and precipitates it into solid silicon dioxide. For almost a century, it has
been known that diatoms are comprised of silicon (Richter 1906), but it was first proven
that diatoms uptake silicon from their external environment in 1955 (Lewin, 1955).
2
Later, it was demonstrated that this uptake was carrier-mediated (Paasche, 1973a,b and
Azam et a!, 1974). Carrier-mediated silicon uptake can be classified as either growthrelated uptake or surge uptake. In growth-related uptake, silicon is transported from the
external environment into the cell at a rate equal to the rate of biosilicification and cell
wall formation. In surge uptake, which occurs after a silicon-starved cell is exposed to a
large concentration of silicon, the diatom takes up silicon from its environment at a rate
much faster than the rate of biosilicification (Sullivan. 1977), leading to an accumulation
of soluble silicon within the cytoplasm before the accumulated silicon is eventually
converted into cell wall.
Soluble silicon in the form of Si(OH)4, crosses the cell wall and membrane, and
then enters the cytoplasm where it is transported by molecular diffusion or by silicic acid
transporters to the silicon deposition vesicle (Hildebrand et a!, 1997). The silicon
deposition vesicle (SDV) is a specialized cell compartment located along the central axis
of a diatom midway between the epivalve and hypovalve, which are the two Petri dish
shaped halves of the cell wall. The SDV is bound by a membrane called the silicalemma,
and it is within this specialized compartment where silicon is polymerized (Drum and
Pankratz, 1964).
G2A
iI
I'i-
Gi
II
I
II
'I
- - . a
__Il_I____I____!tih
I__
G2.B
l'i
Figure 1.2. Diatom cell division. Within the SDV the hypotheca (inside
fitting half of the cell wall) is completed and the first generation diatom
divides. The second generation diatoms G2-A and G2-B have newly
fabricated hypothecas. The epitheca (outside fitting half of the cell wall) of
G2-A was the epitheca of Gi. The epitheca of G2-B was the hypotheca of
Gi.
Within the SDV are specialized proteins called silaffins (Kroger et a!, 1999, 2002). A
peptide sequence common to the proteins silaffin 1-A and 1-B, believed to be the active
site of silicon condensation, is presented in Figure 3. The silaffin proteins catalyze a
hydrolysis reaction that links silicic acid monomers together to form solid silicon dioxide
(Si02) by the creation of siloxane bonds (Sumper, 2002).
4
Ser Ser Lys--Lys--Ser GlySer Tyr--OH
OH
OH
OH
) \CH3 CH3
I
/
CH3
n=4-9
CH3
NH2
Fig 1.3. The active site of silaffin proteins. Amino acid backbone and lysine
modified residues that make up the active silicon condensation site of silaffin 1-A
and silaffin 1-B.
Si(OH)4
(b)
(a)
.S
.
.
.
Fig 1.4. Silicic acid monomers, Si(OH)4, and a silaffm protein within the
Silicon Deposition Vesicle. (a) Two silicic acid monomers come in contact
with the active site of the silaffm protein. (b) The silicic acid monomers on the
active site under go a hydrolysis reaction and become linked by a siloxane
bond (Si-O-Si).
The silicon oxide is formed into nanospheres embedded with the silaffin proteins. The
nanospheres assemble into patterned microstructures that become the cell wall to become
the diatom cell wall (Kroger et al, 1999).
5
(b)
(a)
V V V V V
a -
-
-
r V V -v
L S S S
Fig 1.5. Silicic acid polymerization and nanostructuring. (a) Silicic acid
monomers undergo hydrolysis and polymerize into solid silicon dioxide, Si02.
(b) Si02 formation around the silaffin protein to create a nanosphere.
(a)
(b)
a
II..
j
I
I
I
Fig 1.6. Silicon oxide nanospheres embedded with silaffin proteins forming
into diatom cell wall. (a) Nanospheres pack together to form a contiguous
cell wall inside the SVD. (b) Expanded view of the entire SVD and cell wall
formation along the inner side of both SVDs.
In Vitro Biosilicification
Biosilicification can be achieved outside of the diatom cell (Kroger and Sumper,
2000).
Without silaffin proteins (silaffin-1A, 1B, or 2) to catalyze the silicon
condensation reaction, metastable silicon will stay in solution for several hours.
However, upon the addition of any individual silaffin protein or mixture of silaffin
proteins, the soluble silicon begins polymerizing within seconds.
The
in vivo
silaffin mediated silicon condensation reaction produces silicon
nanospheres of remarkable uniformity, with relative deviations in sphere diameter
reported as low as 1.6% (Volcani, 1981). The
in vitro
silicon precipitation reaction
produces silicon spheres with a much larger size distribution. Kroger and Sumper (2000)
report that using silaffin-1 A produced closely attached or fused silicon spheres with
diameters ranging from 500 nm up to 700 nm. Using a mixture of silaffin proteins it was
possible to produce silicon particles with an approximate diameter of 50 nm, but these
particles were formed as aggregates, not as true nanoparticles. In contrast, the
in vivo
production of silicon produces spherical particles as small as 10 nm (Parkinson and
Gordon, 1999). The small size and accuracy in silicon sphere reproduction in vivo makes
whole cell production the preferred platform for nanostructuring of silica. No studies
have examined the effect of silaffin proteins on substrates other than soluble silicon.
Soluble Germanium in Diatom Cell Cultures
Silicon is closely related in physical and chemical properties to germanium, the
group IV element directly below silicon on the Periodic Table (Jolly, 1966). Because of
7
their similar properties, it was postulated by Lewin (1966) that gennanium could act as a
silicon analogue in diatom metabolism.
Lewin's research also tried to determine if
germanium could act as a diatom growth inhibitor to aid in creating axenic strains of
macroalgae. Lewin introduced soluble germanium into diatom cell culture while silicon
uptake was growth-related.
Lewin's experiments were conducted in nonaerated or
shaker-aerated vessels with vessel volumes of 4 to 50 mL. Lewin found that germanium
inhibited the growth of heavily silicified organisms more than less-silicified organisms
and had no effect on nonsilicified organisms. Lewin also reported that silicon dampened
the effect of germanium related growth inhibition. This first study of the interactions
between germanium and diatom cells led to many other diatom/germanium
investigations.
The first investigations of germanium/diatom interactions considered
gennanium uptake and growth (Azam and Volcani 1974, Sullivan 1976, Sullivan 1977,
Markham and Hagmeier 1982), the incorporation of radioactive germanium tracer atoms
(Ge68)
into the diatom cell wall and organelles (Azam et al 1973, Mehard et al, 1974),
and the effect of germanium on cell wall morphology (Chiappino et al, 1977).
Azam et al (1973) used radioactive germanium (Ge68) to trace the uptake of
germanium into the diatom cell and to test the hypothesis that germanium is a metabolic
analogue of silicon. Azam et al (1973), like Lewin (1966), exposed diatoms to soluble
germanium while silicon consumption was growth-related. While holding silicon levels
constant, Azam et al (1973) varied the molar ratio of germanium to silicon from 0.01 to
1.00 mol Ge/mol Si (mass ratios of 0.0039 and 0.39 g Ge/g Si). Azam et al (1973)
determined that as the Ge/Si ratio in the bulk medium was lowered the greater the percent
of the soluble germanium present in the bulk medium could be taken up and incorporated
as a metal oxide into the biogenic silica spheres. For example, at a Ge/Si mass ratio of
0.0039 g Ge/g Si in the bulk medium, 60-80% of the soluble germanium consumed by
the cell was incorporated into the diatom cell wall. However, at a bulk medium Ge/Si
ratio of 0.039 g Ge/g Si, only 14% of the germanium consumed by the cell was
incorporated into the diatom cell wall. At a Ge/Si mass ratio of 0.39 Azam et al reported
diatom growth was completely inhibited and the organisms became incapable of silicon
uptake.
* Ge(OH)4
(a)
(b)
*
(d)
(c)
**j*
*..
*. .
*.*. *
*
.
Fig 1.7. Germanium incorporation into solid metal oxide. (a),(c) Low
and high Ge:Si ratios, respectively. (b) A low Ge:Si ratio leads to a
high percentage of cytoplasmic germanium incorporated into solid
metal oxide. (d) A high Ge:Si ratio leads to a low percentage of
cytoplasmic germanium incorporated into metal oxide.
*
Mehard et al (1974), Azam and Volcani (1974), and Sullivan (1976) conducted
experiments with silicon-starved diatoms, under surge-uptake conditions. Surge uptake
occurs when a silicon-starved cell is suddenly exposed to an ample supply of silicon or
gennanium. Surge uptake is not growth related, so the surge uptake rate is not coupled to
or limited by the rate of growth of the organism. Surge uptake is many times faster than
growth related uptake, and leads to a large intracellular pool of soluble silicon andlor
germanium. Diatoms are capable of consuming enough silicon in two hours of surge
uptake to complete one cell division (Sullivan 1976). The time scale for a cell division is
measured in tens up to hundreds of hours. Mehard (1974) followed surge uptake for one
hour after the addition of silicon doped with a radioactive germanium tracer to cultures of
the diatom
Nitzschia alba
and found the uptake rate to be constant (zero-order). Azam
and Volcani (1974) attempted to quantify surge uptake kinetics.
They measured
germanium uptake with time over 30 minutes, and quantified the intracellular to
extracellular germanium ratios.
After adding the metabolic inhibitors DNP, sodium
azide, and iodacetamide to different cultures, they observed the diatoms were incapable
of germanium uptake after exposure to these agents. This indicated that germanium
uptake requires metabolic energy and that uptake was not due to adsorption effects.
Sullivan (1977) observed silicon uptake over time for a period of 60 seconds and
determined surge silicon uptake to be a saturable uptake process that can be modeled by
Michaelis-Menten kinetics.
No published studies have made observations past 30
minutes after silicon and germanium addition to silicon-starved cells.
10
Chiappino et al (1977), and later Markham and Hagmeier (1982) determined that
germanium concentrations that inhibit cell growth are quite low, ranging from 1.2 mg to
1.5 mg Ge U1. The germanium addition of both investigations was done during growth-
related uptake, but not surge uptake. Chiappino et al (1977) was the first to discover
intracellular granules within the cytoplasm following exposure to soluble germanium.
When
Nitzschia
cells were exposed to equimolar concentrations of Ge and Si, opaque
granules within the cytoplasm were formed with dimensions of approximately 100 x 50
nanometers in size and ca. 20 nm thick.
Chiappino et al (1977) also asserts in
unpublished data that the granules were found to contain silicon and germanium.
Rationale for Current Investigation
Silicon and germanium metal oxides formed into nanoparticles possess highly
valued
properties
that
arise
from
quantum
level
characteristics,
such
as
photoluminescence (Zacharias and Fauchet, 1997, 1998). Only the particles 10-50 rim in
size that contain silicon dispersed with germanium atoms produced by cell machinery
within the SDV may possess these novel properties. Particles produced in vitro are too
large and sizes too disperse to possess quantum properties.
Currently, methods exist to produce nano-sized metal oxide composite materials,
but these methods are highly expensive and involve complex, high temperature, near
vacuum, or high-energy processes such as RF sputtering (Pal, 2003) or ion implantation
(Meidrum et al., 2001).
Since diatom cultivation is inexpensive, conducted under
ambient conditions, and produces little environmental wastes, it would be the preferred
11
platform for Si-Ge nanoparticle production.
The potential of diatom cell cultures to
produce silicon and germanium composite nanoparticles merits investigation.
Previous investigations that examined the addition of soluble germanium to
diatom cell cultures were limited in scope. Until now, no research was conducted that
observed diatom growth more than 30 minutes beyond the onset of surge germanium
uptake. Long-term germanium uptake and diatom growth following exposure to various
silicon and germanium concentrations has not been considered until now. Also, there
have been no previous attempts to examine the operating conditions that promote
germanium incorporation into cell mass.
Finally, no previous work used scalable
photobioreactor systems to introduce soluble germanium to diatom cell cultures.
Research Goals and Objectives
The overall research goal is to determine conditions that will allow the marine
diatom Nitzschia frustulum to take up and retain germanium within its biomass. The
marine diatom genus Nitzschia was selected for study based on literature precedent
described earlier.
This research has four objectives: 1) characterize the growth rates of N frustulum
in 2L and 3L photobioreactors; 2) estimate the growth-related silicon requirements of N.
frustulum; 3) determine the short and long-term kinetic uptake rates of germanium and
silicon by N frustulum following a state of silicon starvation; and 4) determine the extra-
cellular silicon and germanium conditions that allow cell growth and long term
germanium sequestration within the biomass.
12
This study will support the development of a cell culture process for germanium
sequestration into silica.
Characterization of biogenically produced nanoparticles or
nanostructured Ge-Si is beyond the scope of this study.
13
Materials and Methods
Culture Maintenance
The diatom Nitzschia frustulurn (UTEX algal collection #2042 ORIGiN:
deposition: 1/76 by J.C. Lewin as 53-M (Lewin & Lewin 1960)) was maintained in a
14:10 light/dark photoperiod of light intensity 55
/LEm2s1
at the flask exterior by
artificial light (Feit Electric 9 Watt Compact Fluorescent 2700 °K / PL9).
The
temperature was kept at 22 °C in an incubator (Precision Scientific low temperature
incubator 815). Twenty four 500 mL flasks with foam stoppers containing 90 mL N.
frustulum in Diatom Nitrate LDM medium were maintained in the incubator. Each flask
was swirled for five seconds once per day.
Subculturing was performed every two weeks. Diatom subculturing was performed
under sterile conditions inside a laminar flow hood (Baker EdgeGard Hood model #E63252). Three flasks were combined into one parent fiak and allowed to sit for three
hours to allow the biomass to settle prior to inoculation.
For every parent flask of biomass five 500 mL flasks with foam stoppers plus a 100
mL graduated cylinder were autoclaved for 30 minutes at 123 °C and 23 psig.
After
allowing the glassware to cool 80 mL of Diatom Nitrate LDM Medium were transferred
into each of the five flasks inside the laminar flow hood using aseptic technique.
14
Using a sterile 10 mL volumetric pipette 10 mL of culture comprising no more than
1/6 of the settled culture from the bottom of the flask were removed and placed into each
of the flasks containing 80 inL medium. Four of the flasks were placed back into the
illuminated incubation platform and the fifth flask was kept under low light at 10
as a back-up.
15
Medium Preparation
Diatom Nitrate LDM medium was prepared from a natural seawater base (NOAA
Lab, Newport, OR, USA) fortified with Bristol's Salts containing extra sodium nitrate,
sodium metasilicate, PlY metal solution and a vitamin stock. Upon receiving the
seawater base it was pumped via a peristaltic pump (Cole Parmer Model# 50000-079,
Serial # FK 3114, 45W, 10.6 gpm) through a 5
nylon fiber Omnifilter whole house
water filter cartridge into a clean 55 gallon Poly Drum. The drum was then sealed and
only opened when fresh seawater was required for lab use.
Seawater base was
autoclaved (30 minutes at 123 °C and 23 psig) on demand just prior to medium
preparation.
Bristol's Salts were prepared from salt super stocks and then combined into a final
Bristol's Salts solution. Individual super stock solutions were made mixing solute in
deionized water and had composition: 798.1 mM NaNO3; 5.4 mM MgSO4H2O; 42.3 mM
K2HP043H2O; 128.6 mM KH2PO4. The super stocks were combined in deionized water
to form a salt stock with composition: 39.9 mIvI NaNO3; 0.54 mM K2HPO43H2O; 1.26
mM KH2PO4. Stocks and super stocks were autoclaved (30 minutes at 123 °C and 23
psig) for storage.
The sodium metasilicate, PJV metal and vitamin stocks were prepared by mixing
solute directly into deionized water without the use of super stocks. Sodium metasilicate
(Na2SiO3.5H20) was prepared to 200 mM and stored in a polyethylene bottle. PlY metal
solution was stored in a glass bottle with a composition of: 2.00 mMNa2EDTA; 0.36 mM
Fe(SO4)7H2O; 0.207 mM MnCl24H2O; 0.037 mM ZnCl2; 0.0084 mM CoCl26H2O;
16
Vitamins were dissolved in deionized water to make a
0.0 14 mM Na2MoO4H2O.
vitamin stock of concentrations: 7.38 M vitamin B12; 40.93 aM biotin
(C10H16N2O3S);
2.96 M thiamine HC1 (C12H17C1N4O5HC1); 55.49 pM meso-inositol (C6H1206); 7.93 pM
thymine (C5H6N202);
3.53 jiM
benzoic acid (C7H7NO2);
Ca pantothenate
8.19 p.M
P-amino
(C9H16NO5Ca0.5); 0.73 pJ'I
Vitamin stock was
Nicotinic acid (C6H5NO2).
portioned into 10 mL aliquots and frozen at -20°C until use. Frozen aliquots more than
six weeks old were discarded.
Bristol's Nitrate LDM medium was prepared by adding the stock solutions to the
filtered and sterilized seawater base. The final concentrations of nutrients in the medium
were: 3.98 mM NaNO3; 54.1 p.M MgSO4H2O; 42.2 pM K2HP043H20; 128.4 pM
KH2PO4; 534.8 p.M
sodium metasilicate (Na2Si&.5H2O);
Fe(SO4)7H20; 1.11 pM MnCl24H2O; 020 p.M ZnCl2;
Na2MoO4H2O;
0.01 p.M vitamin B12;
thiamine HC1 (C12H17C1N4O5HCI);
(C5H6N202);
(C7H7NO2);
7.47 p.M
14.47
98.8
0.07
10.8 p.M
0.04 p.M
Na2EDTA; 1.92 pM
CoC126H2O; 0 08 pM
p.M biotin (C10H16N2O3S);
p.M meso-inositol (C6H1206);
Ca pantothenate (C9H16NO5Ca05);
pMnicotinic acid (C6H5NO2).
1.30
5.28 p.M
14.1 p.M
thymine
pM p-amino benzoic acid
17
Bioreactor Operation
Bubble Column Photobioreactors
Three different bubble column photobioreactors were used for all long-term
diatom culture growth and Ge uptake studies.
Table 2.1. Bubble column photobioreactor dimensions
Bubble Column
Photobioreactor
3L#1
Height (cm)
Innei- Diameter (cm)
Volume (L)
9.8
11.4
7.9
3
[
48
36
48
3L#2
2L#1
3
2
All vessels were jacketed and the temperature was maintained at 22°C by the circulation
of water from a cooling reservoir through the jacketing. Illumination was provided by
four fluorescent bulbs (15 W Sylvania Cool White F 1 5T 1 2/CW) positioned parallel to the
vertical axis of the reactor. Two bulbs were on each light stage placed so there were 10.6
cm between the central axes of each bulb. Two light stages per reactor were set facing
each other with a distance of 22.9 cm between the central axes of opposing bulbs. The
reactor was placed centrally between the two light stages. In all experiments the light
intensity
at
the
inner
surface
of
each
bubble
column
was
60 E m2s1 as measured by photon detection (Li-COR QuantumlRadiometer/Photometer
Model LI-189).
FI
A timer maintained light/dark cycles of 14 hr on and 10 hr off (14:10 LD photoperiod).
In all experiments 0.5 L air U' culture mini was passed through an autoclaved 0.20 m
Gelman filter and then bubbled through a sterilized humidifier and then introduced to a 4
cm glass fit placed at the bottom of the bioreactor vessel. Carbon dioxide in the ambient
aeration gas
(Cc02
= 350 ppm) was the sole source of carbon for biomass growth.
15 W Fluorescent
Fig 2.1. Overhead view of a 2 L or 3 L bubble column photobioreactor. The
vessel is centrally located between two light stages that hold two 15 W
fluorescent bulbs each.
19
0.25 in. sample
Tube Clamp
Filter
2
C
Fig 2.2. Schematic of a 2 L or 3 L bubble column photobioreactor.
20
Stirred Tank Photobioreactors
A 500 mL Belco a-Carrier spinner flask (# 1965-00500) served as the stirred tank
photobioreactor vessel.
The height and inner diameter of each
stirred tank
photobioreactor were 15.2 cm and 10 cm respectively and the working volume was 800
mL of culture. The impeller speed was 150 rpm and the impeller dimensions were 2.5
cm height by 5.5 cm width. The vessels were jacketed, and the temperature was
maintained at 22°C by the circulation of water from a cooling reservoir through the
jacketing.
Illumination was provided by two fluorescent bulbs (9 W Feit Electric
Compact Fluorescent 2700 °KIPL9) positioned perpendicular to the vertical axis of the
reactors. One bulb was placed on each side of the reactor. Light intensity at the inner
surfaces of Belco#1 and Belco#2 were measured to be 100 E m2s1 and 200 jiE m2s'
respectively for all experiments.
In all experiments 0.5 L air U1 culture
min1 was
pumped (Fritz aquarium air pump Ultra 30/80) through an autoclaved 0.20 m Gelman
filter and then bubbled through a sterilized humidifier and then introduced to a 6 mm
stainless steel ID tube which passed through the headplate and charged the gas into the
medium via a stainless steel fit at the bottom of the reactor. Carbon dioxide in the
ambient aeration gas
growth.
(Cc02
= 350 ppm) was the sole source of carbon for biomass
21
Light Stage
9 W Fluorescent
Fig 2.3. Overhead view of a stirred tank photobioreactor. The vessel is centrally
located between two light stages that hold one 9 W fluorescent bulb each.
0.25 in.
sample port
22°C H20
Cooling :
filter
Fig 2.4. Schematic of a stined tank photobioreactor.
23
Bioreactor Inoculation
A cleaned and sterilized bioreactor vessel assembly was placed in its holder or on
its stir plate and attached to the cooling water and air lines. The lights, cooling jacket, air
and impeller were checked for proper working order. After the lights, air, and stir plate
were on, the reactor was filled with Diatom Nitrate LDM medium to its capacity volume
less 100 mL for the bubble columns and 30 mL for the stirred tanks. The headplate was
removed and held slightly above the top of the vessel and medium was poured into the
vessel through its top. While allowing the medium to equilibrate with the inlet for a
period of four hours the reactor was regularly checked for proper working order.
An inoculum culture (parent flask) was selected from the incubator based on a
visual inspection. Generally, cultures with the darkest color were selected. The selected
flask was moved to the laminar flow hood (EdgeGard Hood model #E6-3252) and using
aseptic technique 1 mL was removed for cell density measurements via hemocytometer.
A volume of 100 mL inoculum was added to bubble colunm reactors and an inoculum of
30 mL was added to stirred tanks
24
Sampling
A sample of the liquid suspension culture within the bioreactor was removed for
sampling through a 0.250 inch I.D. stainless steel sampling port fitted with a barb and
silicone tube. A sterile sampling syringe (20 mL Norm-Ject, Henke Sass Wolf GMBH
DIN/EN/ISO 7886-1) was drawn full of sterile air (in a laminar flow hood) and then
attached to the sample port of the vessel headplate. The sterile air was pushed through
the sample port to clear the sampling port tube of stagnant medium and accumulated
biomass. A fresh sample was then drawn using the sampling syringe. The volumes were
10 mL for the soluble silicon assay, 5 mL for soluble germanium assay and
spectrophotometric assay, and 20 mL for cell mass density measurements.
25
Experimental Design for Bioreactor Experiments
After the addition of cells to the reactor, initial values of cell mass density, cell
number density, soluble silicon concentration and pH of the culture suspension were
determined.
The pH, cell density and soluble silicon concentration were monitored
throughout Phase One, the initial growth phase. Phase One of the reactor experiment was
to grow the cell culture to a high cell mass density while depleting the liquid phase
soluble silicon. This was to study both the growth-related silicon uptake and to deplete
the liquid phase of soluble silicon. Specific growth rates () were determined from the
least squares slope of the natural log of dry cell mass density versus time data. Depleting
the liquid phase soluble silicon puts the diatoms into a state of silicon starvation which
prepares the diatom cellular machinery for rapid (surge) uptake of silicon and/or
germanium.
When the Silicon concentration reached a level below 50 M for two consecutive
readings 24 hours apart or the silicon concentration had stabilized for three readings the
reactors were given a one time addition of silicon and/or germanium, called a pulse.
Immediately before the silicon and germanium pulses were added, measurements of dry
cell mass density and soluble silicon concentration were recorded as final data points for
Phase One. After the addition of silicon and/or germanium, which marks the beginning
of Phase Two, the cell culture experiences a sudden change in external soluble silicon
and germanium concentration. When silicon starved cells are suddenly immersed in an
environment with ample silicon and/or germanium the cells rapidly uptake the soluble
26
Si/Ge by surge uptake. Surge Ge/Si uptake is non-growth associated uptake that is many
times faster than growth-related uptake.
Experiments to determine Ge surge uptake kinetics were conducted in the
800mL Belco stirred tank reactors. These experiments were short term (t < 0.44 hr). The
short term surge uptake experiments were conducted with a Ge only pulse.
The Ge
pulses were varied in magnitude so that the experimental set would have a distribution of
pulses from 0.5 mg Ge U' to 15 mg Ge U'. '['he method of analysis is described in the
results and discussion.
The experiments to determine long term Ge uptake, Ge incorporation into diatom
biomass, and cell culture growth rates were conducted in the 2.0 L and 3.0 L bubble
columns. These experiments were long term (>3days afler Ge/Si pulse). Data for soluble
Si and Ge were collected every 15 to 40 minutes during surge uptake until a stable Ge
concentration was established. Data were then taken every three to four hours until Ge
effluxed back into the medium or a steady state Ge concentration was established
(approximately four to 12 hours). Dry cell mass density measurements were taken daily.
The cell culture growth rates and kinetic substrate uptake rates were determined by the
methods described in the results and discussion.
The end of an experiment was
determined to be when a steady state was maintained in the reactor for at least two days
or until the algae population was predominantly in cell death phase. At that point the
bioreactor was shut down.
27
Bioreactor Shutdown
At shutdown, the bioreactor vessel was removed from its platform and washed.
First, the vessel was detached from the air and water circulation hoses and the headplate
was removed. The head plate, sample ports and sample tubes were washed with soap and
tap water and then thoroughly rinsed with DI water. Two capfuls of household bleach
were added to the remaining reactor contents. After the bleach killed the biomass (algae
turned white) the broth was poured down the lab sink with a high flow of tap water. The
interior of the empty vessel was then scrubbed with a bottlebrush using soap and water.
The reactor was thoroughly rinsed with tap water followed by DI H20.
If biomass persisted in the reactor more stringent washing methods were used.
For persistent biomass the vessel was first rinsed with undiluted bleach followed by a DI
H20 rinse. When biomass still remained after the undiluted bleach rinse the vessel was
allowed to stand overnight filled with 10% v/v nitric acid to dissolve the remaining
biom ass. To neutralize the acid, 10 g of sodium bicarbonate was slowly added to the
vessel and it was allowed to stand until the evolution of gas ceased. This process was
repeated until the evolution of gas was negligible. The neutralized vessel contents were
emptied down the lab
sink
while concurrently running tap water. The reactor was rinsed
again with DI H20 and allowed to dry.
Periodically the vessel cooling jackets and glass bubble column frits needed extra
treatment. Every three months the inside of the vessel cooling jackets were treated with
10% v/v nitric acid to remove rust deposits. The nitric acid was neutralized and disposed
of as previously described. Following three reactor runs, the glass fits were coated with
Rain-X to maintain a small layer of air between the flit and reactor medium to prevent the
accumulation of biomass within the flit pores.
After the vessel was cleaned and treated, it was sterilized. First the headplate was
reattached. Next, all the head plate openings were covered with tinfoil and secured with
autoclavable tape. The reactor was then autoclaved at 123 °C for 30 minutes at 23 psig.
29
Analytical Techniques
Silicon Assay
The concentration of soluble silicon per volume was determined using a
spectrophotometric assay where Si was complexed with ammonium molybdate
((NH4)oMo7O24*4H20) to form a yellow compound detectable at 410 nanometers
(Farming and Pilson, 1973).
The assay reagents were 6N HCI and an ammonium molybdate color complexing
agent. To prepare the ammonium molybdate color complexing reagent, 10 g Ammonium
molybdate ((N}{4)6Mo7O24*4H20) was dissolved in 75 mL warm DI water by stirring.
After the ammonium molybdate was dissolved, it was diluted to 100 mL with DI water
and allowed it to cool.
The solution was placed on a stir plate with a pH meter
submerged into the solution. Saturated sodium hydroxide, NaOH
(aq),
was added drop
wise until the pH was 7.5 +1 0.5. The reagent was stored in polyethylene.
A 10 mL culture sample was removed from the vessel in accordance with the
vessel sampling protocol and the liquid medium was separated from the biomass via
filtration before the spectrophotometric analysis.
The tip of the sampling syringe
containing the culture sample was inserted into the syringe filter holder (VWR 25 mm cat
# 28144-104) containing a syringe filter (Pall Life Sciences Versapor membrane disc
filter 3p.m pore, 25mm diameter cat # 28149-612, or Cole-Parmer MFS mixed cellulose
ester membrane filters, 3 0 j.tm pore size, 25 mm diameter cat # A300 A025A
Lot#41ALBA) and the 10 mL sample was pressed through the filter into a clean assay
vial. Next, a 5.000 mL sample was removed from the 10 mL aliquot and placed into a
iIi
separate clean assay vial. In rapid succession, 0.100 mL of 6N HCI and 0.200 mL of
ammonium molybdate reagent were added to the sample in the second assay vial. The
sample was allowed to stand for 10 minutes. The absorbance at 410 nm was measured
with
a
spectrophotometer
(Hitachi
Model
100-10
Spectrophotometer)
The
spectrophotometer absorbance was first zeroed at 410 nm with deionized water.
To form a calibration curve, stock solutions of 1.00, 2.00, 3.00, 4.00, 5.00, 7.00
and 10.00 mg soluble silicon U1 were assayed using the assay protocol and the data was
fit to the following empirical relation.
= a1[A410r ±as12[A410]+a513
Where
a51,3
A410
(2.1)
is the sample absorbance at 410 nm measured in mAU., and as1,i, as1,2, and
are empirically determined constants. The measured vessel sample absorbance was
substituted into the model equation to calculate the soluble silicon concentration.
31
Germanium Assay
The
concentration
of
soluble
germanium
was
determined
using
a
spectrophotometric assay where Ge was complexed with phenylfiourone to form an
orange compound detectable at 525 mn (Luke and Campbell 1956).
A 10% (vol/vol) HC1 solution, 25% (vol/vol)
H2SO4 solution
and a
phenylfiourone reagent made by fully dissolving 0.0500 g phenylfiourone (2,3,7tnhydroxy-9-phenyl-6-flourone) in 50 mL methanol and 1 mL 12N HCL in a 100 mL
flask. This phenylfiourone mixture was then transferred to a 500 mL flask and filled to
500 mL with additional methanol. The final phenylfiourone solution was transferred to a
screw cap Pyrex bottle and stored in the dark at 4 °C. A sodium acetate buffer was
prepared by adding 900 g sodium acetate trihydrate (NaC2H3O2*3H20) to 700 mL H20
in a 2 L beaker and dissolving the solute under heat and agitation. The dissolved contents
were transferred to a 2 L flask containing 480 mL 12N acetic acid and were then diluted
to 2 L with distilled water. The reagents were allowed to cool before use.
A 5 mL culture sample was removed from the vessel in accordance with the
reactor sampling protocol and the biomass was removed by filtration before the color
complexing reaction was performed on the medium. The sample was filtered using the
same procedures described for the soluble silicon assay and the 5 mL sample was pressed
through the filter into a clean assay vial. Next, a 1.000 mL sample was removed from the
5 mL aliquot and placed into a separate clean assay vial. To the 1.000 mL sample 0.300
mL H2SO4 solution, 1.000 mL sodium acetate buffer and 1.000 mL phenylfiourone
reagent were added in rapid succession. The sample was allowed to stand for four
minutes before 1.700 mL 10% HC1 solution was added as the final step. The sample was
32
immediately added to a cuvette and the absorbance at 525 rim was measured with a
spectrophotometer (Hitachi Model 100-10 Spectrophotometer). The spectrophotometer
absorbance was first zeroed at 525 nm with deionized water.
To form a calibration curve, stock solutions of 1.00, 2.00, 3.00, 4.00, 5.00, 7.00
and 10.00 mg soluble germanium U1 were assayed using the assay protocol and the data
was fit to the following empirical relation.
CGe
Where
A525
=
aGCI[A525r +aGe2[A52J+aGe3
(2.2)
is the sample absorbance at 525 nm measured in mAU., and ache,!, aGe,2, and
aQe,3 are empirically determined constants. The measured vessel sample absorbance was
substituted into the model equation to calculate the soluble germanium concentration
33
Dry cell weight determination of samples larger than 30 mL
A membrane filter (Whatman 42 Ashless 110 mm cat# 1442 110) was weighed
and placed into a Buchner funnel and vacuum flask assembly. A seal was formed
between the funnel and filter by first wetting the filter and forming a vacuum in the
vacuum flask.
A sample of 200 mL volume was collected in accordance with the reactor
sampling protocol and poured onto the filter paper.
The liquid was pulled through the
filter into the vacuum flask under vacuum. After the liquid was completely removed
from the solids, 50 mL of sterile filtered seawater was added to funnel and pulled through
the filter by vacuum to wash the sample. After the aspirator was turned off, the filter and
biornass were removed with tweezers and allowed to dry in a weighing dish for 24 hours
in air at 22 °C before the final weighing.
A filter mass calibration curve was prepared. First, five filter membranes were
weighed and separately placed into the Buchner funnel assembly, and 200 mL of 5 m
filtered sterile seawater was added. All of the seawater was pulled through the filter into
the vacuum flask. The five filters were dried for 24 hours in air at 22 °C. The masses
were recorded and the initial filter mass (Mf) was plotted against the dried filter mass (Mf
+ Ms). The salt correction factor for the filter, defined as
s=Mj+Ms_i
(2.3)
M
was estimated from the least-squares slope of this data. The dry cell mass density was
then determined by the equation
34
MDwfS M1(1S)
V
DW
Where XDW is the dry cell mass density,
(2.4)
vc
MDW+f+s
is the mass of the filter with cell mass
cake and salts, and Vc is the culture volume used in the measurement.
35
Dry cell weight determination of samples smaller than 30 mL
A membrane filter (Cole-Parmer MFS mixed cellulose ester membrane filters, 3.0
.im pore size, and 25 mm diameter cat # A300 A025A Lot #41ALBA or Pall Life
Sciences Versapor membrane disc filter 3 .im pore, 25 mm diameter cat # 28149-612)
was weighed and then inserted and secured into the membrane filter holder (VWR 25 mm
cat # 28 144-104) in preparation for separation of dry cell mass from liquid medium. A
20 mL sample was collected from the vessel in accordance with the reactor sampling
protocol. The sample was filtered using the same procedures described for the soluble
silicon assay. In addition, after filtration the syringe was drawn full of air and the air was
pressed through the syringe filter holder assembly to remove any excess liquid from the
filter. After clear liquid was observed in the sample vial (if the liquid contained biomass
the whole process must be repeated) the membrane filter and biomass were removed
from the filter holder with tweezers and allowed to dry for 24 hours in air at 22 °C before
weighing.
A filter mass calibration curve was prepared. First, five filter membranes were
weighed. Then, each filter was separately placed within the filter holder assembly and 20
mL of 5 jim filtered sterile seawater was passed through each. Air was then pressed
through (20 mL sterile air) to remove excess liquid. The filters were removed and dried
in air for 24 hours at 22 °C. The masses were recorded and the initial filter mass
was plotted against the dried filter mass
filter defined as
(Mf f
(Mf)
Me). The salt correction factor for the
36
s=Mj+Ms_i
(2.5)
Mf
was estimated from the least-squares slope of this data. The dry cell mass density was
then determined by the equation
MDw4f. M(1S)
'DW
(2.6)
vc
Where XDW is the dry cell mass density,
MDW-f+S
is the mass of the filter with cell mass
cake and salts, and Vc is the culture volume used in the measurement.
37
Hemocytometer Cell Count
A 5 mL reactor sample was collected in accordance with the reactor sampling
protocol. Five drops of the sample and one drop of 0.33% wt phenosafranin were placed
onto a one inch by one inch square of Para film and mixed together with a Pasteur
pipette. A small amount of phenosafranin was drawn up into a Pasteur pipette by
capillary action then injected into the culture sample. The two fluids were thoroughly
mixed by drawing the liquids up and down within the pipette tube. Two drops of the
well-mixed liquid were placed into the central chamber of the Hemocytometer (Fuchs
Rosenthal Ultra Plane 0.0625 mm between grid lines, 0.02 mm deep) and covered with a
cover slip. Capillary action drew the liquid over the counting grid.
The hemocytometer was placed onto the microscope stage and focused at 1 OOX
for cell counting. Each large hemocytometer square contains 2x10 mL and each small
square contains 1 .25x I 0 mL of culture volume. The number of cells in each small
square was counted with a tally counter. The counting continued in 5-10 randomly
picked squares or until 150-200 cells had been counted. In cases of dense culture the
reactor sample was diluted 2:1 or 4:1 v/v in a beaker with sterile 5 tm filtered seawater.
The total cell density was calculated by
N
ND
HN
(2.7)
Where XN is cell number density, D is the sample dilution, H is the hemocytometer
chamber square volume and Ns is the number of chamber squares counted.
Results and Discussion
Phase One Growth: Silicon Consumption
Cell mass density and silicon concentration versus time data for cultivation of
Nitzschiafrustulum in a bubble column photobioreactor are presented in Figure 3.1.
a) BC-Ni-7
-----------------------------------------q 16
0.8
-4- Cell Mass Density
Silicon Conrentration
0.7
14
12
-f
/
P5
V 0.3
--
0.2
/
8
0
-
0.1 -
2
0.0
0
20
0
40
30
60
120
100
Phase One Time (hr)
bI BC-Ni--It
0.40
12
-
0.5
-4--Cell Mass Dencity
- --Silicon Concentration
S-.
10
-
0.30
8
0.25
J
Sc')
0.20
0)
° 0.15
4
1.
-
23
2'
0
50
0
100
150
Phase One Time (hr)
C) BC.Ni.41
0.40
12
0.35
10
-+--CellMnss Density
Silicon Concentration
0.30
5,.
8
'V
'V
=5, 0.20
J
6co
\
0,
>. 0.15
4--
,0.10
0
U
x
0.05
0.00
0
50
100
150
Phase One Time (hr)
Figure 3.1. The dry cell mass density and soluble phase silicon concentration
versus time during Phase One for Nitzschiafrustulum.
39
The cell culture in each of the runs BC-Ni-27, 38, and 41, experienced a different lag
phase (20, 75 and 0 hours respectively) but all experiments were similar in that silicon
was depleted in the liquid phase leading to cell culture silicon starvation. In all diatom
cell cultures where growth was observed silicon depletion was always achieved.
The Phase One growth period was between inoculation and silicon starvation.
The nutrient medium formulation was such that soluble silicon in the form of sodium
metasilicate (Na2O3Si) would be the limiting substrate guaranteeing silicon starvation.
Dry cell mass density
(XDW)
and soluble liquid phase silicon concentration
(Cs1)
were
monitored during Phase One in bubble column photobioreactors. The decrease in silicon
concentration was accompanied by an increase in cell mass density. Therefore, Phase
One silicon consumption was considered growth related. The time to silicon starvation,
(C5
0), was approximately 80, 120, arid 40 hours for BC-Ni-27, BC-Ni-38, and BC-Ni-
41 respectively. Since silicon is a required component in the cell walls of .N
the depletion of silicon led to an eventual cessation of growth.
frustulum,
40
Predicted cell mass density and silicon concentration versus time data for
cultivation of Nitzschia frustulum during growth related silicon consumption in an ideal,
well mixed, batch photobioreactor are presented in Figure 3.2.
0.25
Xg/L
0.2
f
F
12
I
J
0.15
6
0
50
0
100
150
Phase One Time (hr)
Figure 3.2.
The diy cell mass density and soluble liquid phase silicon
concentration versus time during growth-related silicon consumption in an ideal
reactor. Assumes well-mixed batch photobioreactor with XDW,O = 0.03 g DCW U
a specific growth rate OfLmax = 0.02 hf',Monod constant of K
0.12mg Si U
and a silicon yield coefficient of Yxjs =20 mg DCW mg Si1.
Material balances on cell mass and silicon substrate in the cell culture are
VpXAt = VXDw((f) VXDWI
(3.1)
-
(3.2)
At =
VCs(+&) VC1
/ Si
where V is the culture volume (liters), Yxisi is the silicon to biomass yield coefficient (mg
DCW mg Si') and
is the specific growth rate (hf'). The resulting differential
equations are
dXDW
= 1K; t =0, XDW = XDW,O
(3.3)
41
;t=O,Cs=Cs,o
dt
(3.4)
with p. defined by the Monod model as
/4nlax
where
K,L
CSi
(3.5)
is the Monod half saturation constant for Si (mg Si L) which corresponds to
the silicon concentration at which the specific growth rate is half the maximum (Pmax).
The system of equations were solved numerically by a
order Runge-Kutta method
using MATLAB.
The Phase One growth rate versus the initial Phase One silicon concentration is
presented in Figure 3.3 and Table 3.1.
0.030
1
0.025
0.020
0
I-
0.015
0
0
0.010
0)
0.005
0.000
0
5
10
20
15
Initial Silicon Concentration, Csj,o,i, (mg Si
L1)
Figure 3.3. The initial Phase One soluble silicon concentration versus specific
growth rate. Cs1,1,o, is plotted versus the Phase One specific growth rate, p.. The
solid line represents the expected growth rate from Monod parameters. K, was
estimated from literature as 0.12 mg Si U1 (Martin-Jezequel et al, 2000).
42
Table 3.1. Phase One specific growth rates and silicon yield coefficients for
bubble colunm photobioreactors growing Nitzschiafrustulum.
Experimental Run
BC-Ni-27
BC-Ni-36
BC-Ni-37
BC-Ni-38
___BC-Ni-41
Phase One Specific
Growth Rate, i, (hr ±
S.D.)
Silicon Yield
Coefficient,
Yxis, mg DCW
mg Si1
28.2 ± 13.3
biomass silicon
concentration, C1,x,
jmo1 Si g fresh
0.018±O.0018(n=6)
0.017±0.0018(n=6)
13.5±3.2
13.3±1.8
989±234
1003±134
0.018 ± 0.0027 (n = 9)
16.8 ± 5.8
794 ± 274
0.021±0.0041(n7)
41.2±7.2
323±56
0.021 ± 0.0015 (n
1
10)
weighf'
445 ± 210
For Si-limited, well-mixed batch growth during Phase One, the biomass material balance
yields a differential equation analagous to Eq. 3.3
dXDW
dt
iiIXDW
with t = 0, XDW= XDW.O
(3.6)
and
Pmax
C,
(3.7)
K±
Literature values for K, for diatoms range from 0.62 pg
U1
to 240 pg L' (Martin-
Jézéquel et a!, 2000). If K <<Cs then equation (6) is approximated by
dt
/JmaXXDW
with t =0, XDW XDWO
(3.8)
and when solved for XDW yields
XDW =
where the Phase One specific growth rate
XDWOeX(
(ni)
(3.9)
was determined from the least squares
slope of the natural log of dry cell mass density versus time daia. From initial silicon
concentrations of 7.47 to 14.82 mg Si U' (266 jiM to 528 jiM) the specific growth rate
43
was nearly constant at a value of 0.019 ± 0.0041 hf'. The growth rate over this range
was constant because in this range the silicon concentration was far above the saturation
level, i.e. Csi>> K.
The soluble silicon concentration and dry cell mass density measurements were
also used to calculate the silicon yield coefficient (Y,cjs), and biomass silicon
concentration
(Cs1ix).
Yxisi and Csi,x values are presented in Table 1 .Yxisi is defined as
X,1,. XDo
csiIo csiIf
where
XDW,1 ,
Csi,i,o and
and
CS1,1,f
XDW,1 ,o
DW
(3.10)
AC1
are the final and initial Phase One cell mass densities, and
are the initial and final Phase One silicon concentrations.
In previous studies, Brzezinski (1985) determined that the Si content (Cs,) for
two Nitzschia species was 200 and 250 mol Si g fresh weight. Brzezinski's Nitzschia
were cultivated in fY10 and fY20 medium (Guillard and Ryther, 1962) having initial silicon
concentrations of approximately 0.45 and 0.23 mg Si U'. These concentrations were
much lower than the 7.47 to 14.82 mg Si U1 presented in Fig. 3.2. Claquin et a! (2002)
showed that increasing the amount of Si substrate in the environment tends to increase
the relative amount of Si in the cell mass. Therefore, the cell mass silicon composition
determined by Brzezinski (1985) and the silicon composition determined in this study
(Table 3.1) are found to be in reasonable agreement.
Phase Two Growth: Si and Ge Consumption in a Bubble Column Photobioreactor
Phase Two began after silicon starvation was achieved and a one-time addition of
soluble germanium andlor silicon, referred to as a pulse, was added to the
44
photobioreactor cell culture. The one time addition of silicon and germanium at the
commencement of Phase Two was conducted in four different regimes, which were
classified as quadrants. The initial Phase Two germanium and silicon concentrations are
presented in Figure
3.4
and Table 3.2.
CGe,2,0
(mg Ge Ld)
15
.
.
I
III
fl
t
o
csi,2,O
'---------
i
(mg Si U')
-110
T.
.
1*
0
II
iv
Figure 3.4. Initial silicon and germanium concentrations for Phase Two. The
experiments were conducted in four regimes, low Si I high Ge (I), low Si / low Ge
(II), high Si I high Ge (III), high Si / low Ge (IV). Individual data points signify
each bubble column experiment.
Table 3.2. Initial silicon and germanium concentrations measurement during
Phase One and Phase Two.
Quadrant
I
Run #
Stage 1 Csi
1,0
(mg Si L)
Stage
1
r20 (mg Si U')
2
CGe,2,0
(mg Ge L)
14.82
0.00
7.16
12.94
0.00
7.45
5.71
0.00
3.34
8.21
0.00
1.85
II
BC-Ni-27
BC-Ni-28
BC-Ni-26
BC-Ni-49
BC-Ni-63
10.62
1.09
1.19
III
BC-Ni-Si
10.75
5.37
11.52
IV
IV
IV
IV
IV
BC-Ni-36
BC-Ni-37
BC-Ni-38
BC-Ni-41
BC-Ni-62
10.88
5.52
1.60
11.32
5.22
1.69
11.45
5.42
3.04
7.47
6.34
0.00
10.89
8.91
1.51
I
II
II
45
Quadrant I experiments had an initial germanium concentration
(CGe,2,0)
above 5
mg U' (high germanium) and an initial silicon concentration (Cs1,2,0) below 5 mg U' (low
silicon).
Quadrant II, III, and IV experiments had initial Phase Two Si and Ge
concentrations of low Ge / low Si, high Ge / high Si and high Si / low Ge, respectively.
Growth Observations for Quadrants I-IV
The cell culture specific growth rates within all bubble column photobioreactors
during Phase Two are presented in Table 3.3.
Table 3.3. Phase Two growth rates tabulated by quadrant.
Experiment
Quadrant
Phase Two Specific Growth Rate,
(hf' ± 1 Standard Error)
BC-Nj-27
BC-Ni-28
BC-Ni-26
BC-Ni-63
BC-Ni-51
BC-Ni-36
BC-Ni-37
BC-Ni-38
BC-Ni-41
BC-Ni-62
I
0.00015±0.0016(n=6)
I
II
II
III
IV
N
N
N
N
-0.0030 ± 0.0032 (n = 5)
-0.0010 ± 0.00012 (n = 3)
O.0065±0.00064(n=11)
0.0054±0.00031(n=6)
0.011±0.00080(n=10)
0.0032±0.0019(n =7)
0.011 ± 0.00016 (n = 7)
0.0087 ± 0.0025 (n = 7)
0.0050±0.00030(n=17)
-1
XDW,2,O (mg DCW L
0.83
0.52
)
XDw,2,f(mg DCW L
.065
.040
n/a
n/a
0.26
0.32
0.18
0.19
0.22
0.29
0.30
0.58
0.74
0.71
0.24
0.62
0.41
0.83
Doubling
time, td (hr)
4620
nla
n/a
106
128
63
216
63
79
138
Time Range
(hr)
0-169
0 - 93
0 141
0-118
0-158
0-124
0-98
0 - 92
0-44
0-191
46
47
The cell culture within bioreactors which received only germanium in Phase Two
did not experience growth while the cell culture within bioreactors which received silicon
experienced growth regardless of the level of germanium addition. No cell culture
growth kinetics post surge germanium uptake (non-growth-related uptake occurring after
silicon starved cells experience an abundance of environmental silicon) has ever been
quantified. However, Lewin (1966) reports a zero growth rate for diatoms exposed to
germanium in the absence of silicon during growth related uptake, as do Azam et al
(1973), and Markham and Hagmeier (1982).
It was also noted in each of those
investigations that the Si/Ge ratio plays a role in dampening the growth limiting effects of
germanium. However, Azam et al (1973) reports that at Ge/Si ratios above 0.1 the uptake
of silicon by diatoms still fell to less than 5% of that in diatoms not exposed to
germanium. Silicon uptake is coupled with cell wall development and overall growth.
Lewin (1966) reports that at an initial liquid phase Ge/Si ratio of 0.3 mol Ge mol Si', that
growth is negligible and no growth was possible at a ratio of 0.6 mol Ge mol Si',
regardless of actual concentration. Contradictory to this observation is that experiments
BC-Ni-63 and BC-Ni-5 1 had initial liquid phase molar Ge/Si ratios of 0.42 and 0.83 and
the cell culture of both experiments grew. With doubling time defined as
in 2
td =
I!
(3.11 )
BC-Ni-63 and BC-Ni-51 grew with doubiing times of about 106 and 128 hours
respectively (Table 3.3). The difference between Azam, Lewin and this investigation is
that in this investigation the cells were exposed to germanium after silicon starvation and
not while Si consumption was growth related.
Quadrant I Experiments
The intracellular and liquid phase germanium concentrations for cell culture
within the quadrant I experiments BC-Ni-27 and BC-Ni-28 are presented in Figure 3.5.
a) BC-Ni-27
8
7
6
.5
04
E
U
2
I
0
50
0
100
200
150
Time After Phase Two Ge Mdition (hr)
b) BC-Ni-28
9
8
7
6
05
0
2
I
0
0
20
40
60
80
100
Time Alter Phase Two Ge Addition (hr)
Figure 3.5. Soluble germanium concentration versus time curves for runs BC-Ni27 and BC-Ni-28 (Quadrant I).
49
c) BC-Ni-27
8
7
6
E
0
C)
2
1
__L___.L_.__j_____j_
0
_IL_j._
_.L_L_L_____L___S_..._L..J_ ______
o
4
3
2
i
Time After Phase Two Ge Addition (hr)
d) BC-Ni-28
9
1
8
7
$-- Liquid Phase Ge
w
CD5
w
C,
2
I
0
0
1
2
3
4
5
Time After Phase Two Ge Addition (hr)
Figure 3.5. Soluble germanium concentration versus time curves for runs BC-Ni27 and BC-Ni-28 (Quadrant I).
50
e) BC-Ni-27
9
8
06
'4
01
0
50
100
150
Time After Phase Two Ge Addition (hr)
0
200
f) BC-Ni28
14
12
0
10
6
n
0
20
40
60
80
100
Time After Phase Two Ge Addition (hr)
Figure 3.5. Intracellular germanium concentration vs. time for the germanium
pulse experiments BC-Ni-27 and BC-Ni-28. (e) Calculations based on a cell mass
density of 0.76 ± 0.06 1 g dry cell mass U' (f) Calculations based on a cell mass
density of 0.46 ± 0.11 g U' (Quadrant I).
51
The diatom cell mass in the Quadrant I experiments BC-Ni-27 and BC-Ni-28
experienced a rapid uptake of germanium immediately after the addition of soluble Ge02
to the culture suspension. The rapid uptake of germanium observed immediately after the
addition of soluble germanium to silicon-starved cells is hereafter referred to as surge
germanium uptake. Surge germanium uptake was followed by a germanium efflux back
to the culture liquid. At the end of surge uptake, the germanium partitioned to the
intracellular component over the bulk liquid phase.
The partition coefficient,
K,
is
defined as
K
=
CGe/ X
Pceii
(3.12)
CGe
where
and
K
Pcell
is the density of the fresh cell mass itself (g fresh cell mass L fresh cells1),
has units of L culture L fresh cells1. At the end of surge uptake, diatom cells
effluxed their intracellular germanium back into the liquid phase. It can be inferred that
the intracellular germanium remained unbound or unpolymerized and was therefore
osmotically active. The energy expenditure required by the cell (Azam and Volcani,
1974) to maintain this high intracellular to extra cellular concentration gradient could no
longer be maintained. Therefore, efflux occurred.
The slow uptake of germanium following surge uptake was not accompanied by
cell growth. The specific growth rates were j.i = 0.00015 ± 0.0016 hf' and
= -.0030 ±
0.0032 hr', for the diatoms in runs BC-Ni-27 and BC-Ni-28 respectively. Azam et al
(1973) observed that diatoms were capable of forming large intracellular germanium
poois by continuing to uptake Ge in the absence of cellular growth. For run BC-Ni-27,
from 20 to 169 hours following Ge addition, the cell suspension continued to partition
germanium from the bulk liquid into the cell mass. Run BC-Ni-28 maintained an
52
equilibrium partition coefficient of at least K = 1200 L culture volume L cell volume'
from 50 to 96 hours. The diatoms were likely capable of maintaining a K > 1 because a
limited amount of germanium can be condensated along with silicon (Azam et al, 1973)
or some of the germanium could have formed an insoluble non-biogenic complex within
the cytoplasm.
Quadrant II Experiments
Experiment BC-Ni-26 was carried out such that two germanium additions were
added to the cell culture. The first germanium addition resulted in a liquid phase soluble
3.34 mg Ge L. After the first Phase Two
germanium concentration of CGe,2a,O =
germanium addition the cell mass germanium concentration (CGC,x) was calculated using
the relationship
CGe/X =
CGe200
CGe2a
XDW
0t3,42hr
(3.13)
Just prior to 3.42 hours after the first germanium addition, the soluble germanium
concentration was CGe,2a,f = 0.0 mg Ge U1. At 3.42 hours after the first germanium
addition a second germanium addition was added which brought the soluble germanium
concentration up to CGe,2b,O = 3.90 mg Ge U1. After the second germanium addition the
cell mass germanium concentration was calculated by the relationship
CGe2aO + CGe2bO
CGe/ X
=
XDW
CGe2b
t
3.42hr
(3.14)
53
The measured soluble germanium concentration immediately prior to the second
germanium addition was nearly zero and so it was approximated that all germanium had
partitioned into the cell mass fraction.
The intracellular and liquid phase germanium concentrations associated with the
quadrant II experiment BC-Ni-26 are displayed in Figure 3.6.
The intracellular and
liquid phase germanium concentrations as well as the cell mass density of the quadrant II
experiment BC-Ni-63 are presented in Figure 3.7. The surge uptake and germanium
efflux for the quadrant II experiment BC-Ni-49 are shown in figure 3.8.
a) BC-Ni-26
5.0
4.0t
-:.
3.0
2.0
-4.- LIquid Phase Ge
C.:.
1.0
0.0
-1.0
50
0
100
150
Time After Phase Two Ge Addition (hr)
b) BC-Ni-26
4.5
4.0
3.5
3.0
2.5
o 2.0
E
1.5
1.0
0.5
0.0
-0.5
0
Time After Phase Two Ge Addition (hr)
Figure 3.6. The soluble liquid phase germanium versus time for the experiment
BC-Ni-26 (Quadrant II).
54
C) BC-Nl-26
7.0
6.0
5.0
0
4.0
0)
3.0
E
2.0
C)
TTTTGI_____________________
1.0
0.0
0
20
40
60
80
100
120
140
160
Time After Phase Two Ge Addition (hr)
d) BC-Ni-26
8.0
7.0
6.0
a)
CD
5.0
4.0
a)
0
1
2
3
4
5
Time After Phase Two Ge Addition (hr)
Figure 3.6. The intracellular germanium concentrations versus time for the
experiment BC-Ni-26. (c,d) Intracellular Ge concentration based on a cell mass
density of XDW = 0.61 ± 0.14 g dry cell weight U1 (Quadrant II).
55
a) BC-Ni-63
1.4
0.8
-4-- Liquid Phase Ge
-S- Cell Mass Density
1.2
0.7
1.0
0.6
-J
0.8
0.5
0
c,
0.6
AA
-
!0.4
0.3
0.2
0.2
0.0
0.1
C.)
C..)
0.0
50
0
100
200
150
250
Time After Phase Two Ge/Si Addition (hr)
b) BC-Ni-63
1.4
1.2
- 1.0
0
-4- LIquid Phase Ge
0.8
0.6
0.4
-.7
0.2
0.0
0
5
10
15
20
30
25
Time After Phase Two Ge/Si Addition (hr)
c) BC-Ni-63
6.0
0.7
5.0
0.6
°5L
/
4.0
0.4
3.0
0.3
E 2.0
c
a
0.2
--- Cell Mass Ge
1.0
C.)
u-
0.1
Cell Mass Density
0.0
o.o
0
50
100
150
200
250
Time After Phase Two Ge/Si Addition (hr)
Figure 3.7.
The cell mass density (XDw), liquid phase soluble germanium
concentration (CGe), and intracellular germanium concentration
time for the experiment BC-Ni-63 (Quadrant II).
(CxJG),
versus
56
BC-Ni-49
2.0
1.8
Liquid Phase Ge
1.6
1.4
1.2
1.0
E
0.8
0
00.6
0.4
0.2
0.0
0
5
15
10
20
25
Time After Phase Two Ge/Si Addition (hr)
Figure 3.8. The initial germanium uptake and germanium efflux for the culture
within the experiment BC-Ni-49 (Quadrant II).
The cell culture within the Quadrant II experiments BC-Ni-26 and
BC -Ni-63
both
consumed germanium by surge uptake, but the germanium efflux differed slightly
relative to Quadrant I experiments. During run BC-Ni-63 only a slight germanium efflux
occurred and a low liquid phase germanium concentration was maintained, less than 0.20
mg Ge L' until over
150
hours after Si/Ge addition. The culture in
experienced growth, while the culture in
BC-Ni-26
BC-Ni-63 also
did not. This is most probably
attributed to the fact the BC-Ni-63 received some silicon
(Cs1,2,0 = 1.09
mg Si U1) at the
onset of Phase Two.
The culture within BC-Ni-26 went from an intracellular germanium concentration
of
CGe/X = 5.52 ± 1.31
mg Ge g DCW' to
C0eix = 5.52 ± 1.47
after the second
germanium addition which took the bulk concentration of soluble germanium from
57
CGC,2a,f
= 0.0 mg Ge U' to
CGe,2b,O
= 3.9 mg Ge U'. The diatoms did not take up a
statistically significant amount of additional germanium after the second germanium
pulse. This would indicate that an equilibrium effect was not the dominant phenomena.
Had an equilibrium effect been dominant, a soluble liquid phase germanium increase
from 0.0 mg Ge U' to 3.9 mg Ge U' would have produced a measurable increase in
intracellular germanium concentration.
Further investigations regarding germanium
reactions within the cell, and the relationship between germanium concentration in the
bulk liquid, cytoplasm, and silicon deposition vesicle would need to be examined to
propose what other phenomena effect
K
and Ge uptake. Only limited work has been
previously done regarding Ge partitioning between the liquid medium and the cell mass.
Azam et al (1974) calculated intracellular to extra-cellular germanium concentration
ratios of up to
K
3500 L culture volume L cell volume' In the future, a medium
perfusion experiment with medium containing germanium may be carried out to better
understand these phenomena.
The culture within BC-Ni-49 experienced the smallest germanium pulse of all the
Ge-only experiments (CG,2,o = 1.85 mg Ge U') and still effluxed nearly all of the soluble
germanium consumed during surge Ge uptake. The diatom's inability to permanently
assimilate such a small amount of germanium indicates that diatoms are not able to
metabolize germanium when supplied without silicon.
Quadrant III Experiment
Liquid Phase and intracellular germanium concentrations in the cell culture for
the Quadrant III experiment BC-Ni-5 I are presented in Figure. 3.9.
59
a) BC-Ni-51
14
12
11i
04
Liquid Phase Ge
2
0
50
0
100
150
200
Time After Phase Two Ge/Si Mdition (hr)
b) BC-NI-51
14
12
10
-±- Liquid Phase Ge
'Li
0
5
10
15
20
Time After Phase Two Ge/Si Mdltion (hr)
c) BC-NI-51
12
10
-+- Solid Phase Ge
0
0,
0
0,
0
0
0
50
100
150
200
Time After Phase Two Ge/Si Addition (hr)
Figure. 3.9.
The liquid phase soluble germanium concentration
(CG),
and
intracellular germanium concentrations (CGe/X) versus time for the quadrant III
experiment BC-Ni-Si. (c) Cell mass germanium concentration calculations based
on XDW = 0.52 ±0.19 g DCW U' (Quadrant III).
The diatom cell mass of run BC-Ni-5 1 experienced surge germanium uptake,
germanium efflux, and growth. The culture for run BC-Ni-5 1 also experienced a high
concentration of germanium and silicon at the onset of Phase Two,
U' and
high,
Cs1,2,o
C0e,2
CGe,2,Q
= 11.52 mg Ge
= 5.37 mg Si U'. The liquid phase germanium concentration remained
> 7 mg Ge U', throughout the entirety of Phase Two and had a specific
growth rate of j.i = 0.0054 ± 0.00031 hf1. However, previous work by Lewin (1966),
Azam et al (1973), and Markham and Hagmeier (1982) showed that initial Ge
concentrations of 6.9 mg U' completely inhibited growth.
Quadrant IV Experiments
The liquid phase germanium concentration and cell mass density versus time for
Quadrant IV experimental runs BC-Ni-36, 37, and 38 are presented in Figure 3.10. The
liquid phase silicon concentration and cell mass density for the Quadrant IV experiment
BC-Ni-41 is presented in Figure 3.11. The liquid phase and intracellular germanium
concentrations as well as the cell mass density for the Quadrant IV experiment BC-Ni-62
is presented in Figure 3.12.
61
a) Ni-36
0.8
1.8
1.6
0.7
Liquid Phase Ge
U Cell mass density
1.4
0.6
0.5
U
1.0
0.4
0.8
Time Alter Phase Two Ge!Si Addition (hr)
b) BC-Ni-37
0.30
2.0
.
1.4
1.0
p0.6
/4
0.25
0.15
0.io
Liquid Phase Ge
04
0.05
N Cell mass density
0:2
0.00
0.0
40
20
0
60
80
100
120
Time After Phase Two Ge/Si Mdition (hr)
c) BC-Ni-38
3.5
0.8
3.0
2.5
0
2.0
E
1.5
J
1.0
0
0.7
Liquid Phase Ge
Cell mass density
0.6
0.5-'
0.4
0.3
.
0.2
0.5
0.1
nfl
w.V
0.0
0
20
40
60
80
100
Time After Phase rwo Ge/Si Mdition (hr)
Figure 3.10. The cell mass density (XDW), and liquid phase soluble germanium
concentration (CGe), versus time for experiments BC-Ni-36, BC-Ni-37 and BCNi-38 (Quadrant N).
62
d) Ni-36
1.8
1.6
1.4
-4-Liquid Phase Ge
1.2
1.0
0.8
0.6
C)
0.4
0.2
0.0
0
5
10
15
25
20
30
Time After Phase Two Ge/Si Addition (hr)
e) BC-Ni-37
2.0
1.8
1.6
'
1.4
1.2
Di 1.0
!08
0.6
0.4
0.2
0.0
5
0
10
15
20
Time After Phase Two Ge/Si Addition (hr)
f) BC-Ni-SB
3.5
3.0
-+- Liquid Phase Ge
I
2.5
2.0
Dl
E 1.5
j1.0
0.5
0.0
LLJS
0
5
10
15
20
Time After Phase Two Ge/Si Addition (hr)
Figure 3.10. Concentration of soluble liquid phase germanium (CGe) vs. time for
the experiments BC-Ni-36, BC-Ni-37, BC-Ni-38 (Quadrant IV).
63
The cell culture of experiments BC-Ni-36, 37 and 38 all experienced growth with
doubling times of 63, 216 and 63 hours respectively. The slow doubling time of BC-Ni-
37 and its nearly complete efflux of germanium indicates the difficulty in achieving
repeatability in living system experiments.
BC-Ni-41
7.0
0.50
0.45
6.0
0.40
5.0
-'
--
4.0 I
0.35
0.30
-4- Liquid Phase Si
0.25 0
Cell mass density
E
C)
3.0
0.20
U)
C.,
0.15
2.0
><
0.10
1.0
0.05
00 I__L___j_
0
1__.I_.J_......r _ I
20
I
40
I
-
0.00
I
60
80
Time After Phase Two Si Addition (hr)
not
Figure 3.11. The soluble liquid phase silicon concentration
(Cs1),
and the cell
mass density (XDW), versus time for experiment BC-Ni-41 (Quadrant TV).
A one time addition of silicon in the absence of germanium was given to the Quadrant IV
control experiment, BC-Ni-41, at the start of Phase Two
(Cs,2,o
= 6.34 mg Si U'). BC-
Ni-41 experienced an increase in cell mass density and a depletion of soluble silicon.
The soluble silicon versus time plot for run BC-Ni-41 (Fig. 3.11) indicates that silicon
uptake was surge uptake and not growth related.
64
The doubling time for BC-Ni-41 was 79 hours (Table 3.3). However, the silicon uptake
took place within five hours. This is in agreement with Sullivan (1977) who showed that
diatoms are capable of taking up enough silicon in less than two hours to complete one
cell division.
a) BC-Ni-62
1.8 .-- -------------- -------
0.9
.*
1.6
0.8
1.4
0.7
1.2
_J
02
1.0
o
05
O08
E
0.6
c3
0.4
0
*
Liquid Phase Ge
Cell mass density
50
0
100
150
0.4
0.3
200
250
Time After Phase Two Ge/Si Addition (hr)
b) BC-Ni-62
1.8
1.6
1.4
-*- Liquid Phase Ge
L1.2
0
0
0.8
0
0
0.6
0.4
0.2
nfl
U.',
0
5
10
20
15
25
30
Time After Phase Two Ge/Si Addition (hr)
C) BC-Ni-62
4.5
0.9
0.8
$____a-
ç4.0
3.0
0.6 -'
/
r
0.3
Solid Phase Ge
Cell mass density
1.0
0.5
0.2
><
0.1
0.0
0.0
50
0
100
150
200
Time After Phase Two Ge/Si Addition (hr)
250
Figure 3.12. The cell mass density (XDW), liquid phase soluble germanium
concentration
and intracellular germanium concentration (CyJG), versus
(CGe),
time for the experiment BC-Ni-62 (Quadrant IV).
The culture within the quadrant IV experiments BC-Ni-36, 37, 38, and 62 all
consumed germanium by surge uptake with no germanium efflux at the end of the initial
germanium uptake period. For runs BC-Ni-36, BC-Ni-38, and BC-Ni-62 germanium
uptake was permanent while for run BC-Ni-37 germanium efflux occurred after two
days.
All four cell cultures were able to grow regardless of initial germanium
concentration.
Phase Two: Silicon and Germanium Incorporation Into Biomass
The changes in soluble liquid phase silicon, soluble liquid phase germanium and
the dry cell mass density for selected experiments during Phase Two are listed in Table
3.4 and Table 3.5.
Table 3.4. Initial and final Phase Two silicon and germanium measurements. Error based on duplicate assays.
Experiment
BC-Ni-36
BC-Ni-38
BC-Ni-41
BC-Ni-62
Cs,2,o (mg Si U') ± 1 S.D Cs1,2,f (mg Si U') ± 1 S.D
CG,2,o (mg Ge U') ± 1 S.D
CGe,2,f (mg Ge U')
± 1 S.D
5.52±0.05
0.28±0.20
1.60±0.02
0.10±0.01
5.42 ± 0.02
0.00 ± 0.03
3.04 ± 0.02
0.34 ± 0.01
6.34 ± 0.06
0.97 ± 0.05
0.00 ±
8.91 ± 0.56
0.48 ± 0.11
1.51 ± 0.07
0.00
0.00
± 0.00
0.02 ±
0.02
Table 3.5. Phase Two initial and final dry cell mass measurements and Phase Two change in measured values. Error based on
duplicate assays.
Experiment
BC-Ni-36
BC-Ni-38
BC-Ni-41
BC-Ni-62
XDW,2,O(rngXDW U')
XDW,2, (mg XDW U')
AC1 (mg Si U')
ACGe (mg Ge U')
AXDW (mg XDW U')
180± 30
710± 30
5.24± 0.21
1.50± 0.02
530± 42
220 ± 70
620 ± 80
5.42 ± 0.04
2.70 ± 0.02
400 ± 106
290±50
300±0
410± 10
5.37±0.08
9.92±0.57
0.00±0.00
1.49±0.07
530±20
830±20
120±51
67
Silicon is a required substrate for diatom growth but germanium is not (Lewin
1966).
But are diatoms capable of using Ge as a substrate when it is available?
Assuming silicon and germanium limited growth a material balance on biomass, silicon,
and germanium is
XDw2I XDw2O =
where
Yjs1,2
and
YXJGC,2
c21)+ YXIGe2(CGe2O
CGe2f)
(4.15)
are the Phase Two silicon and germanium to biomass yield
coefficients with units of mg XDW mg Si, and mg XDW mg Ge1 respectively. By
multiple linear regression,
Yxjs,2
111.15 ± 58.57 (n = 4, 1 S.E.,
r2
and
YXJGe,2
were determined to be 36.42 ± 19.29 and
= 0.37) alternatively calculated as 1023 ± 542 g DCW
mM Si' and 8074 ± 4268 g DCW mM Ge' respectively. The yield coefficient values
reveal that germanium can be available for diatom uptake and incorporation. In this case
germanium is found in lower cellular levels than silicon, but was also administered in
lower levels than silicon.
It was previously known that germanium was capable of
entering diatoms, but only at the tracer level, because radioactive germanium has been
detected in diatom cell walls (Azam et al, 1973) and been used to trace silicon
metabolism (Mehard et al, 1974, Azam et al 1974). Germanium as a substrate has not
been quantified until now.
Germanium and Silicon Specific Surge Uptake Rate Comparison
Liquid phase material balances for Si and Ge during surge uptake over a discreet
period of batch operation, assuming constant cell mass density, constant culture volume,
and well mixed reactor contents are
In
out + gen
accum
0+0+ VR, At =
vc
O+O+VRGeAt = VCGef+t
where V is the culture volume in liters and
Rs1
(3.16a)
Si 1
VCGet
(3.1 6b)
(3.16c)
and RGe are the observed uptake rates.
Assuming. constant volume, dividing by At, and letting At -+ 0, the material balances
become the differential equations
with t
R5 =
R Ge
where
Rs1
dCGe
dt
0, Csi = Csi,o
with t =0, CGe
CGe,O
(3.17a)
(3.17b)
and Re are defined by Michaelis-Menten kinetics as
R5 =XDW
RGe =
R51,
K, +
Rtex CGe
(3.18a)
(3.18b)
KGe + CGe
where
R'j,max
and
R'Ge,max
are the specific maximum uptake rates and Ks and KGe are the
Michaelis-Menten half saturation constants which correspond to the concentration of Si
70
and Ge at which the uptake rate is half the maximum.
It
is imperative to note
mathematically that when
KGe >> CGe
RGex CGe
(3.19)
, RGe
KGe
CGe
>> K;
,
XDwR;eix
RGe
(3.20)
where Eq. 3.19 represents a first order uptake rate expression and Eq. 3.20 represents a
zero order uptake rate expression. Substituting Eq. 3.19 into Eq. 3.17b yields
dCGe
-x DW
dt
RGexCGe
with t
0,
CGe = CGe,O
(3.21)
KGe
with solution
lfl(CGe) =
X DW R'Gemax
ln(Creo)--
(3.22)
KG.,
Substituting Eq. 3.20 into Eq. 3.l7b yields
dCGe
dt
=
with t =0,
CGe = CGe,O
(3.23)
with solution
CGe = CGeO
XDWRet
(3.24)
Taking the natural log of both sides of Eq. 3.24 yields
ln(CGe) = lfl(CGeO
XDWRet)
A Taylor expansion on the right hand side of Eq. 3.25 about t =0 yields
(3.25)
71
lfl(CGe) = lfl(CGeO)
(XDW Reniaxt)
(XDW Remaxt)2
(XDW Rex
3f3
2
CGCO
where t is small and
CGe,O
t)3
+
(3.26)
Ge,O
is large so the higher order terms are neglected reducing the
equation to
ln(CGe) = ln(CGCO)
XDWReX
(3.27)
CGeO
which is now in the same form as 3.22. As a result, at short times the data analysis can
be conducted as if the cell culture has a first order Ge uptake rate, even though the
Michaelis-Menten uptake rate expression is parabolic. Analytically solving both cases at
short times gives a solution of the form
CGe =
CGe,oe_XDwkt
(3.28)
which is the solution to
dCGe
where
RGe
= XDWkCGC= RGeO with t = 0, Ce CGe,O
(3.29)
has been converted to the initial uptake rate (RG,o) because of the time
constraint. Eq. 3.28 is equivalent to
lfl(CGe) =
lfl(CGeO)
XDWk 1
(3.30)
where t is small and k is the maximum specific first order uptake rate constant (L g
DCW' hf1) and by using Eq. 3.30 can be determined from the least squares slope of the
natural log versus time data divided by dry cell mass density. The initial specific reaction
rate is found by dividing the right hand side of Eq. 3.29 by the cell mass density.
R;eo
= k'CGeO
The same manipulation is done with the silicon material balances.
(3.31)
72
The natural log of germanium and silicon concentration versus time for the first
0.6
hours for run BC-Ni-36 is presented in Figure 3.13.
0.50
1.75
0.45
1.73
0.40
:-
-J
()
0.35
.11
0)
0.30
U)
CD
,
lG W
0.25
U)
U)
U)
U)
0.20
a-
1.67
0.15
0.10
1.65
0.05
L.L._LJ_LJ 1.63
0.00 '0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Time (hr)
Figure 3.13. The natural log of germanium and silicon concentration versus time
for experiment BC-Ni-36.
The initial specific uptake rate constants for each bioreactor experiment where
both silicon and germanium were added in Phase Two are presented in Table 3.6.
Table 3.6. Phase Two maximum specific silicon and germanium uptake rates.
Experiment
BC-Ni-36
BC-Ni-37
BC-Ni-38
BC-Ni-51
BC-Ni-62
BC-Ni-63
k'1 (L g DCW' hr') ±
S.E.
k'Ge
(L g DCW'
1 S.E.
') ±
k'j/k'e±
1 S.E.
0.78 ± 0.09
1.91 ± 0.30
0.41 ± 0.080
0.70±0.22
1.69±0.31
0.42±0.15
0.85 ± 0.06
1.50 ± 0.44
0.56 ± 0.17
-0.23±0.73
0.58±0.20
-0.39± 1.25
0.96 ± 0.12
1.21 ± 0.13
0.80 ± 0.13
1.23 ± 0.52
9.31 ± 1.83
0.13 ± 0.062
73
In all cases, the ratio of specific maximum uptake rates (k'sj/k'G) was lower than one,
making germanium the preferred uptake species. In previous work
Ge68
was used as a
tracer to determine silicon uptake rates (Sullivan, 1976). In these studies it was assumed
that during surge uptake the diatoms did not distinguish between Si and Ge. Since Si and
Ge are not perfect analogues it is possible that the silicon surge uptake rates reported by
Sullivan are in error.
Kinetic Rates of Surge Germanium Uptake
The initial specific germanium uptake rate (R'G,o, mg Ge g DCW' hi'), versus
the initial germanium concentration (CGe,O) data for stirred tank photobioreactors is
presented in Figure 3.14. The reaction rate data is presented in Table 3.7.
100
r
90
80
070
C.)
60
40
C)
30
20
0I
P
0
5
10
15
20
25
Initial Ge Concentration (mg Ge
30
35
L1)
Figure 3.14. The maximum germanium uptake rate versus initial germanium
concentration in stirred tank photobioreactors.
74
Table 3.7. Germanium concentrations and initial germanium uptake rates.
Experiment
STR-Ni-61
STR-Ni-60
STR-Ni-57
STR-Ni-46
STR-Ni-45
STR-Ni-42
STR-Ni-55
STR-Ni-58
STR-Ni-59
STR-Ni-54
CGC,O
(mg Ge U')
Rcje,
(mg Ge g DCW' hr')
0.21 ± 0.02
0.34 ± 0.04
0.02-0.08
10.71 ± 1.85
0.03 -0.12
0.03 -0.43
0.02 -0.20
1.30 ±0.30
1.42 ± 0.02
2.65 ± 0.04
7.78 ±0.12
11.42±0.15
12.36 ± 0.02
22.94 ± 2.81
18.78 ± 0.97
10.71 ± 0.80
77.26 ± 4.56
59.48 ± 2.08
77.57 ± 10.69
16.15±0.92
51.40± 11.67
73.90 ± 5.49
24.21 ± 0.16
Time (hr)
0.64± 1.11
0.07-0.33
0.05
0.40
0.05 -0.33
0.07 --0.32
0.07-0.32
0.05
0.33
Where at short times from Eq. 3.31 and Michaelis-Menten kinetics
R Ge,niax C
_ Ge,O
Ge,O
'Gc,O"Cie
(
KGe
By non-linear regression of the CG,o
VS R'Ge,Q
i
.
2
CGeO
data, KQe and
R'Ge,max
for
N frustulum
were found to be 5.02 ± 3.17 mg Ge U' and 90.5 ± 18.9 mg Ge g DCW' hr' respectively
(n
10, 1 standard error,
r2
= 0.80). The uptake of Si and Ge across the cell membrane,
through the diatom cell wall, and into the cytoplasm is believed to be an active transport
mechanism (Azam 1973). Active transport mechanism kinetics are commonly modeled
by Michaelis-Menten kinetics. The germanium uptake kinetics for the species
Nitzschia
alba at concentrations above 0.073 mg Ge U' and durations below 0.50 hours were
described by Azam (1974) as having a half saturation constant (K) of 0.37 mg Ge U'
and a maximum specific uptake rate
(R'Ge,max) of 97 mg Ge g DCW' hf'.
75
The KGe value reported by Azam is significantly lower than the KQe determined in this
investigation.
Azam was able to accurately collect uptake data at lower Ge
concentrations by using radio labeled germanium (Ge68). Therefore, the extremely low
'Ge
value determined by Azam may be a good indication of the actual parameter.
76
Conclusions
Experimental investigation of diatom cultures taken to silicon starvation and then
given a pulse addition of soluble silicon andlor germanium to the cell culture medium
showed that: 1) silicon is required for diatom growth;
2)
in the presence of soluble
germanium, diatoms grow and divide in the presence of soluble silicon; 3) germanium is
incorporated into the diatom cell mass when introduced to diatom cell cultures in the
presence of silicon.
A two phase experiment was conducted such that during Phase One, diatom cell
culture grew to a high density and in the process depleted the culture medium of soluble
silicon. Phase Two of the experiment began afler all of the soluble silicon was depleted
during Phase One and varying concentrations of silicon and germanium were added to
the silicon-starved cell culture. Four different Ge/Si addition schemes were developed
and classified into four different quadrants: Quadrant 1, high Ge/low Si; Quadrant II, low
Ge/low Si; Quadrant III, high Ge/high Si; Quadrant IV, low Ge/high Si. Phase Two
cultures that received germanium only with no silicon (Table
cultures to experience no Phase Two growth (Table
3.3).
3.2)
were also the only
However, the control
experiment BC-Ni-41 received only silicon at the onset of Phase Two (Table 3.2) and
experienced growth (Table
3.3,
Figure
3.11).
When silicon was not present, diatom cell
culture did not grow.
The culture within the Phase Two experiments that received an addition of
germanium only without the presence of silicon effluxed the germanium back to the
liquid phase at the end of surge uptake (Figures
3.6, 3.8).
The cultures that received
77
silicon with germanium at the onset of Phase Two retained the germanium consumed
during surge uptake (Figures 3.10, 3.12). To maintain soluble germanium within the
cytoplasm may require a large expenditure of metabolic energy by the diatom ôells.
Diatom cells are not able to expend this hvel of energy indefinitely, and so soluble
germanium in the cytoplasm will eventually diffused back into the liquid medium. The
concentration of soluble germanium in the cytoplasm can be reduced by polymerizing the
soluble Ge to Ge-oxides. The germanium inside the Quadrant I (Figure 3.4, Table 3.2)
cell cultures that received germanium only did not condense the soluble germanium into
germanium oxides because the germanium effluxed back to the bulk medium. Since the
germanium within the Quadrant IV (Figure 3.4, Table 3.2) Ge/Si co-addition cell cultures
did not efflux germanium back into the bulk medium, these cells must have been able to
polymerize the germanium.
Therefore, a silicon addition was administered with a
germanium addition to maintain germanium within the cell mass after surge germanium
uptake.
The cell cultures of experiments that received both silicon and germanium
(Quadrant III and IV experiments) at the onset of Phase Two increased in cell mass
density with time, which is indicative of growth (Tables 3.2, 3.3). In addition, only
cultures that received silicon during Phase Two experienced growth. As long as silicon
was present, the cell cultures grew regardless of the germanium concentration or Ge/Si
ratio. Cell cultures were given initial Ge concentrations as high as 11.52 mg Ge L (BC-
Ni-Si) or Ge/Si ratios as high as 0.83 (BC-Ni-Si) and 0.42 (BC-Ni-63) mol Ge mol Si'
and still experienced growth with doubling times of 128 and 106 hours respectively. The
Phase Two culture that received silicon only had a comparable doubling time of 79 hours.
Phase One doubling times were approximately 36 hours.
The culture within the Phase Two experiments that received an addition of both
Si/Ge, consumed the available germanium, retained the germanium in the cell mass
phase, and all experienced growth (Tables 3.2 - 3.5). The cell culture within these
experimental runs consumed and retained all of the available silicon and germanium
(approximately 1% of cell mass as germanium) and experienced growth.
This is an
exciting prospect because nanospheres created by cell machinery within the SDV that
normally contain silicon dioxide could also contain germanium.
It has been shown that growing the marine diatom
Nitzschia frustulum
until
silicon starvation and then applying a one time co-addition of silicon and germanium will
produce a cell culture that contains both silicon and germanium permanently affixed
within the biomass. However, any nano structure or material analysis of this biogenically
produced Si/Ge combination is beyond the scope of this investigation.
Future Work
To create a silicon and germanium nanocomposite material within diatoms
requires the addition of a combination of silicon and germanium to silicon starved cells.
It appears that the amount of silicon and germanium in the composite material could be
"tuned" by controlling the silicon and germanium content in the liquid medium around
the cell mass.
It may even be possible to create stratified layers of Si and Si/Ge
composite material by alternating the addition of Si and Si/Ge to the cell culture.
Ongoing investigations will optimize the levels of Si and Ge addition efficacy of various
addition protocols. A characterization of the nanostructure, nanophase composition and
material properties of the composite materials will also be conducted.
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Appendix A: Spreadsheets
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CUPLOR,
NOoulipop.
C.N 1/n.r.C.:
Ag. olinoculu.,,
Ni-4-I2.02
5.00...
MediC. .0/05.:
InonIdul VOlum.'
M..oin.d inooliunr SnoOp:
110.101.1.4/0,100,0.
Pho,pn,t. cnn..
M.d/non ,.4402.nr.,4 rot.:
CO. mthnn
IHnn.0onit,oftY
7 d.c.
54101. 1DM 9001.1
2000 nI
100 ml
5.200006 4/mI
Is mg Sun.
0.2 nIM
Dm114.0
L,nrppl000rnonl
Dolt
22 0C
3071 ppm
1000-00 mg So///I nrA))')
0,0544 mg 5.///I nrAU)
-0.1651 (nrg 0./il.
0. Int.pbn.no. 0.15 S)AO.y
0
75 00/rn'-...
40 lOw R,00..corO snip. )FISTI2/CW)
45 Inn. I,.n, 0.00.1 001., 01.1.0.. SqU.l. lch
l4I0N/
1/102004
101.0FF
2,6 (in)
pO,.4o.
doom.,
110 (inn,)
0.6 R.t.ntio.mtlon P.060,
dOt. 2/16/2004
26.16 (mAU)//mgS.)
5,n
0040
M.400,.d End of 0,05th Ph.., P.,.m,.t.,.
54001006 0./k/mI
0.11 Numb.r DorOl)), Ni
0.9001000 0./S/mI
Ni, ISO.
0,01 nrgDCW/L
0.1/Moo, D.,orty. 1.
A// ISO.
0,14 740000,1
SI/lou, 5/In... On,
-0.02 5951/I
On,, ISO.
000 nrgSr/I
5/010.0.0.0 D.n.Ay2460nn,
ro
No.0°
-1,41160/04 / (ml nlUU0)
12651 0.II.//,rin03U)
000I4/mI.
01100,101 Plo,,... mold co.SboI,nt. V.,.,
551-Coo-C.,
573 moSlIl
US/ISO,
All 0,-City
SIC XI/0,I.01V
V... 150
--------..
0.15 nrgSi//.
0.07 nrgOCW/1
0,0121 /SgOCW//)mgSr/
0.0256 /mg0003//)mgSi/
000 /mgSrtI
Si uplake. k0
First Order Reaction Coettloleet
Ge uptake. k.
Run Identlllcenor,
Corc, C
k9
ide coraitions After Pulse
000 095461
334 /cngSe//I
ISO
flItS ReactIon Ret.
SII,CO
003 (rogGoit
41-26
I 50
Rune
Peel Puke Dale
C000
k05 sr
Gemnaclum Coot, Cc,0
157PM
061 IQDCWW61
Ge Reactor, Rc.
665 L(tCW to)
010 U(CW 01
000 L/(CW In000 LJ(CW In)
610 rroG./)g0CW In)
033 lIlOGe/I900W Ill)
000 ro9SilI9OCW 01
000 m9S1)ODCW Al
-
rIrz,pawrIc,?s,nlzwp,ntT!
COkl SD
712612003
Cell Mass DensIIyX
547 /mgGsy(950W)
014 IQDCW4&
Pulse
000 /mgSIy(ODCWI
SI
.eornna_.fl6d,rtSRflauJs
Re. ISO
S/ Reactor. Rs
R50 IS/I
°c.on I 62
NormeOzed
l71-lIflIC-',,t-U]t
-
NoImsierd Ge Pulse
-
Pulse edded
Gernre,rlum Pulse
500 mg Ge/I
20 ml
lOmgGe
00 mQ SI/I
5020 mo SI/I
Slock Ge cons
Volume added
Geedded
SIlICOn PUlse
added
SI cone
SI
Slocu
_tlC
ikOriWtPIOtinslI.Is
.tee1cfl.sIA1S,p8V,.se/np
SuIw,06p.cRsd00esposeren,sak
.dee4Infl.fl05I9PflI.5IW,
.aee1Ien,pSaOaern.I
ounsI9rese.tI
c5IeIr.aGstIrRrpG
.cieNuewpsc,sar_Its__.rI
twLITtW.Ir46ryppSp
u4useflsI*Alerpe..5
.tUtT.1pnoefrpn.w
coriatRPJu1u6I9ra5W8.at,
.e.4IsonInInrespIcseeu
I.trAI5fl.*2,1i,pflI6I.55
I4ee43RruR7,suLr!6..4
I4e4ISRflcJflIsIi9reya*
Iw1I5rr5.PpGI9rfltIsas
I4rstaErflPp.l.1u,peIs4cas
flIW1I5kflr/IS7IO7atssisI.1.T
1e,4/I4W1I5fiIrJ7flte!5I.1.
adeeuIaptfls.s:.nr.4.nnIou,
-
wsnsps
I1!1r.FlIlil.
.
F4IIr1w1!1'lTnrrTTu7rnrT
- 1.
..
-'W
61,111 MSIt(O1,.lI*I
6.03
6p.,00ph303lm.IAI
6/ 88.*p0&lIbIMl0ll
PA.
8.0.3/Ill 4.1,0.004011
All 801310.5/, .804,1
1.41.40.
SI. 9,99'. 3/4011
rIm. SolId,
3:00 PM
0.1.3003.4:
8/5/2003
A/I 80*01.18,
S/N
20376
0818:
1500 mL/n,m
8*,
00,8010*8181 ..0Ag
1000.05(11,9
OS, 53310.1*1 SAl
T8l0,.tWI'
N/all-AZ
:
II1,,0.,SAII,
/081.37.
I8oi,ot0i.mp8
638.18 LOM ,...11
3000 IA.
3.84/1,1,,
0.0.:
0.4.401,4 448.1
11,0.13
11
71 /E/II'1.83
All5W5W,,..,dA,,,p.(FI5IlVCW(
450,11,3,,, .... Io,I,,31013/.,.q,a,.p(l.fl
4.005/-OS (09540)1 olA1'(
0.005(1,9 4.//lL .,.AU)
00243 (11458//I.
8. IoI50i.18ll33/5l*SIA.y
a.!.
,.dk.
doIs43
I 505/1093/2,1
0.01891,0048. D..141y4950,,,.
2428/lOS 3/Ill
Ml.
Ill
lSll1gS/L
5/8,2003
.191780011.1 (ml mAU'(
12651 5.4.1/mI 0*0)
M.d/WI
42 mM
1.18'
0 oUJd
25/mm,
6.016.3.66.11 F3/te,
0.11 N1,m3.l O.,40Iy, N,
6105/105 661./mI
N,, IS.O
0.005/100 1.14/OL
0.3 Ms.. 0.1.11/.0,
/1,10,0
S/I/tI, Comc., 0,,
C1,, 150,
0.09 IlgD22I//L
000 o450W/L
10.82 13512/
0.04 mgS.t
M...W.d 5/Id If 010*55/
Ph,., P.1.00.1.,.
Wl,M1,.I 42 04/PM.
043.
Pot. cIa.
d1.m.I.,
Ste/h/mI
US/I'll)
110
3.SR.1.OSIo.tIlI P.3/I,
2/18/2004
II d.,oki:
P406508/s 3013.
41.1,050
8/17/2003
28.16 (.000)/(,IgS.)
M..11fld
3(1W,)
38 8*883 0.1161,313/I
22 '0
00,1500*4813.
Cd 148/0
5/01185/85
-857 (moSOl.
00, 531.1.1..
04141,. LoSI,,g
SO/O. Il-AU1)
0.012 (log 5.1,/I .oOU(
9*1
P.O L118 Sl/.IC.V.l,.pll-1000
10P
1/23/2003
S..
(1011)
C.SNIIIS.10,O'lI*,N,
N,. ISO,
0.1114.,, 0.0011/. 5,
2SIEION 3./A/mt
0003030 ..14/mL
050 mgOCPIR.
I/I. ISO,
8/1*0,0000., 01,5
0.190903/8/2/
0.25 mIS//I
0.,, ISO,
0.05 mgSU.
5003., to 01110*.. 53./a Oo.ltI*,.* b,
14.55 logS/I.
2/Si. Co,.C.,
/141.18.0.
0,07,,9SIL
/12, I/pOX,
XX,IS.O.
SI 3.Xy1.4
5/15,55.0,
0,40 mgOCW/L
0,16 mq000/l.
10,1,
V//.
0.0262 (0900W)/(I1OS/(
0,0133 )mgOCWyPogS,)
Run Idantiftoslon
RunS
mOld CoditInno Mt., Pul..
91.27
Po.tPuReOata
Poise sddad
8(913)03
10:22 PM
G.rrrm.nhtmm Pulse
0,00 )rngSi)t
Ge uptake, k0.
1.05 LJ(OOCW hi)
CoI 5,0,
0.00 ),ngStWl.
k'o.,so
0.07 IJ(SOCWSI)
G.rrnaniurn Co. Co.0
Si uptake, k
1JOI3CW hi)
C0.. ISO,
7.16 )n,gOe)4.
003 ),ngtae)lL
k'c,ot
i/)500W hI)
Cad Mean 000aityX01,,0
0.50 (500W)t
itoe,,.
500 na 0.11
StOck Ga conIc:
35,28. added:
251,11
0. add.d:
First Order Reaction Coaflfctant
Silicon ColiC Cu,,.
19.0.
Nornnd,zad Ga PuS.
Norntadzed Si Polo.
010 IOOCW$.
1,4.28 (,rGeY)O0CW)
0,00 )lnaOl/152CW)
nOd Reaction Rate
Ge Reatton, Rfl..
12.5 mrs Ge
8un. 50.0
Stock Siconc.
50201(90011
Si Ra0oi, P00
8.o 150.
Vtlum. added
Sledded'
OOn(9Sdt.
Silicon Puts.
Old.
--
-
I
'n
-
.- I '01
a. .
_
,.
-5 .
ZEV'hi
____________--_____
-5
.*..u,0
'8
-__.R.
- 5t..
- 5.5..
- (9th,
-----------------.. .r
0,48 mgG&)900W hI)
000 maSV)QDCW iv)
tn900lgDCw hi)
c1 r'1.-7-
.
..I1.e
-
1162 ngct&)ODCW hn)
.
'a. .
0C.,'
a
.
'r'it
c.
o.a. co
p.j
.
rsI.1
2o0
2
0
2
8
g
"
O
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
S
S
2
r-
0
z
r
,
0
0
0
d
0
0
0
0
2222
5
2222
C) 0000-,,00*C,C
2
-
0
2
o
0
0
2
0
0
0
0
0
0
0
0
0
0
S
S
S
S
S
8888
S
88
288888888 222
888882222288828288222222 888
8 2d
2
0
52
E
882228
S2882S8 8888
0000
R, I4.fltIfloMI000
440
66.0.00.430.0007 303500 P0... Ps,.00.lao.
ColIb00610000
NI-as
9100. 00.1,000*
Sp.cCopflot3000.640
NUiu
Oh, ko000.I.,.011009
B40..0I00 4.600100000'
01 94541$ 0440,0
2100. 30.00.4
SI 50.95 5.115,0000
27
50,6000,0.0., 5144
90000100004
55 0
CO floo.t., ..0.,g
000$000
50,5*.
5)04
Clt,.o. 1054)0009
03500.
Cd 1903 0.
Ag. ci 00004900:
MoOs.o:
500d114000300.:
1,0000400000*00.
063144
0o0
Ig,a000090fl 0600973.
36,001DM 15.0000
2000000.
0.7.:
100001.
7500006 0)0,1
CSOouSOt.d 0491 0.11 SOOi3:
S.SOE005 6l000L
iS 00951.1
0.00200,9
M.4i,co1*.0fl.o4 .0.'
0.2 00,66
25(0700)
2.000005 00(4/01
0000+000.13/001
0.40,090000(1
0,, 15.5.
0,03 ogOCWR.
SO/COO Coos .00.
02.44 009541.
5.,, ISO
.0.S7 (009 SOIl
5/1712005
007,09341.
004 00 000500fl
0.005mg 3.(I(L 04(0)
4o 15 W 64000.o.,4 Isoop. (FINTIOIC.S)
1.00pp13000,.70
4500007001100690Io,400 .000..., $504.0S POOh
140003/
10000FF
0.0240
(009 00S51.
05 100SOlsOOflO$
sold, SI Assay
dOt.
2/16,2004
6000=
Salt (WAU5ØOO900(
00491,030, 40 50050=5
PflO$0
700.00.190,
COIl 514,0051 5.m.Xj, N,
0.0010000 09100/mI
P00 .106
2,5(140)
No.15.0,
0.000040 0.14/mI
4.10.0.,
110(0,00)
0.1166... 0.0./tv, 0,
0010.
0910 0605060015100 Facto,
4.002/1512009
Xi, 13,0,
S6/coossos.,5.,
C.,, 03.0.
060,090000/1
0.07 O900W/L
0.71009314.
0.06 09541
Coil tloomi0300.0011y49600000
WiloonbO #000,... 21.14 Cc00014aot.
000 0
.1.91760*54/ )mL o40U°(
l26S1 O5II4/(707L0004U(
PI0O.05 00000.
4.00.0.,
SO/IL OOU(
4.000-05(009 S35(C,OAU'(
75000l70'-sOO
INlao.,.Oor04op.
Ph56073Ood'
1.000-OS (0009 So(/5) ,00U')
CoO 34045' 000.55. Nc
N3, ISO.
Coil Mo., 500574, Xc
3.5 mId,,,.,
00, 06000060600:
7400*
56.6.00.4000004000,4.0.051
loSal 3,00,404000000
9000
22 C
500,004 op.
31.2.16-4
3100)
P005 *70=
3:00 PM
COO
FouLlY. S00$000 V.(0.400.3400
lOpS
0400=
00911./,01
0 mUd.5
05/, C000,
aSh, 150.
AX. 0,-OX,
AX, 03.0.
SIOoXy'odCo.l7, 2,,,
V,,00. 03.0
W11
V007
1244 009S47,
0.10 09540.
00200950W/I.
007 0740014/1
0.0005 (,O900W(/(m4Si(
000S6 (,O900WY(045,(
Ron id.ctiflca500
SunS
ntis ConditIon. MIen Pul.e
Silicon ConG. 0.
Ni-20
Post PuS. 055
Iso
Pin. .dd.ci
Oenncaniun,
01/2003
10:02 PM
Cca
Coon., Cc..o
10.0
Cel MetS OntilyXcocas
OenmeniumPui..
510050. 00cc:
ynoolSO,
505 3,9 SM.
Odin. .44.4:
IS
fit
NcncSiaad S. Pal,.
Ncnn.ilo.d Si Puke
0,00 imoSiSi.
0.00 (,nqSiYl.
7.45 (000.11
031 )mgSe(t
050 )000W$.
007 )90CWII.
12,30 ),ngSa)l)OOCW)
0,00 (n,9SiY(900W)
7.S 0,90.
9. added'
SOOn nig Ski.
Si addS:
0,0 nng SilL
-.,. -
0,00 IJ)SDCW hi)
flitS Reaction Rae
Se Re.odon, Ro.o
iSO.
R,c
7,79 mgGa,0900W hr)
0,70 n.gO&M0CW hi)
0,00 nngSi/SDCW hi)
0,00 055c0900W hi)
IUL:.j2.c
1t
.0
-II!l.e
__.a
,
.I51ain
- o. S .1om
-S
-S ..
S__0.
- .0.5
k',
Ro10
0 it
..1--.=-
- na.,.
0.10 I./(O0CW hi)
0.00 1j(O0CW hi)
Si 5.50303,
Slack Si cant:
Volon,. added
051/)00GW hi)
5IcplS, 4
k'0.
Scc ISO.
Silicon P0100
.
Find OS., Reaction Coalficilat
S. uplak., 4.
............
-------__________
91
Sample Identification
Date
Measurements Continued
Rung
Cultivation
Time After
Sampie#
Time
Time
Ge Pulse
Trial/i
hr:min
(hr)
(hr)
Silicon Concentration continued
Lu C
C+1-lS.D.
(mgSi/L)
8/9/2003
81912003
Ni-28-Ia-1
Ni-28-la-2
819/2003
14j-2g-2a-1
8/9/2003
8/912053
Average
Germanium Concentration
Ln
Liquid
a-i-
1S.D.
A525
DilOtiOfl,Du
PhaseGe
Cvl-1S.D
5C0
AC,a-v/-
(mL sampiel
Conc.
mL mediomi
(mgGelL)
(rugGelL)
(mgGeIL)
(mgGe/L)
(mgGe/L)
7.45
0.314
0.000
0.444
10:02PM
103
0.00
855
1.0
7.22
103
000
892
1.0
7.e7
103
0.23
002
Ni-28-2a-2
lo:ie PM
I016 PM
103
0.23
809
1.0
8.99
Ni-2g-3a-1
18,33 PM
104
0.52
685
1.0
5.33
8/9/2003
Na-28-3a-2
10:33 PM
104
0.52
697
1.0
5,45
819/2003
Ni-28-4a-1
10:55 PM
104
088
578
.0
4.23
1.0
66l
919/2003
Ni-20-4a-2
10:55 PM
104
0.88
596
l.0
4.33
8/912003
Ni-28-5a-1
11:29 PM
104
1.45
396
1.0
2.63
8/9/2053
Ni-28-5a-2
11:29 PM
104
1.45
389
l.0
2.56
8/912003
Ni-28-6a-1
11:55 PM
lOS
188
348
1.8
2.23
'1.88
337
1.0
2.16
358
1.0
Average
LnC
Ln C*I-
Ca-ve, Solid
ISO.
Phase Ge
trngOe/900W/ (m0Ge/9DCW
1.98
2,01
0.042
2.04
5,057
0.798
0319
5.39
0.809
2.056
0.329
4.28
0.060
3.166
0.32t
al-
1 S.D.
Conc.
1S.D.
665
Ca-
1,89
0.00
0.00
16.32
lOS
0009
1.75
leg
0.017
4.51
1.45
o.ole
0.06
0.015
l0.62
2.75
0.82
I.90
1.67
1.70
1.44
l.47
j_
_iv__
2.60
0.040
4.842
0.316
2.20
0.049
5.247
0.317
0.79
0.022
1150
2.97
2.31
2.31
0.000
5.134
0.314
0.84
0.84
0,000
11.28
2.90
2.57
0.075
4.874
0.322
0.92
0.94
0.028
10.69
2.77
1.14
0.002
9.49
2.48
1.75
0.011
3.71
1.16
1.43
0.003
7.19
1.93
1.41
0.013
7.34
1.97
8/9/2003
Ni-28-6a-2
11:55 PM
105
8/1012003
Ni-28-7a-1
12:45AM
106
8/10/2093
Ni-28-7a-2
2.72
Ni-28-8a-1
12:45 AM
1:24AM
106
8/10/2003
106
3.37
382
1.0
2.52
8/10/2003
Ni-28-8a-2
1.0
2.62
453
1.0
3.11
157
337
400
499
395
Ni-28-8a-1
1:24AM
2:02AM
2:02AM
l06
8/10/2003
454
1.0
3.12
122
18.97
72/
1.0
5.71
/22
18.97
729
1.0
5.80
155
570
568
1.0
4.17
1.0
4.l8
8/1012503
Na28-9a-2
8/10/2003
Ni-28-10a-1
8/I0/2003
Ni-28-lOa-2
8/12/2003
8112/2003
Ni-28-lla-1
Ni-28-llu-2
5:00PM
5:00PM
2:00AM
2:00AM
155
51.97
51.97
8/13/2003
Ni-28-l2a-1
7:00 PM
196
92.97
558
1.0
4.06
8/13/2003
Ni-28-12a-2
7:00 PM
196
92.97
566
1.0
4.14
OIl 3/2003
Ni-28-12a-3
7:05PM
196
92.97
0.08
Lu C0
C,
(mAU)
10:02 PM
107
Average
0.97
0.95
0.80
0.77
0.96
3.11
0.008
5.75
0.061
1.693
0.320
4.16
0.0l4
3.281
0.3l4
4.331
0.314
1.13
1.16
1.74
l.76
1.43
1.42
4.10
0.054
3.347
0.3l0
1.40
1.42
92
Run ./6tM,,
PIStOn P.,.m21075
60-00
OW .p.rpr
Bi71S50O7d$lpb2
SI. 8786/. 0,147171
Th,.Slo,704,
0.5
ts 7,0
SPst510Pl108st0t
26
7.41.4,,
Ow/100m.S,S/14
17731S
745PM
001
- 7.0,5,4.4 /.0tu04,, 0.ty:
0.104/41.4065/7411 0.n,
7/26/2003
700-OS /7775 51)/IL II1AU')
sat
l4/slllOtp
00, tOlt.l,1l80t,
60.2.18-3
lIjls3a, ,,4.0,ily:
8t.,.
76 IE/ll"SSt
05 48 W fl,t,..t.0 Isnlpo (Fl 611 2/CW)
NIOOIeLOUIn,,lt,
280045
I.tl,lppl.t,1.l,l
Phtthp.102.
46 n's has tnO& t47l 0,0.,,., s641O pftah
/45,06/
105/OFF
0.0248 (l,,g 05)/C
/op.
7=1502.
0.5.45,
0.72 /t54101084t/
Wh.tn,.n42 005.48
2.8//Is)
110/mInI
M..541700 £500600035 Ph... P.,,ssts,.
Cd NtsS, 0St523N(
3770700 aslI./SL
NI, ISO
CS/I Ms.. Dlsty, 0,
2.807404 0018/sI
0,78 l,1SOCW/L
Ie,: 150
002
003 II500W/C
S/V401
Ct,,., 057
C, ,, ISO
'O OS ms41l.
076/2004
28,16 (,MU)/)sgS.)
CsO 67441040, 0.tt.0y 02600,..
7.S9E*04 8/45.
041.
0 n4/d.y
0.00 II8OCW/L
0s I,I.00,.41t. .4th SI Ass.,
47/6/2403
I
Msd04mmpI.c.mt,.I.'
0,15,0.
0.75 11/1/2000
S,,-
4000.06(71435W/C MU')
0.008 (17,9 3.)/(L ,,00)
1207s06 #/t
P555-tan,
Ms..U,54 86/flap O47tMfl Ph.., P.1541=7.,.
Cd 647,15., 0.1556/N,
7,007700
ls/l,,L
1.7774030,18/1711
N,, ISO.
004 7190CW/I
C.8M.ssOsnsSo,X,
0,072 (4715 54)/IL 1140U)
.0,0087 )n.g SO/I
050n.y 03115,51/sn
0475,. LQ.dlnQ
0.4578 00, 00.47577404,5
054.:
OtI =
8t'
,1.l.,..IOng
CO, 100,4.7.1 S/N
Flits, Mass C.tb,stltfl
P5)/LW. StI.,,. V.l,spt,-4000
lops
Pt,. lAO
3/U")
5,,,-
0.OS 71SSWL
Oil/ton 108/ ..... 11510 Ct000/0n0
V
.7.9116 n.lS / )I,IL 1,040')
00508/mL
00,07.0;
00. ISO.
0007/58 t006. 1,,,,
On,,,
1S.0
0,48 l,lsnOW/L
0,03 71I5OCW/L
0.0738 (SOOCW(/(sgSl(
00032
I,I490CW(/(SSSI(
---------------
93
Run IdetSIIIuetIon
11*1.1 CondItIon. Mar Pulse
810,38 Con,.. C00u
552 )SI)/L
Post Pm 0/.t.
C,, 10.0
005 (nns*3YL
P.O.. added
G.nnan,un Con,,, On,,,
7.60 (mgO.)t
RunS
51-38
650PM
10/23/3803
C,, 155
0/dMa.. OjIyXoo..
GermanIum Pup..
Xoo, 15.0.
5*01.0. COn,
505 trig 0.7.
CoIn added:
0,8 Itt
Seadded'
NotpnSIoedGa PUS.
NOnnStz.d Si Pu/on
61041 Onto, ReactIon CoalItcIotS,
0. uptake, ,.',.
791 7./3/DOW hI)
0.30 L/(QDCW Or)
S/upt.ke, k'a
0,76 IJ(900W Pr)
0.02 (miCe)17.
018 (9OCW)1t.
0.09 7.1)90/OW tn)
003 (9OCW)1L
8.70 (nigue(/(9OCW)
14101.1 R.0000n Rate
0. Reacdon, R,
2987 (rngSi)/(QOCW)
4.4tt.gGS
3,054190.1(900W hr)
049 tngO&/900W flr(
4,31 mgSi/(900W hr)
0.50 .tCSI/(900W flt)
Rn., ISO,
91110041 FIllSe
Si ReocSon, R
5*31.5384,.
5820m950L
R, 70.0.
4 it
CO/urn, added
5357.9807.
Sledded'
/,',rrrprr'ri .
I.tlnnrnr."rnnn" 10/I.
_3/'___
- n,.. ....,
Co'.1'
_
------------------
-'.4.,...
-'.3/-n .
.0.0.'.
._p,
- ..4.1/r1r.Iot.
5.
- '4. '..
6
..
.7.3/....
- .04.04.. 1'S_..-'- .0..4..'.1 .3
- .04/n,.,'
'047'n.' ..5n'
-
- '.,t'..=_
06'
= '41 .
.i7.
rip 5. ..p=1.
O'i'
- .41
04
--fl
_____
R'a.Z'.P'
.aflsoc0.I'
.- ,.
1'flflflfl..
.'
i7.-flflfl..
.- aI'flflaea4i..P
.pI.. 5.L
.90. anC..
!2.iu
ot
I'.
.
eC90.
.
.
ti.
C'
. u-n.
.04. .
0' -- J"C"
-
"no. . ..'
a
.
....
a.
c-.a. '--''n' -s..
5.
p. --
r so
a
e
'51.
-flfl
fl-rn
flflflfl
flOflflfl.'
.. .n'c.'
fl.
a,'
aflC
.- a,"
so-
r-..a' -
.r
-.90. -flfl
.__R
so. pp.
.
*t.51.
t so' -flflflfl
.p..
so.:
c.
Sample Idenlificatlon
Date
Meanurements Continued
RunS
Cultivation
Time After
SampleS
Time
Time
Ge pulse
TnaIlt
hr:min
(hr)
(Or)
Silicon Concenteatlon continued
Lu C,
Cv/-IS.D.
Average
Ln C
Gemnunlun. Concentration
Ln C+/ISO.
(mgSVL)
10/23/2003
Ni-36-la-1
6:55PM
95
0.08
10/23/2003
Ni-36-la-2
6:55 PM
95
0.08
10/23/2003
Ni-36-2a-1
708PM
95
0,30
10/23/2003
Ni-36-2a-2
7:08 PM
95
0.30
10/23/2003
Ni-36-3a-1
7:25PM
96
11.58
10/23/2003
Ni-36-3a-2
7:25 PM
96
0.56
10/23/2003
Ni-39--4a-1
7:45 PM
96
5,92
10/23/2003
0.05
1.70
1.71
0.010
1.72
0.00
1.69
1.69
0.000
1.64
0000
1.56
0.007
1.39
0.003
1.24
0.010
0.95
0026
0.012
1.69
000
1.64
1.64
0.03
1.56
Ni-38-4a-2
7:45 PM
96
0.92
10/23/2003
Ni-36-5a-1
8:20PM
97
1.58
10/23/2003
Ni-36-5a-2
9:25 PM
97
1.56
10/23/2003
Ni-36-6a-1
9:25PM
98
2.58
10/23/2003
Ni-36-6a-2
9:25 PM
98
2.58
10/23/2003
Ni-36-7a-1
99
4.09
007
10/23/2003
Ni-36-7a-2
99
10/24/2003
Ni-36-9a-1
10:55PM
10:55 PM
1:45AM
102
4,08
6.92
0.01
-0.31
-0.31
10/24/2003
Ni-36-8n-2
1.45 AM
102
692
10/24/2003
Ni-36-9a-1
6:00AM
106
11.17
0.01
0.06
0.010
10/24/2003
Ni-36-9a-2
6:00 AM
406
11.17
-0.30
0.05
0.06
10/24/2003
a-I
10:00AM
ItO
15.17
0.01
0.38
0.38
0.007
10/24/2003
6-lOa-2
0.07
0.016
15.17
a-
10:00AM
1:00PM
110
10/24/2003
113
18.17
10/24/2003
a-
100PM
113
18.17
10/24/2003
0
121
26.17
10/24/2003
a-
9:00PM
9:00PM
121
26.17
10/25/2003
a-
145
50.17
10/25/2553
a-
145
55.17
10/26/2003
a-
169
74.17
10/26/2003
a-
9:00PM
9:00 PM
9:00PM
9:00PM
169
74.17
10/27/2003
9:00 PM
493
98.17
10/27/2003
6-lOu-
9:00PM
493
98.17
10/28/2003
6-16a-
10:30 PM
219
123.67
10/28/2003
6-168-
10:30PM
219
123.67
1,55
0.01
1.39
1.39
0.04
1.24
1.25
0.93
0.96
0.39
0.02
0.08
0.06
0.00
0.01
0.01
0.18
0.20
-1.97
-0.86
-1.42
0.790
CO3l.iqoid
Averafte
Ln Ca
Cu-/- IS.D
0C,
AC*I-
Dilution, D.
Phase Ge
(mL samp/e/
Conc.
(mAU)
mLmedium)
(mgGe/L)
(mgGe/L)
(mgGe/L(
(mgGe/L)
(mgGe/L)
263
1.0
1.62
1.60
0020
0.000
0.028
259
1.0
1.59
234
10
1.41
234
1.0
1.41
223
1.0
1.34
223
1.0
1.34
203
1.0
1.20
206
1.0
1.22
/88
1.0
189
174
175
1.0
1.02
157
1.8
170
1.0
92
A525
C
0.188
0020
0.264
0.020
1.21
0.014
0.388
0.024
1.11
0.005
0.493
0.021
1.0
III
III
4.0
1.02
1.02
0.005
0.583
0.021
0.02
0.91
0.95
0.058
0.653
0061
-0.10
1.0
0.99
0.46
046
0.004
1.138
0.020
63
1.0
047
0.50
1.5
0.57
1.5
0.54
90
1.0
0.5/
75
1.0
0.42
79
1.0
0.44
17
1.0
0.11
27
1.0
0.16
15
1.0
0.10
12
1.0
009
14
1.0
0.10
17
1.0
0.11
19
1.0
0.12
9
1.5
0.07
14
1.0
0.08
13
1.0
0.09
1 S.D.
047
0.013
0.00
0.00
0.35
0.000
1.02
0.22
0.29
0.000
1.43
0.29
0.49
0.012
2.11
0.41
0.10
0.004
2.68
0.51
0,46
0.000
1.0
Cv/-
Phase Ge
rrngGn/uocWr (rngGnlgDCWr
0.46
0.000
96
Cru, Solid
IS.D.
Conc.
1.34
89
Ln C+/-
Lu Cu
ISO.
1.41
101
Average
0.35
0.35
0.29
0.20
0.19
0.20
0.10
0.11
0.02
0.004
3.17
0.60
-0.05
0061
3.55
0.74
-0,77
0.009
6.18
1.16
-0,63
0.091
5.79
1.12
-0.65
0.046
5.85
III
-0.04
0.037
6.35
1.19
-2.0l
0.271
7.96
1.50
-2.38
0.117
4.07
0.76
-2.27
0.000
3.16
0.59
-2.38
0.000
2.60
0.49
-2.46
0.000
2.61
0.49
0.02
-0.01
-0.77
-076
0.54
0.049
1.066
0.053
0.52
0.024
1.078
0.032
0.43
0.016
1.169
0.026
-0.69
-0.56
-0.61
-0.68
-0.86
-0.81
0.14
0.037
1.465
0.042
-2.20
-1.82
0.09
0.011
1.509
0.023
-2.30
0.10
0.011
4.499
0.023
-2.35
-2.46
-2.20
0.10
0,036
/507
0,041
-2.11
-2.66
0.09
0.007
1.017
0.021
-2.52
-2.40
95
Ron ld*0OS*.nIo,n
n///O
Rio,.
di, 07519i
4.00,5307
*,l000,mi.l.,6,74
Iii,,. SloltOd.
0.12 Sb/l.a
- solon It/i
Oil 8000llu.Ig,o0/00l9
Sp.OS'ophonon,.Ooio
01007 MOOs C.lIbn.OIOn
8) MnmCsIISIMIom
lyp.
InSIs
81,:
745 PM
1,000.00 (719 5l)/)L 0l3O)
10/18/2003
*50 *
0, flmonn, S/nd
C0a.
N000/asp.
C.ILn./D.
74/-S-IS-I
80.4/On:
07,7740,1.:
ln00n 202*.:
120.0*0.4/720/4/0* d.70y
CiooO/.184 03.1001 O,*/10:
ni/S 3/ un.di//m cone.
C0i0070.moOOn
II//.n475400 (1.7.50:
7d.ys
6001.1DM 1.000/n
2800 nIl.
570 lu
1.300*05 #/,n,L
4.48*0044/nit
05mg Si/I
M.d//On 0.41*0201.04.1.
0.27712
0 mL/d.y
008IIOS/n*lc.V.,npm.5000
0//oIl)
d,m.S,
251mm)
SOt R.0.nOon 5.0107
0.0.
8/17/2503
350 pu
0*1 i *
40050081703.0(1 mOO')
0.005 mg 0.)/(Lsn.50)
00248 mg 0.71.
75
IIl.*875101 1*nIpO
4*15W 800,.so.,4/.mp. (FISTI2/CW)
L*21pp/*0.nt.71
Pfl0007Ilod:
45 mm (loIn 0.270 0/01*, 5*070061 oqoon. pOol,
0
00.00
SO Imt.,00n.noO 060, SI 0.0.7
dot.
20.16 (tnAU)/)lngGg)
COIl NUnS., D.n.iIyMnOnnn
400
17/6/2703
0
-181700.1(0 /(mL mOO')
goo,,/l
12651 cOIl, / (,mL 7001/)
0005/nl
1.20=0
04,, ISO.
7 01700/ 0,110/mI.
000 m0DCW//.
001 /7UOCW/L
C.IIMOcoO,n,ily, 0*
0,, IS,O
Si100n 0070,, Cs,
0.09
11.02 mi/Sot
0.30 nigS.
/0,0/3/0851/
P.n,nI.l0l.
2.6450000.0/mt
4607*04 006/lIlt
MOOS//l.a End 05070*007010.0
00170710.742 O.flI.
typ,
2O
801 R.t.ndom0oo
2,5(00)
0-0000,
0/16/2004
0*15'
COIIOd//unb.0D.lOiby,S.
Con, ISO.
Si'
7*1mm/n
22 '0
01000
P012*717518 0050.
0012 mg 3/)/)L 7000)
p072*/nO
-0.8*80 (*19 3/10.
00.6_M.
Ag00ltn000hJn,
7/20/2003
S,.
0,040
0.4 6/nesS, 0.tu4/Iy,6/,
5,, 15.0.
C.UM008 30*0/10,31
0.19 n,SOC/0/L
07100,
SOot/n (007*., CO3
002 mgOCW&
000 mgSO.
cs,, IS-C,
007 n,gSOI.
6/Soon to Blonn.o, 1104 Co00EoIsn'0
/Si, 3,,-C/
ASi,
0.0.
10, 31-COt
01.70.0.
Si 000.2 0007, Yco
V,,0. 150.
0113 mglot
0.31 tugS/I
0.16 mOOCW/I
002 mCW/L
02003 )m400WO77QSI)
0.0018 (m0OCW)/)mgn,)
flIt, 14.09100.400
1,11.1 Co,dltio,.. Nt., PtIs.
RI
90.37
FOOt 0,4.0 R..000t, Co,ffiol.ot
5900,, Co.,... C...
S 22 (,,,sSi7I.
0. ..plok., k,,
1.60 1l(OOCW I',)
P0.IPIJI.. 0.1.
CS,, I S.D
0.04 (.090711
k,, ,o
031 L/90000 lo)
Pt16. .44.4
5.8.0.0.0,, 80,0,, C..,,
Co.s I S.D.
C.l SM.. 0.0,110 Vt,o.,
1.60 (mgS.(l1
Si oplA.,
.1. os
650PM
10129,2003
0.00.010,,,
PsI..
ISO.
StoolS.
Volt,,. .44.4
5000,2 S.L
56 ml
44mg 0.
S..ddS
No,o,00o.d5. PItS.
640004.0.45, P0..
0.05
(o,aS.(t
070 11(2_DOW fl7
022 L1(900W 00)
0.39 (900WWI.
0.02 (90003111
805 (,0sS.Y(OOCW)
2774 )095iy)SDCW(
1801.1 6.sttIofl Rats
auto., P.41..
Stool St 00o
4
5620 mOS4L
0. P0.0000,0,,.,
265 .0905J)SDCW 62)
Rb,, 15,0,
0.60 m900)SOCW1.(
S. R..olo,, R,
3.67
gSW(90CW 1.)
1.67 msSV(90000 Po)
Rb, 1S0.
_
..
________
_
--------------------..
S.
0.
l0. .-'!!
2IF!'
2
c,.__J..
.=_3.
______
91
0
j91j
C.
.2_I. .4
.2I. 0
W'0
. -
.C'2_" .0'.1
.09'
,,..
.91'
.
'.50'
-
..,.
..09.
....
.09
.01.
11.09'
91 .51'
v.
. .,.,
t''.fr4
.
'1'
''0'
'
jr.
,'
...
'j
.,.-,
41.
'80
.--...jt..
20.
41
97
Sample Identillcation
Date
Meanarennentu Continued
RunS
Cultivation
Time After
SampleS
Time
Time
Ge Pulse
TrialS
hrmin
(hr)
(hr)
Silicon Concentration continued
Ln
C0v/-l5,D.
Average
LnC
Germeniom Concentration
Lu
C0 riiSO.
Ni-37-la-1
6:55PM
95
0.09
10/23/2003
14i-37-la-2
6:55 PM
95
0.08
/0/23/2003
Ni37.2a1
708 PM
95
0,30
10/23/2003
Ni-37-2a-2
7:08 PM
95
0.30
10/23/2003
Ni-37-3a-1
7:25 PM
96
0.58
10/23/2003
Ni-37-3a-2
7:25 PM
96
0.58
10/23/2003
Ni-37-4a-1
7:45 PM
96
0.92
10/23/2003
Ni-37-4a-2
7:45 PM
96
0.92
10123/2003
Ni-37-5a-1
8:25 PM
97
1.58
/0/23/2003
Nr37-5a-2
8:25 PM
97
1.59
10/23/2003
Ni-37-6a-1
8:25 PM
98
2.58
10/23/2003
Ni-37-6a-2
9:25 PM
98
2.58
10/23/2003
Ni-37-7a-1
10:55 PM
99
4.08
10/2312003
Nj-37-7a-2
10:55 PM
99
4,59
10/24/2003
Ni-37-Oa-1
Ni-37-8a-2
1:45AM
1:45AM
502
10/24/2003
102
6.92
50/24/2003
15-37-ga-I
6:00AM
6:09AM
106
/1.17
159
11.17
10:09AM
10:09AM
110
15.17
III)
15.17
113
18.17
6.92
10/24/2003
Ni-37-9a-2
10/24/2003
Ni-37-lOa-1
10/24/2003
Ni-37-lOa-2
10/24/2003
Ni-37-lla-1
10/24/2003
Ni-37-11a-2
1:00 PM
1:00 PM
113
16,17
10/24/2003
Ni-37-12a-1
9:00 PM
121
26,l7
10/24/2003
Ni-37-12a-2
9:09PM
121
26.17
10/25/2003
Ni-37=l3a-1
9:00 PM
145
10/25/2003
Nl-37-13a-2
9:00PM
145
10126/2003
Ni-37-14a-1
9:00 PM
569
50.17
50.17
74.17
10126/2003
Ni-37-I4a-2
9:00 PM
169
74,17
50/27/2003
10/27/2003
Ni-37-15a-1
9:00PM
9:00PM
593
98.17
193
98,17
Ni-37-15a-2
0.04
1.66
1.65
0.009
1.65
0.04
1.66
1.66
0.008
1.59
0.000
1.67
0.00
1.59
1.59
0.00
I.52
1.52
0.000
1.52
0.03
1.30
5.30
0.003
1.24
0.028
lii
0,012
5.33
0.10
1.26
0.04
3.12
1.22
1,10
0.10
1.06
1.03
0.034
1.13
0.00/
5.22
0.015
1.23
0.009
1.18
0,037
1.97
0.018
2.20
0.015
1.01
0.00
1.13
0.05
1.23
1.13
1,21
5.03
1.21
5.22
0.05
1.17
5.39
0.13
1.96
1.99
0.33
2.21
2.19
0.12
2.33
2.35
2.34
0.012
Dilution, D
Phase Ge
Ln C,
Average
C0
C'r-/- IS.O
tC0
(mLsample/
Conc.
mL mediumi
(nrtGe/L)
(mgGe/L)
(nrgGe/L)
(nrgGe/L)
270
1.0
1.67
1.69
0.030
0.000
276
1.0
1.71
250
1.0
1.52
245
1.0
1.49
240
1.0
1,46
234
1.0
l.4l
230
1.0
l.39
220
1.0
1.35
227
1.0
1.37
220
1.0
1,37
587
1.0
5.10
196
1.0
1.16
187
1.0
1.10
181
1.0
1.06
(mAU)
(mgS'LIL)
10/23/2003
Liquid
A525
158
1.0
163
1.0
0,95
161
1.0
0.93
157
1.0
0.91
168
1.0
0.98
160
1.0
lOS
156
1.0
554
1.0
153
168
229
0.91
Average
Ln C0
L/Cr+/
Ln Cr v/-
C,, Solid
iSO.
Phase Ge
ISO,
Conc.
(mgGe/L)
0.043
C00 v/I S.D.
lmgCe/tOCW1 InmGeIODCW
0.01
0.52
0.018
0.00
0.00
0.54
1.51
0.025
0.501
0.039
0.42
0.41
0.016
0.96
0.22
1.43
0.029
0.253
0.042
0.40
0.38
0.35
0.36
0.020
1.35
0.25
1.37
0.024
0.319
0.039
0.33
0.31
0.018
1.69
0.26
0.31
0.004
1.69
0.22
0.30
1.37
0.005
0.319
0.03/
1.13
0.042
0.559
0.052
0.12
0.037
2,97
0.38
1.08
0.027
0.608
0.041
0,10
0.06
0.08
0.025
3.23
0.36
0.93
0.022
0.758
0.038
-0.09
-0,07
0.024
402
0.41
-0.08
0.019
4.07
0.41
0.31
0.32
0.10
0.35
-0.06
0.92
0.018
0.767
0.035
1.02
0.054
0,672
0,062
-0.02
0,02
0,053
3.57
0,46
0.90
0.90
0.009
0,792
0,032
0.11
0,010
4.23
0,41
1.0
0.89
0.88
-0.lO
0,12
0.93
0.067
0,757
0.073
-0.12
-0.07
0.072
4.02
0,53
1.0
0.98
1,0
1.38
1.37
5.0/0
0.315
0.032
0,32
0.32
0.007
1.93
0,26
227
1.0
1.37
251
1,0
1.53
1,59
0.060
0.100
0,086
0.46
0.000
0.36
0.31
267
1.0
1.64
246
1.0
1,50
0.42
0.000
0.65
0.23
255
1.0
1.56
-0.07
-0,30
0.05
0.02
0.31
0.43
0.50
1,53
0,045
0,160
0.054
0.40
0.44
Run 490663.613,
0.4/918.42,.
84.6
M6p8wn
51-56
891,484/un d..u.9uun'
991+. +0,16614
l.b,.do
SC 90919/. 13181,4,
T/fl. S/md
1000PM
031.0122.4
11/1/2005
=40.868... 06)14,6425
9p.3tu.phOtnn,.4n10
A,, 061.8,81., nuing
4/ A.ayC.Iibn=ti3n
Al, 020,6.1., S/N
tfl,8
20579
/500 nflUn00
416 600/2/8;
S
881+
0811N0058, 08+843,Ne
P88 LII. S,l.S28V.188P,n-5000
8/145)
7418 9/28
1.006-OS 089 SO/IL n,0U/
N, ISO.
Cd Mass 08630+, 08
Xa.IS,0.
6S6R.I.l,Ii,
0.012 1,69 SO/CL n,40)
SlI+1,nCn,
8.606404+8/91,/ni
7,07E4031.191/s,.
003 SI9OCWIL
0.16 n19DCW/L
-
C
61,45 830,6.
-0.8461 /,ng Si/fl.
9. A...yC.Iib,.II8n
220
0.8914,. ID.:
6+991nW8.p.
00, 3unn.n0.Oun
51.4-194
/6.8,91.34,42.48113/
209+,.
438211/23491+8
94,8/. LOM ,..Au,
MediAn,
2900+8/
43+314,4 lu/In.
94.0881.41,0,14., 0.n.iy.
0.4,0916841316911+6114.480+:
lnRM Si +86491+8
184,0./u. 0,108
L.n,p pI.,u.nOnI
Ph+6+p.,l00:
8
76
45
7,0,, 2.8.8/ 24/81 .0,6.00,
144,064/
0,8
6/67/2003
68.38/0.0 End 35 0,25465 Ph... P6,651834,.
4,006.05 yng 0./I/C n,A0)
aqu.,. 9040
101,03/P
4,048
WI,4/m.,42 424848
lop.
0005 (+19 G$/(L 614/U)
4415W 6u+,..,e,3 A,nps /715712/0W)
72169/2*
2.5
III'S)
0.0248 (189 0./fl.
0. Int.nl.n.nS.*1* Si A...0
08/.
4.,. 2115/2004
2116/2004
28/4 /.nAU)/(mgo.)
Cu/i Mumb.tO.n.9y2862nn,
82.88
2200+06 2/,nL
7.306304 2/n,L
IS m9346.
.,n,,n.1,9176 00191/nt nhU1)
6910.1.
Cu/I NAnb.n08184f, N,
2,406400481911+81
N,, 66.0,
000220088/91/811
CS/I 94s 036.60, Dl
022 UOCW/L
0110,0
313341 Cu,,, C,,
0.07 ODCW/L
C,,, /50.
002 ISOS./L
AS1,Cu,.C+,
241,65.0.
62651 +8418/ (+81800)
Pl,0.l.
M.d..+n
08/.'
360 9026
AX. (4.004
.880041616
4/2.10.0.
0 'nC/day
Si luDyi9 .086, 0,2
0,,. ISO.
17I
l7'!I
W!1O
Th7
I_
007 790./I
11/7+8930/
0.36 +835.6,
0_lu 300W/I
007 900W/I
02/66 /gocwy/m9S1.
0,0052 /900w//(m9S)
Rio, id.nIISoSIo,,
FIn.l 00., 95.08/on Co.tflcsnt
Antis CondltIooa AR.n PUS.
S/boon Conc, C,,,
5.42
OnoSiVi.
P0,/Pub, Oat.
C,.,t SIt.
0.02
(mgSiyL
PU/a. .ddad
0./man/urn Con,. C,.,,,
3.04
RunS
N/AS
81)8/2003
Co.,, I S.D.
12.00 AM
Colt US. Dendly 0ntoo,
Oe,rn.ntumPttt..
Xoo,oIS.0
Stook 0.00,,,,:
Volume added.
S.
.44,4'
mQ 0,/i.
7 05.
500
35 fib
0. up/ak., k,,
ku. to,,
(rngC.5.
0,02 (mgO.)&
022 I000W$.
Volum..dd.d
SIrS.
15.9 nb Ski.
81.44.4,
It)
0081/0002051)
007 (OOCW)ll.
NomiS/ned 0, Pul,.
83,88
NoInl040ed Si Pub.
2477 (noS,y(,SCw)
(tngS.YI000w)
fl/S/a R0.080fl Ret.
0. Rotten, 8,,,
4.55
9,,,, ISO.
U/lion, PUS.
5820 55505.
0.85 IJI9OCW
450
95
Sitcic Si con,,;
IJ(QOCW nj
044 iJ(gOCW At)
1,50
SI up/ak., K.,,
8,04
In005)(900W tin)
maCs/U/DOW
SI)
Si R.a000n, 99,,
4,59 rngSW(OOCW tn)
Ran 15.0
0.30
rnQSuS900W tn)
----
---- ___8 ----________-------___
=Io_nfl_e.
't'.I'. !"ftt
flflfly, '-...
=n_nfl_7._.aa..ft. 'ft'
___fl_y,
.1
...
.fl'?fl
5, .2..55 .747..
- ft. .2--C'
.0a'.!'
.0'.,.
C'e'
eO..V.
'
5'' .U!.
/8.
'd'
'20
.0'
.0'
C.
.(
.. nv.-
ft
a0..
.0,.
/8.
!"100 0I?.
.0'
.5I 0' .St.
'C.
1s .05.
.0ft.'
4a .55.
._
St
- .-_z-
...,
rn
I4IZ't'.R.5. . -!'flflflfl_y.
.r...c.
'I' .0,' -.20.ftnt Z..2. ' z..flflflfl_,.
s'S, .n.ar.aa.
'b.'.Oj.
.50.
-.
.
n.,--P.l
0-5'
,
-
P5/81.
.
R"O'
100
Scorpio Identification
Date
Meoeuronrentu Continued
RunS
Sample#
Tr/al#
Time
hr mm
Cultivation
Time After
Time
Gn Pulse
(hr)
(hr)
Silicon Concentration continued
Ln C
C,n-i.tS.D
Gennanium Concentration
Average
Ln C00 *1-
Ln C,
ISO
(mgSifL)
Ni-38-la-1
Ni-30-la-2
12:00AM
146
0.00
11/8/2003
12:00AM
146
0.00
11/8/2003
Ni-38-2a-1
12:20 AM
146
0,33
11/8/2003
Ni-38-2a-2
033
Ni-38-3a-1
147
0.83
11/8/2003
Ni-38-3a-2
12:20AM
12:50AM
12:50AM
146
11/8/2003
147
11/8/2003
Ni-38-4a-1
11/8/2003
Ni-38-4a-2
11/8/2003
Ni-38-5a-1
11/8/2003
Ni-38-5a-2
11/8/2003
11/8/2003
11/8/2003
1:45AM
1:45AM
148
083
170
148
1,70
149
2.75
149
2,75
Ni-38-6a-1
2:45AM
2:45AM
4:40AM
151
4,67
Ni-38-63-2
4.40 AM
151
11/8/2003
Ni-38-7a-1
3:30PM
162
11/8/2003
Ni-38-7a-2
3:30 PM
162
11/8/2003
Ni-38-8a-1
6.00 PM
164
11/8/2003
Ni-38-8a-2
467
1550
1550
1800
1800
11/10/2003
Ni-38-9a-1
211
11/10/2003
86-38.54-7
6:00PM
5:00PM
5:00PM
241
65.00
65,00
11/11/2003
86-38-ISa-I
730 PM
238
91 50
11/11/2003
'4i-38-lOa-2
730PM
238
11/11/2003
Ni-38-lSa-3
7:30 PM
238
5150
9150
164
0.02
1.69
1.69
0.003
1.61
0.003
1.53
0,002
1,69
0.02
1,61
161
001
1.53
154
0.04
1.36
1.35
0.010
1.19
0002
102
100
101
0.010
-241
-2,43
1.35
0.01
1.19
1.19
0.03
0.00
-2,46
0.00
0.05
0.03
0.033
CO3
A025
Liquid
Ln C0.0
C000/- IS.D
Dilution, D
Phase Ge
(tnLoarnpie/
Conc
(mAU)
mLmndiumt
(nrgGe/L)
(mgGe/L)
(mgGn/L)
(rngGe/L)
(mgGe/L)
443
1,0
3.02
3.04
0.018
0.000
0.026
446
10
3,05
365
1.0
238
357
1,0
2.32
352
1.0
2,28
349
1.0
226
277
1.0
1.72
282
1.0
1.75
255
1,0
1,56
252
1.0
1.54
223
1.0
1.34
235
1.0
1,42
49
1.0
0.28
51
1.0
0.29
41
1.0
39
1.0
51
tO
024
023
025
83
1.0
0.47
60
1.0
0.34
62
r5
0.35
CO3
1CO3,,
tC0,0-v/-
Average
Ln C,.. ni-
C05,, Solid
Cve, *1
Ln CO3
IS.O
Phase Ge
t SD.
1S.D.
Cony.
1.11
vrrgGeigOCWi /mgGsJgOCW/
Ill
0.006
0.00
0.00
0,85
0.019
3.14
0,96
0.82
0.007
3.51
1.05
0.55
0.015
5.95
175
0.44
0.010
6.80
2.03
0.32
0.042
7,57
2,27
-126
0.027
1258
3.75
-146
0.033
9.38
2.80
-100
0,337
9.43
1.64
1.07
0.023
431
1.29
1,12
235
0.045
0686
0.048
2.27
0,017
0.769
0.025
0.87
0.54
052
0.81
173
0.026
1.303
0.031
0,54
0.56
155
0.015
1.488
0.023
138
0.058
1.650
0.061
0.44
0.43
0.29
0.35
028
0.008
0,23
0.008
2806
0.020
0.38
0.125
2659
0.127
2.753
5.020
-1.25
-1.24
.144
-1.49
-1.24
-076
0.34
0008
2.693
0.020
'.1 08
-1.05
101
0.41.75,,.
6111*
Nl.4I
M...0!.0 S!.30p 51,6066 Ph... Pt
01119619$!
Call NO.56.! 0m.,5.N,
36
31 bubbl. CAlm!,
A!! IIC.$!n.t.! SIN
S000!,g
071,.
pm.,..
20379
0.005000 mg Oil/Il mOO')
1500 !!!L)!n$!
0.t.St.0.d:
11111/2003
6.
CO, 66nn.t., SIN
*96 ,lin,m11b,m:
M.d/u,,
M.d/Ion WE.!,.:
11.!,4Iam mon.:
l.mp.,51un.
lOSnd*.p.
00, COn2.1,Al5$n'
1.1.4.20.1
Ithomin.Oan nO.004:
14 d.y.
Nd12I. LOM 03*61
2400 ml
100 ml
M.nW.dln00Ijumd.nfly.
06)0111*60*5* C.Ild.n650
InNS SI m.d.,,!,
INumbA, bmp.
L!!!P Ct$011!!4
PImbp.,04
00101 (ma Si/IlL rn/IS)
.05605(mgS,)IL
22 'C
SS
0,15W 6110,2.04,3 Amp. (P05222/0W)
06 n!m I!, 0.fl.I 0,4., .11,1.2., .qo.,. p/IA,
145/ON!
105/OFF
0.!.
I
10,, ISO,
006 m,00WIL
S!,on Con, .4,
1.47 mgS,I.
C,,, ISO.
dat. 11/1/2003
2.'
I,.,
6/17/2003
4.005-06 mg 030/IL mA/I')
0005 mg Cs/IlL mAUl
02245 1mg 5./IL
O.lnImI,10,n. 004! 61 A.03y
0.04 mgS.IL
M.,.or.d End .10mm/In PS,..
Wfl.lm.n42 4,51.6.
lOP.
1,611 $126
2.5 1pm)
dim.0.!
110 1mm)
SaIl 4.1.n6,mtbt. F.nS!
465 0)5/2004
20.16 )106U)IlmgC.)
Call Numb., 0.00/ION,
6,150.
Call Mm. 0.5010,0,
11, 15,0,
P,!.m.I,!,
0072/005 1.09/mI
2,166005 2.10/mI
0.29 maCOW/I
SOS 0400W/L
5100! 0,00.. C,,
0.65 530500,
0,,ln,O,
001 mgS,/L
0*01.0mb.! D.m.ltyAN6O,m.
Slut,! I. B/on!,.. 55.lO Co.tn,b,!5 7._I
ilSl,C6.00
46411,45/IL
IS !!!g 54.
10651 CaIb! (ml m,AU)
Pho.p6616 COn!,.
251mm)
Salt 630.1,00111,0,1,!
SIlO
0.11 M.,. 0.5,24, 0,
5,0mb
CO,
Cdl!,!. LOSIng
Cut/fl:
C.0 1kw/C,
Pd Lii. SA.n04Vflpn,.3000
3 I/mI
OS,, Iso.
AX, X,-0O3
00.10,0,
SJtoOy.goo.I,V1.0
vn,, 16,0
0.04 mgSft
0.27 maCOW/I
0.05 m600W/L
0.0410 lmgOOW)/(mgS,I
0.0070 /n,900WYIn'gS,)
102
EilohI:nI:IIigI:I:I:IIII
IIOHHHHIIIIIFJ1FIII
iiiiiioiiiioiiiu:iii
iEIIIIHIIIHIIIIII::::III
HHHIIHHIIH]]H
:iooioioi0000:ii
iiII
EEEE
IIIIIIHIIOHHIIHII
iiiiiiioiiiiioiiiiiii
if IIIOIIHHIHHIIT1III
XIIIHIHHOHHIW1III
EEEE.O,EE
iiioiuiioiiiioiii
iHHHHIIHHHIIIIIIII
*
'
j
2
Sample Identification
Date
Measurements Continued
Run#
Cultivation
Time After
Sample#
Time
Time
Ge Pulse
Trial#
hr:min
(hr)
(hr)
11/16/2003
Ni-41-la-1
8:06 PM
123
0.02
11/16/2003
Ni-41-la-2
8:06 PM
123
0.02
11/16/2003
Ni-41-2a-1
8:15 PM
123
0.17
11/16/2003
Ni-41-2a-2
8:15 PM
123
0.17
11/16/2003
Ni-41-3a-1
8:30 PM
123
0.42
11/16/2003
Ni-41-3a-2
8:30 PM
123
0.42
11/16/2003
Ni-41-4a-1
8:48 PM
124
0.72
11/16/2003
Ni-41-4a-2
8:48 PM
124
0.72
11/16/2003
Ni-41-5a-1
9:12 PM
124
1.12
11/16/2003
Ni-41-5a-2
9:12 PM
124
1.12
11/16/2003
Ni-41-6a-1
9:45 PM
125
1.67
11/16/2003
Ni-41-6a-2
9:45PM
125
1.67
11/16/2003
Ni-41-7a-1
10:25 PM
125
2.33
11/16/2003
Ni-41-7a-2
10:25 PM
125
2.33
11/16/2003
Ni-41-8a-1
11:35PM
127
3.50
11/16/2003
Ni-41-8a-2
11:35 PM
127
3.50
11/17/2003
Ni-41-9a-1
8:00 PM
147
23.92
11/17/2003
Ni-41-9a-2
8:00 PM
147
23.92
11/18/2003
Ni-41-lOa-1
4:30 PM
167
44.42
11/18/2003
Ni-41-lOa-2
4:30 PM
167
44.42
11/19/2003
Ni-41-lla-1
Ni-41-lla-2
Ni-41-lla-3
9:15PM
9:15PM
196
73.17
196
73.17
9:15 PM
196
73.17
Silicon Concentration continued
Ln C
C1,+/-1S.D.
Average
Ln
Ln
+1-
1s.D.
(mgSi/L)
11/19/2003
11/19/2003
0.06
1.85
1.85
0.009
1.86
0.004
1.73
0.005
1.56
0.003
1.29
0.000
1.00
0.005
0.67
0.007
0.22
0.006
-0.23
0.000
-0.03
0.051
1.84
0.03
1.85
1.86
0.03
1.73
1.73
0.01
1.56
1.56
0.00
1.29
1.29
0.01
1.00
0.99
0.01
0.68
0.67
0.01
0.21
0.22
0.00
-0.23
-0.23
0.05
-0.07
0.01
104
MW,
1l,tItle,lIo,,
Sp.0t7-pfl010l'.*td,
81016861., *.7-1ip691.
1016o.m,.S,..Ith,g
M6.e,l.IS/l4
ci! 9*'..I.:
061* St,0.d:
SI A*O6yC6Iib!8tI77
20379
4.000-05 (7.5 5(14(1 CO.U')
0,012 )m9 S(l)L '.40)
CO. e*l7*811084117'
19-9-al-a
-
6044,,, e.à'.l.
5.640.1.0071, =812,1
60l
-0,6681 (179 51(11
0. 046.7 CIIIIMM,,
0.4.:
611712003
CWt0,. Lh79
0.811*10
P.H1,I6S10*,'..V.,.,poo-0000
0'.,.
75 /lE1W'-6e.
204*0.
1977040101 5 7776
M...,,S j0.im. d.,,*6y.
6.500006 #/,71
0.900942089666 d.,ay.
d.5901050/WI
1118515/ med9'.,.,.
Is ,eg 514.
PI0W,.I. eø'.e.
0.491, ,.pSe,mCO,W.:
0,2 mU
0 mI/day
P000.p.,i.d.
141110N1
4 OOE-00 ('.9 5.(1(I 01010)
0,005(1795.1411 ,04UI
0,0206 I'70 G.)0
4, 35W 6777.80.0 Io'.p. (FISTI2/CW)
45,7,7171170866*1001016617806,659018
2900 '.L
200 71
Me,*ur.d Sl..I..p 57.055 Ph.,. P.,.m.tn
0.6 N/],7b., 0.*15,N,
5 900*05 0.16771
9.16,0.
4247004 1,lS1'.L
0.11 MO,,
500 l7Lll7i,,
111211200S
Ag*&ooW,9-l7:
lope
tth
0, 1708r89en.. 0490 SI A.y
10170FF
26,16 (I7AU)l(,790.)
0*0
09199,76.! D.e.67A460,m
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000 7QOCWIL
0,01 7,900000.
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621 77951(1
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lyp
7776.10*
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0.6.9,84=74*0.770.05
Cot N/77b.! 0*74.79,
0.000*00 0.60/O1L
2,5(107)
4*7.1*1
150 1,77,)
9,15 R.t.,lt1.0100, Feet'.!
48.6115/2004
C.IIM.,0 0.,,ilo, 0,
Z,IS.0
04,0170*,.,, C,.,
C..,. ISO
0.16 71900W/I
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-
000 .79S4I.
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001 m9OCWIL
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00015 ),gOCWy),795i)
h UI
o
ll:OH1OIHOIOIOIII1IOIIIIO1I III
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.
IOOOIOOOI1I011OIIIIOONOIIH
llllHIIIIHHHIHIIHHHHHIHHIII
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IIIIIHHHIIHIHHHHHHHHHhIIH
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p$b
a
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flu
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Sample Identification
Date
Measurements Continued
RUSS
Cultivation
Time After Silicon Concentration continued
SampleS
Time
Time
Ge Pulse
Triaot
hr:min
(hr)
(hr)
Ln C
Cvv+/lS.D.
(mgSi/L)
Average
Ln C
Germanium Concentration
Ln C
Liquid
0/-
1S.D.
A525
Dilution, D5
Phase Ge
Average
Ln C0
C00
mU/ IS.D
5C0
ACv0/
(mLSample/
Conc.
(mAU)
mL medium)
(rngGe/L)
(mgGe/L)
(mgGe/L)
(moGelL)
(mgGe/L)
1.85
0.003
0000
9:28 PM
99
007
365
1.0
1.85
Ln C0
0.004
0.62
9:26PM
9:28PM
99
0.07
365
1.0
1.85
0.003
052
Ni-49-la-3
99
0.07
366
1.0
1.85
0.003
0.62
11/25/2003
Ni-49-2a-1
9:41 PM
100
0.28
274
1.0
1.39
0,004
0.33
11/25/2003
Ni49-2a-2
9:41 PM
100
0.28
274
1.0
1.39
0.003
0.33
11/25/2003
Ni-49-2a-3
9:41 PM
100
0.28
273
1.0
1.39
0,053
0.33
0.008
-0.42
0.003
-0.43
0.003
-0.44
0.006
-1.04
0.003
1.05
0.003
-1.02
1.39
0.003
0.458
11/25/2003
Ni-49-3a-1
10:00 PM
100
0.60
127
1.0
0.66
14/25/2003
Ni-49-3a-2
10:00 PM
100
0.60
125
1.0
0.65
11/25/2003
Ni-49-3a-3
10:00 PM
100
0.60
124
1.0
0.64
11/25/2003
Ni-d9-4a-1
10:27PM
100
1.05
66
1.0
0.35
11/25/2003
Ni-49-4a-2
Ni-d9-da-3
10:27 PM
100
105
65
1.0
0.35
10:27PM
100
1.05
67
1.0
0.36
11/25/2003
Ni-d9-5u-1
11:02 PM
101
1.63
62
1.0
0.33
0.004
-1.09
11125/2003
Ni-49-5a-2
11:02 PM
101
1.63
61
1.0
0.33
0.003
-1.11
11125/2003
Ni-49-5a-3
11:02 PM
101
163
62
1.0
033
0.003
.109
11/25/2003
Ni-d9-6a-1
11:30 PM
101
2.10
78
1.0
0.41
0.006
-0.08
11/25/2003
Ni-49-6a-2
11:30 PM
101
2.10
80
1.0
0.42
0.003
-0.86
11/25/2003
Ni-49-6a-3
11:30 PM
101
2.10
79
1.0
0.42
0.003
-0.87
11/26/2003
Ni-49-7a-1
2.63
59
4.0
0.32
0.011
-1.14
Ni-49-7a-2
102
1.0
030
0.003
-1.19
Ni-49-7a-3
55
1.0
0.30
0.003
-1.20
11/26/2003
Ni-49-8a-1
403
263
263
327
56
11/26/2003
12:02AM
12:02AM
12:02AM
12:40AM
102
11126/2003
52
1.0
0.28
0.013
-1.26
11/26/2003
Ni-49-8a-2
12:40AM
103
3.27
49
1.0
0.27
0.003
-1.31
11/26/2003
Ni-49-8a-3
12:40AM
103
3.27
47
1.0
0.26
0.003
-135
11/26/2003
Ni-d9-Sa-1
2:10AM
104
4.77
45
1.0
0.25
0.013
-1.39
11/26/2003
Ni-d0-9a-2
104
477
42
1,0
0,23
0.003
1.45
11/26/2003
Ni-49-9a-3
104
4,77
40
1.0
0,22
0.003
-1,49
14/26/2003
Ni-49-lOa-1
117
17.60
296
1.0
1.50
0,012
0.41
11/26/2003
Ni-49-lOa-2
2:10AM
2:10AM
3:00PM
3:00PM
117
17.60
292
1.0
1.46
0.003
0,40
11/26/2003
Ni-49-lOa-3
3:00 PM
117
17.60
292
1.0
1.40
0.003
0,40
11/26/2003
20.60
292
1.0
1.48
0.057
0.40
120
20.60
310
1.0
1.57
0.003
045
120
20.60
289
1.0
1.47
0.003
0.39
11/27/2003
Ni-49-12a-1
146
46.60
269
4.0
1.37
1027/2003
11/27/2003
Ni-dS-I2a-2
Ni-49-12u-3
6:00PM
6:00PM
6:00PM
8:05PM
8:00PM
8:00PM
120
11/26/2003
Ni-49-llu-1
Ni-49-lla-2
Ni-49-lla-3
146
46.60
46.60
267
1.0
267
1.0
11/26/2003
146
0.65
0.35
0.33
0.42
0.31
0.27
0.24
1.49
1.51
1.36
0.008
0.005
0.003
0.005
0.010
0.013
0.013
0,012
0.057
0.006
1.200
1.497
1.518
1.432
1,543
1.580
1.615
0.360
0,342
0.006
0.31
1.36
0.003
0.31
1.36
0.003
0.31
0.488
Solid
Phase Ge
Cxv /I S.D.
Conc.
11/25/2003
102
IS.D.
/rngGnlgDCWl (mg0n/gOCWI
11/25/2603
11/25/2003
Ln Cm,r '1
15.0.
N/-49-la-1
14i-49-la-2
11/25/2003
Average
0.62
0.002
0.00
0.00
0.33
0,002
2.85
0.13
-0.43
0.012
7.46
0.33
-1.04
0.014
9.30
0.41
-1.10
0.009
9.4d
0.42
-0.07
0.012
8.90
0.39
-1.10
0,034
9.59
0.43
-1.30
0.046
9.82
6.44
-1.44
0.053
10.04
0.45
0.40
0.000
2.24
0.12
0.41
0.037
2.12
0,37
0.31
0.004
3.04
0.14
107
O
I
I
2
iiii
g
!
1
2
82
.
lii
8
E
fflhIiiH!i
I
nI
U
4
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'-4
!IIOOOHOOOOOII
II IHhIOIHHNOHOHii
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IIHflhIIIIOHIIHIICI
iOIOIHHhIOOHOOOI
I[HHIIOOIIHOHIOH
IHIHIIIllHHIIHH
EEEE
1IIIHIIIIIIIIIIIIIIIE
IHIIOOIIIOHIIOOOII
LEIIHIIIIIIOHIHIIIHIIII
IJLIHIIIIIIIIHIHHIIOIIO
IIIIIIIIIIIllhIHIIIIIIIIII
4400
E E E
4
j
E
FLIIIHIIIIIIOIOOIHOIOII
I011lllOOOhIOHOHII
Sample identifIcation
Date
Measurements Continued
Run#
Cultivation
Time After Silicon Concentration continued
Sampte#
Time
Time
Ge Pulse
moOt
hr:min
(hr)
(ho)
Ln C
C,+I-lS.D.
Auerage
Ln C,
3ermanlum Concentration
In C
IS.D.
(mgSi/L)
10:50 PM
25
0.08
1/5/2004
Ni-51-Ia-1
Ni-S1-Ia-2
10:50 PM
25
0.08
115/2004
Ni-51-2a-1
11;15 PM
25
0.50
115/2004
Ni-51-2a-2
11:15 PM
20
0.50
11512004
Ni-51-3a-1
11:40 PM
Ni-5i-3a-2
11:40 PM
25
25
0.92
1/5/2004
116/2004
Ni-51-4a-1
12:35AM
26
1.83
1/612004
Ni-51-4a-2
12:35AM
26
1.03
1/6/2004
Ni-SI-Sn-i
1:45AM
27
3.00
1/6/2004
Ni-51-5a-2
1:45 AM
27
3.00
1/6/2004
3:15 AM
29
4.50
1/6/2004
Ni-51-6a-i
Ni-51-6a-2
3:15AM
29
4,50
1/6/2004
Ni-51-7a-1
7:15 AM
33
8.50
1/6/2004
Ni-51-7a-2
7:15AM
33
6.50
1/6/2004
Ni-SI-Ba-I
1:00 PM
39
14.25
1/6/2004
Ni-51-0a-2
1:00 PM
39
14.25
1/6/2004
Ni-51-9a-i
Nl-51-9a-2
3:30PM
41
16.75
1/6/2004
3:30 PM
41
16.75
1/6/2004
-51-l0-I
7:30 PM
45
20,75
1/6/2004
-51-lOa-2
7:30 PM
40
20.75
1/7/2004
-51-I/a-I
1:00 PM
83
38.25
1/7/2004
-51-I la-2
-5I-I2a-1
1:00PM
11:00AM
63
38.25
1/9/2004
105
84,25
119/2004
-51-12a-2
1100AM
lOS
84.25
1/12/2004
-51-13a-I
-51-13a-2
12:30 PM
182
157.75
12:30 PM
182
157.75
1/5/2004
1/1212004
1.65
1.44
1.66
0.312
1.88
0.34
1.81
1.76
0.059
1.72
1.12
0.92
1.06
1.72
0.201
1.57
0.38
1.75
1.70
0.069
1.62
0.113
1,97
0.019
1.65
0.57
1.70
1.54
013
1,99
1.96
0.79
2.13
2.06
0.100
1.99
0,48
2.00
1.95
0.069
1.90
0.97
2,02
1,92
0.143
1,81
2.12
2,17
0.05
2,16
1.96
0.296
2.08
0.106
2.35
0.070
1.68
0.048
1.75
2.01
0,73
2.30
2.40
0.32
1.85
1.91
C, Liquid
0/-
Average
Lv Cern
C,,,0*I- iSO
0C,0/-
Dilution, D5
Phase Ge
(mLsarnplel
Conc.
(mAU)
mL rnediuml
)nigGelL)
(nogGelL)
(mgGeIL)
(mgGeIL)
(mgGelL)
621
4.0
11.81
11.52
0.413
0.000
0.584
595
4.0
1123
543
4.0
10.07
530
4.0
9.79
550
4.0
4.0
10.23
517
471
4.0
8.51
460
4.0
8.29
426
4.0
7.56
428
4.0
7.56
430
4.0
7,64
439
4.0
7,83
513
4.0
9.42
495
4.0
9.03
507
4.0
9.29
497
4.0
9.07
504
4.0
4.0
9,22
9.05
9,14
496
520
4.0
9.57
9.64
526
4.0
9,70
492
4.0
8.96
496
4.0
9,05
453
4.0
8.13
432
4.0
7.69
528
525
4,0
9.75
9,60
A525
4.0
CO3
4CO3
Average
Ln
Lv
CO3
C/
iSO.
ISO.
0.201
1.586
0.459
C00, 01-
Phase Ge
1 SD.
Conc.
(ng0e/gDCW) (mgOeIgDCW)
2.47
2.44
0.030
1.42_
9.93
Cve. Solid
31_
0.00
0.00
22.22
2,30
0.020
3.06
1.43
2.29
0.052
3.19
1.73
2,/3
0.020
6.02
2.37
2.02
0.000
7.63
2.92
2.00
0,017
7,29
2.81
2,22
0.030
4.42
1.89
2.22
0,017
4,51
1.88
2.21
0.013
4.59
1.80
2,27
0.010
3,63
1.56
2.20
0.007
4.84
1.95
2.07
0.040
6,56
2.75
2.27
0.005
3,49
1,51
20
9.87
0.510
1.651
0.600
9.51
31
,2S
8.40
0,166
7.56
0,000
3.120
3.957
0.413
774
0.134
3.778
0.434
0.445
2.14
2.11
2.02
2.02
2.03
2,06
9.22
0.275
2.293
0.496
2.24
220
9.18
0.153
2.337
0.440
2,23
2,21
0.122
2.300
0.431
2.22
2.20
0,092
1.891
0.423
2.26
227
9,01
0.061
2.518
0,417
7.91
0.314
3.608
0,519
9.7/
0.046
1.80$
0,416
2.19
2,20
2,10
2,04
2,28
2.27
110
51.,, 00,11151.600
M...u..DSI.,lop 0,lfl Ph... P.,.m.t,,,
C.l(8E8t1,00
C.IIN,Imb., 0.1.15,5,
1*1.81110,10104
80,00,1/2 d1.0,iP00,1
3140941.00130111
l80d/2
0,0,. 040.4:
1030 PM
0.1. SIng:
1/28/4004
Di, 04008/., SIN
01..:
2007S
Di, 8/2018/.
0.002/tOO (mg 510(1 0*0')
0.0101
00, 8000.,.l.l S/N
0,1.:
MSj,m,:
0/2/2/21,0000,,.
1.11.40.00,40,., 4.1128y:
C.14,4.IS /23.1 t011 4.1.15
106.1 Si mStjm,o,,,.
5110.1.30,.:
PI100pl/2l. 3010
0.4(10, 156M311100 .1.:
IU,.b0,1
NI-SOlO
202.
1441.1. 1DM
200 '"1.
8/5303000/1,11
0.1.43:
0.2mM
S.It 0.10,15.,, P8413P
S.,.
4015W5,o, ....... p.515012/0114)
L.mp pl30. /2.0/
45p,mI,t.,, 1.03.1301.18/20.1., sqlMl 511*
1410051
101.0FF
'4.,-
mg 0.0(1 ,,/SUt)
0,0000mg 08)/lI mAO)
'O,ISS7(mgo.4l.
0. lnt.p9r8n0. 0/tI,
005 mg000/L
0,, ISO.
0,01 mtOCW/1
5110.0,, 0.03,04,
1089 mOO/IL
C,,
027 mgSl/I.
Iso.
0.15 10,0/110030/
Wfl.tm.n42 4.4...
I/p.
25/1101)
p/20O0
SI *...p
20,86 (AU),(mg0.)
0.11 N0,,,b., 08,305,9,
N,, IS.D
0.11 0... O.'0, 11,
4.1.2/102004
0.1.,
C.IIM30,00p.iIO, 10,
1112/2004
I ODE-OS
IS XC/m'-OOO
81000.0., 1.1,15.
S.
0.048
0,_ISO
9100004,0., C,,
0 3OCt06 6014/mI
1236005 1.14/1,11
030 m200WIL
0000,gOCW/I
1,30 m3SI/L
04,100.
009 /290/IL
0.11 50mb. 0.tO44tyA861004,
01OCt05 8/mi
5mg SIlL
41,10
001(1 mItO)
0, 0300yC.lIbOSi3t,
350 ppm
Ag.tO.o.uIu,,,
(1,19
.0.5980 mg 500.
00,flt.t,.i.
CO/tO,. 109.0,0
0.8 It,. ID,
SlIm)
500/28
2115/2004
01OCt01 1.141/21
P.8111. S;nOOV.I..P0,.t000
951.
0)04,, to Blom...
.3.91751.8.1 (CL mAU')
8'-I=
12681 ,.II.l(mLmOII(
01.14/mi.
0 mI/tI
5*14
151, Co..C,.i
1/Si, IS.0
40,0,-OX,
01100704 off. 0,,,
Co., 5.0
Oo.ISti.mL So.,
8.94 mgSIt
0.28 m9S4L
025 Il/900WII.
00273 (m900WY)mgSi(
0,0015 )m300W)/(m90,(
.TnIr
.....
------
111
:Iff:IgI:KIIIu:IgIgnhiiaIIIIIIIII
ii:::ii:iiiiiiiiii
IIHIIIIIIOIHIIIIHIIH
iiiiiiiiiiiiiiiiigii:i::iiiiiiiiiii
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Sample idenificatlon
Date
Meanarements Continaed
Run#
Cultivation
Time After Silicon Concentration continued
Sampin#
Time
ieee
Ge Pulse
Tdalii
hr:min
(Cr)
(Cr)
LnC
CvrO/lS.D.
Average
Ln C
Germanium Concentration
Ln
CrCi
IS.D.
IL
7:55 PM
/42
0.08
2/3/2004
10-62-la-I
Ni-62-Ia-2
7:55 PM
142
0.06
2/3/2004
Ni-62-2a-1
6:12 PM
142
0.37
2/3/2004
Ni-62-2a-2
8:12 PM
142
0.37
2/312004
Ni-e2-3a-1
9:12 PM
143
1.37
2)3/2004
Ni-62-3a-2
9:12 PM
143
1.37
213/2004
Ni-62-4a-1
10:30 PM
145
2.67
2/3/2004
Ni-62-4a-2
10:30 PM
145
2.e7
2/3/2004
Ni-62-5a-1
/1:30 PM
146
3.67
2)3/2004
Ni-62-5a-2
3.67
Ni-62-6a-1
149
7.17
2/4/2004
N,-62-6a-2
1l:3OPM
3:00AM
3:00AM
146
2/4/2004
149
7.17
214/2004
Ni-62-7a-1
11:00AM
157
15,17
2)4/2004
Ni-62-7a-2
11:00 AM
157
15.17
2/4/2004
/0-62-ga-i
400PM
162
20.17
2/4/2004
Ni-62-8a-2
4:00PM
162
20,17
2/4/2004
Ni-62-9a-i
9:00 PM
167
25.17
2/4/2004
Ni-62-9a-2
9:00PM
167
25.17
2/5/2004
62-iSa-I
9:00 PM
191
49.17
2/5/2004
-62-100-2
9:00 PM
191
49.17
2/6/2004
-62-ha-i
-62-lla-2
207
65.17
207
65.17
235
230
68.17
88.17
0.24
0,58
2/3/2004
2/7/2004
2/7/2004
r-62-12a-1
-62-12a-2
1:00PM
1:00PM
12:00PM
12:00PM
2/8/2004
-62-13a-1
6:00PM
260
118,17
2/8/2004
-62-130-2
6.00 PM
260
11817
2/9/2004
-e2-14a-1
8:00 PM
286
14417
2/9/2004
-62-I 4a-2
8:00 PM
286
144,17
2/10/2004
-62-ISa-I
9-00 PM
311
169.17
2/10/2004
-62-15a-2
9:00 PM
31/
169.17
2/6/2004
2/11/2004
-62-lea-i
6:30 PM
333
190.67
6:30PM
333
190.67
2/11/2004
-62-17aS-
10:00 PM
336
194,17
2/11/2004
i-62-llas-
10:00PM
336
194.17
2/11/2004
'-62-lTas-62-l7as-
10:00 PM
336
194,17
10:00 PM
336
194.17
i-62-l7asi-62-l7as-62-i7as-62-l7as-
10:00PM
10:08PM
336
194.17
336
194.17
10:08PM
338
194.17
10:00 PM
336
194.17
2/11/2004
2/11/2004
2/11/2004
2/11/2054
2/11/2004
2//1/2004
2-16a-2
0.56
2.23
2.19
0,062
2.02
0.012
2.14
0,09
2.03
2.01
0.99
1.77
1.76
0.016
1.75
0.05
1.53
1.52
0.012
1.51
0.07
1.38
1.61
1.21
0.93
0.67
0.80
0.57
0.44
-066
-0.92
-0,59
0.102
363
1.0
1.56
295
1.0
1,22
1.25
0.039
o.2e5
0.082
306
1.0
1.27
236
1.0
0.93
233
1.0
0.91
229
1.0
0.89
205
1.0
0.78
166
1,0
179
1.0
0.63
155
1.0
0.54
0.61
0.24
89
1.0
0.23
0,91
0.016
160
10
0.56
/66
1.0
0,59
0,72
0,075
52
1.0
0,07
61
1.0
0,11
58
1.0
0.09
82
1.0
0,20
55
1.0
0.08
72
1.0
0.16
67
1.0
0.13
60
1.0
0,19
31
1.0
-0.03
36
1,5
-0.01
36
1.0
-0.01
5/
1.0
0.06
30
1.0
-0.03
38
1.0
0.00
45
1.0
0.03
38
1.0
0.00
0.82
0.076
0.49
0115
0.53
0.142
0.54
0.045
0.38
0087
-0.55
0.148
-0.45
0.11
(mgGe/L)
0.000
1.0
0.32
009
(mgGe/L)
0.072
1.0
5,57
0.13
(mgGe/L)
1.51
91
0.63
0.51
(mgGn/l)
1.46
169
5.41
0.43
(mgGe/L)
1.0
0.010
0.77
019
ml. medium)
343
1,22
0.78
0.17
(mA/i)
0.011
0.90
0.16
Conc.
0.018
1.22
0.04
-0.75
0.237
Ln Cv
(mLsample/
1.60
1.59
0.03
Average
PliaseGe
1.36
1.35
0.06
Liquid
Dilution, D5
A525
0.69
C5,
m-r-/ 1S.D
5C0
5C0-r-/-
Average
Lu CovC1
Ln Co
IS.D.
ISO.
Solid
C0 */
Phase Ge
1 S.D.
Conc.
/mgGn4/DCW /mqG./8DCW/
0.38
0.41
0.048
0.00
0.00
0.22
0.031
0.68
0.27
-0.08
0.011
1.96
0.24
-0.18
0.098
2.24
0.36
-0.40
0.035
2.79
0.25
-0.56
0.082
3.11
0.29
-1,43
0,027
4.22
0.24
-0.55
0.035
3,09
0.25
-2.48
0.340
3.29
0.18
-1.99
0.049
2.93
0.23
-2,20
0,450
2.99
0.20
-2.15
0.191
2,56
0,14
2.33
0.11
-2,79
0000
2.04
0.12
-5.83
0.000
1,88
0.10
-4.00
1.737
1.79
0.09
0.45
0.20
0.24
0.92
0.010
0.591
0,073
-0.07
-0.09
0.84
0,082
0.676
0.109
-0.11
-0.25
0.67
0.024
0.842
0.076
-0.37
-0.42
0.57
5.047
0.939
0.24
0,006
1.274
0,072
0,58
0020
0934
0,075
0.09
0.029
1.426
0.078
0.086
-0.62
-0.50
-1.41
-1.45
-0,57
-0.52
-2.72
-2.24
0.15
0.077
1.365
0.106
-2.30
-1.60
5/2
0.054
1.304
0090
-2.53
-186
0.12
0,022
1.394
0.076
-0.02
0.016
1.529
0.074
0.03
0.048
1,485
0.086
-2.01
-2.28
-2.79
-0.01
5.025
1.527
0077
-5.83
0,02
0.022
1,494
0.076
-3,37
-58/3
113
Run ld.nllSnMI.n
Ru,*
818*631. C0*n1*
PD,.8S P.,.n.5.l01,
51.48
l'i/,,.SIwIS
91.ySo**d
1000PM
0.5. 31.344:
5/2812004
10' 85014.1
AS 5*.m.1,,
4/,SOo.,.t., SW
CS)b,rlun.
44.. *010O Ill
8P,,I**plloI*rn,040
4) M..y C.)Ib,.IUo*
084.' 2/16/2004
21534
=
4000 n,L/rn8l
C0 8*wrn.S,..501g
CS, 6unD,.l., S/N
AI00MU. C.Obo,000
Pd L0.Sn0nn,X.0,pn.-5000
ISp,
'806iO4
S//rn)
0.0010*00 (8095///(1 mAL/')
50000 (*15 5/7)1*0*0)
.0 485 (809 Si//I
SS14IM.,8unP1.o
MWIU,.d SSg*l*pGm*Ifl P118*0 P.,.m8*0
6,6010*00 5.11./nI
0.6 Numb., [348047,1..
5,150.
0.05 ,I/90CW/1
x., Iso
005 mgDCW/I
10.42 mg/nI
S16.*n CW,u., 0,
C.., ISO.
4.20 11/1/2003
6350*34 IS/mI
C.I/M888C*W47,/I,
0.20 moSSI.
0, Aa.y C&)b,,tI.n
Cu/nj,. ..o.d),g
006*,.:
5/10*5/asp.
CO3*080*flb*P'
0.110.
5*9.24-I
I9a,.45, l4.,,ily.
ID.
54. *5 Snoulum:
M.d/urn
.4.410, mUon.:
5011.0,100,.:
M.m,nd )*.uuund.n,Iy
0.5,4,4.4/1934. *.IId./U4.
104.18, ,n*t*2
14534.154. ,..u180
1600,1
.4.411., ..pd.o.,,.*d .1.:
l8umfr,.5*/.mp.
L.*p p/580rn.nI
PflSup.,Sd:
350 ppm
/
75 /E/D'98*
.5.,.
4515W IUD,*u.I4I,n,p. (715,112/OW)
45,,,, 4*0!!! *018*50158! sS*t .q,s,. p/lu),
145505/
104.0FF
0.0044 mg 4.7(1 mAO)
5,01.8/34
2,5/uI)
M668*,.d End ,l0,m.l5
0,/I N*ob., 54.08//p. N,
N,. ISO.
.0.1667*4 0./lI
45*...,
110 (mm)
0.1110,,. 0.0*47,0/
0.25 *900W/I
015,0.
0,02 80900W/I
0,/*On CoIn., C.,
1.66*400.
C.,, 13.0
0,06 *190,/I
1/12/2004
1.0010.06mg 0.0(1 n,AA')
0. *1411,101*0 WIt. SI
4.).
M..y
2/16/2004
26.46 (mAU)/)m30.)
6.8010*04 6/,11
CMI
8,100005 6/mI
*814
0,2 *,M
o ,nL,d8P
W081m3o41 4.5...
155,,
6*56 R.300mtAn P905.,
45.
5 *
2/15/2004
0.048
Ph.,. P.wu.I.s,
3 2lE*00 uMb/m1
1,645*00 *51./mI
54*!lt. S.m.)104944nm
11/472003
09,
4 nM
Phs,pSI.
091,.
22 C
,*'
-16114 .I1./ (ml DAU')
I26S1 u.n. / (ml *340)
0 *.b,, ml
5)5***InSIom*.. 41.140*801*1,111. 1,.,
73,, C,,-C,
673048/11
ASi, /5.0,
0.2/ 8090/11
/2,11,-OX.
0.18 090CW/I
*10,180,
0.02 80900W/I
5)tuXy,Idu,.ff,V1.
V,.. 150.
00202 (m905W7(mgn,)
0.0006 /mODCW)//mgS,)
V
114
.iieigi:i:io:iiiiiipiuiugiiiiii
IIHhIINOIIHUiIHIEIEEHIHI
IIIIllIpIphIHI:IgIuiIgIuiIgll
0
EEEE
I
L
IIIIIIIIIIIIIIIIIH
I
I
E E S
S S
-
0
8
H
Sample ldenslficatlon
Dale
Meanwements Continued
Run#
Cusivation
Sarnp(e#
Time
Tnal#
hr:min
Time
(to)
Time After Silicon Concentration continued
Ge Pulse
(hr)
Ln
Cv/-1S.D.
Average
Ln
Germanium Concentration
Ln C0+/iSO.
(mgSi/L)
7:55 PM
142
000
2/3/2004
NL63-la-1
Ni-63-ia-2
7:55 PM
142
0.00
2/3/2004
14i-63-2a-1
8:12 PM
142
0.28
2/3/2004
Ni-63-2a-2
0:12 PM
142
0.28
2/3/2004
Ni-63-3a-1
9:12 PM
143
1.28
2/3/2004
Ni-63-3a-2
9:12 PM
143
1.28
2/3/2004
Ni-63-4a-I
/0:30 PM
/45
2.58
2/3/2004
Ni-63-4a-2
10:30 PM
145
2.59
2/3/2004
Ni-83-5a-1
11:30PM
146
3.58
2/3/2004
Ni-63-5a-2
11:30PM
146
3.58
2/4/2004
Ni-63-6a-1
149
7.08
2/4/2004
Ni-63-6a-2
3:00AM
3:00AM
149
7.08
2/4/2004
Ni-63-7a-1
11:00 AM
157
15.08
2/3/2004
2/4/2004
Ni-63-7a-2
11:00AM
157
/5.08
2/4/2004
Ni-63-Oa-1
400PM
162
20.08
2/4/2004
Ni-63-8a-2
4:00 PM
162
20.08
2/4/2004
Ni-63-9a-1
25.08
Ni-63-9u-2
9:00PM
9:00PM
167
2/4/2004
167
25.08
2/5/2004
Ni-63-10a-1
9:00PM
191
49.08
2/5/2004
Ni63l0av
900PM
191
49.08
2/6/2004
1:00PM
1:00PM
207
65.08
2/6/2004
Ni-63-Ila-I
Ni-63-Ila-2
207
60.08
2/7/2004
65-63-/2a-I
230
89.08
2/7/2004
Ni-63-12u-2
l2:OOPM
12:00PM
230
88.08
2/8/2004
N(-62-13a-1
600PM
260
118.08
2/8/2004
Ni-93-13a-2
260
118.08
2/9/2004
Ni-63-14a-I
286
144.08
21912054
N-63-14a-'
288
i44.08
2/11/2004
Ni-63-105-1
335
193.08
2/11/2004
Ni-63-15a-2
6:00PM
8:00PM
8:00PM
9:00PM
9:00PM
335
193.08
2/12/2004
Ni-63-16a-1
355
213.08
2/12/2004
Ni-63-16a-2
5:00PM
5:00PM
355
213.08
2/12/2004
Ni-63-l7as-
7:30 PM
350
215.58
2/12/2004
t4i-63-l7asNi-63-l7as-
7:30PM
7:30PM
358
215.58
358
215.08
2/12/2004
0/0
0.52
0.08
5.589
0/5
0.01
-0.36
-0.34
0.0/9
-0.33
0.03
-0.46
-0.43
0.041
-0.40
0.0/
0.08
5.03
-0.40
0,78
0.73
0.02
0.93
0.70
0.46
0.46
0.57
0.75
1.53
2.24
2.13
0.020
287
1.0
1.18
81
1.0
0.20
74
1.0
0.17
45
1.0
0.03
47
1.0
0.04
63
1.0
0.12
65
1.0
0.12
42
1.0
0.02
49
1.0
0.05
46
1.0
0.04
0.06
1.0
0.09
59
1.0
0.10
0.72
0.5l3
119
1.0
0.37
I/O
1.0
0.37
136
1.0
0.40
134
1.0
0.44
41
1.0
0.02
53
1,0
0.07
0.93
0.008
0.72
0.033
0.53
0.099
0.47
0.012
0.59
0.038
0.74
0.017
1.56
0.044
1.59
0.69
(mgGelL)
0.500
1.0
0.73
0.21
(mgGe/L)
0.0/4
57
0.62
0.03
(rngGe/L)
1.19
51
0.48
0.07
(mgGe/L)
/20
0.024
0.60
0.02
(mgGe/L)
1.0
0.76
0.75
0.17
mL oedium(
291
0.005
0.92
0.07
(mAU)
0.86
0,7/
2.18
0.077
5C5*f-
Conc.
0.041
0.74
0.03
Ln CO3,
n*/ I5.D
(raL Sampia/
0.005
0.86
0.05
Average
Phase Ge
0.09
-0.46
0.07
Liquid
Dilution, 0n
-0A3
0.09
0.01
C5,
A525
65
1.0
0.12
62
i.0
0.11
70
1.0
0.15
48
1.0
0.05
38
1.0
0.00
49
1.0
0.05
64
1.0
0.12
94
1.0
0.26
151
1.0
0.52
148
1.0
0.51
220
1.0
0.88
235
1.0
0.92
C5.,
5C0
Average
Ln C*1-
Cvs, Solid
C5,/
In Ce
/5.0.
Phase Ge
I S.D.
IS.D.
Cons.
)rnOGOIgDCW) (rngGO/9DCW)
0.18
017
0.0/2
0.00
0,00
-1.71
0.125
3.95
0.36
-3.26
0,164
4.51
0.39
-2.12
0.053
4.19
0.37
-3.48
0.586
4.03
0.40
-3.02
0.333
4.47
0.39
-2.38
0.069
4.30
0.37
-0.99
0.009
3.21
0.28
-0.8/
0.015
2.03
0.18
-3.38
1.030
2.86
0.27
-2.14
0.082
2.67
0.23
-2.49
0.795
2.10
0.23
-439
2035
2.00
0.18
-1.74
0.000
1.69
0.22
-0.66
0.000
1.28
0.11
-0.12
0.058
0.65
0.13
0.17
0.18
0.023
1.008
0.027
-1.62
-1.50
0.04
0.006
1.151
5.515
-3,37
-3.14
0.12
0.006
1.069
0.015
-2.16
-2.08
0.03
0.0/9
/155
0024
0,05
0.016
1.139
0.021
0.09
0.006
1.097
0.0/5
0.37
0.003
0.820
0Th4
-3.07
-3.04
-3.25
-2.79
-2.43
-2,33
-0.99
-1.00
0.45
0.007
0.743
0.0/6
-0.80
-0.92
0,04
0.038
1.146
0.041
-4.11
-265
0.12
0.010
1.072
0.017
0.10
0.070
1.092
0.072
003
0.035
/162
0038
-2.08
-220
-1.92
-3.04
-083
-2.95
0.19
0.097
1.001
0.090
-2.12
1.36
0.51
0.010
0.675
0.017
-0.65
-0.98
0.89
0.051
0.302
0.053
-0,16
-0.08
116
842, Id.l0Iifl2Wi,
5/230
N//dO
Sio,..012,4.sor400,,:
750,11 SIR
Si2Il.4
Dot. SS3.d.
1/110,
CWib,,Kl,.
Sp..Slpflltlm.401
51311/1000 111,1 1211 510
MI ftmOm.t,,..t41
8./Ill
Ml (I*m.l., S/N
500 PM
A/I 221,1.0.:
11/1112003
M.,.u,.451,Oop 01000/, Ph3
Co/I 5.2,10.1 D.m,W. N,
SI M00yC.1110,.lio,
0,0.:
0157004
03,0
1200-054mg 4/i//i mAO',
22
DII 25
250 mlJmd,
CO. 3o.s,l., 0.0
21=
CO. 20,21.1.10,574
Sb,.
0012 (1119 S/i/hi 01AU)
-0.8041 mg S/I/I.
i/p.
P1.1.1*1,.
1.SI030S 1.50/mi
P50 L.I. Si.,.000.l,,po,.3003
25 /.1m(
4/501.1.1
SOIl 001.1,30, P1.10,
6.1. 11/1/2303
Coil 32,,, S.o.4 0,
0,, ISO.
0,12 m300W/L
5/410,00,11., C,7
0,00 III9OCW/L
5.71 mgS,/L
C3*, S.D
0,02 1,135//I
3 0000YC.i1b1W1111
02 'C
ClOIhIOS L0.dO,g
0.1.:
5
COn 141.1.0
Ag. ll/,10142,,,
M.di,o.
42040,1,204200.
hums/si.,, flSls/P4'
P4/4.20-S
Od.y.
P48,1.1.1KM 10.100,
700 ml
100 6O1,CSh
33.40
OOSWPuII.sh011smp.0700 K/PLO)
i.11P04000IIl.l1
PICOCP,li.d
SO ,,m, 010,,, 203..i 12/I.,
401054/
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0.00 mgSOI.
5.8.0000 EM? 0000*0017 Ph.., P000*00.0076
Coil 5,0060,00000*, N,
0.00E0000010/t,tL
0.0024/0,9 501/IC 1,411/
p2480/08
25/0)1)
70,15.0.
.0.1740 (So Aol/I
0t0./.t
1101,0000)
Coil Moo, 0.004,74
0.28 00900W/I.
'1/, ISO,
5400? 0248.,
000 eg000/L
9/0,5) A*..y
2/16/2004
8S *0
30 ,t,L
5.000000*004,0*0.00*7
0.10281.4 i,g0.io.iI d.n.0y
0.0101 )eg SO/CL *1011/
25/0000)
480001.,
-03006 nO S/Al.
S. 088.0 CotIb,08i0,.
002.:
2)30004 'Le,* S.
0 ,,,LjtM?
P0011), S,j.,,o. 20t0.pe-3000
lope
2)77.3004
0.000*00 leg S/OIL rnOU'i
0006000?nfl.,$008tO
0P2512004
50760004 SlOflUp50880tfl P0550 P0,8fl8t,00
CoO Slob., 00000/1. 5*
000E000 0.0,101
Alt., U... ,,.tlb*018t,
SI 0.flyC.iI010iS.
22
25,16 (soUl/logS.)
Soil R.t.00enI/opfooto,
doll 2115/200*
5,o
0,0*8
2,000*00 *010/mI
C01
C., ISO.
1,21 mISol.
2)21 ,,'Q&/L
0011 SOobo, D.,,0400S680nt
dOt.
1,10.15/00000*01,0.
Is ,,,6S01_
000.7. 00,8:
17/5/2003
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P--i.
Slit,?. 0 8/00*80 71014 000IIi*/.0t. 74,
/I5,Co,-Co,
871m90*C
AS,, ISO,
AX, 0,-CO.
5.4/124000000*07.0).:
OX, ISO.
OI00110900.5,Voo
0,. ISO
0,22 0095.).
0,10 o900W/L
000 ,,,900W/I
0,0212 (m.000l/O,'gSi(
0,0006 (n,900W/OegS')
.
135
Rsn ld.,tIftc.ii.n
InIi. Condition. Aft.,
Ronti
N,-6I
PSI..
Siocon Conc. C,,,
0.00 )rngSi)lI.
Foot Psi.. Cot.
C,,,, ISO.
PSI.. .dd.d
G.,nnonionnConc. C,.,.
CO3,. 18.0,
000 )rngSi)&
0.21 )rngG,yL
0,02 (rngo.yL
0.11 Moo. 0,nOIy 8,,,,.
0,26 )9DCW)IL
837 PM
2,3/2004
O.,n,mnkrnn Psi..
X,,,V,. 1
28 ng 0,/I
Stock 13. conc
Volotn. .ddod'
O..dded
S.D
0.79 mgGey)gOCW)
0.00 (ntgSty(SOCW)
Ncnn,.io,d Si Psi,,
0125ncgl3.
Silicon Psi..
Siock 8 conc,
Volo,m,. odd.d
6620 ,ng Sft
Sm .43.4
1nl041 Rooction R.t.
0. Ro,cii,n, 6,.,
6,,, 15.0.
0.64 nmgGn/(9DCW Sm)
Si R.0,t/omt, 6,,
0.00 ,ngSW(9OCW 5,)
R,o IS.D.
000 ,mgSil(9DCW Sc)
lIt nm0&(90CWSr)
'
!4.r3.ja.t'.'t
"
'X'.'
!cotCJl
!.rn,Z?4k.tr:''I'
!2I1CZ1I
:c'ic. .:Ilr!ar,.z'l.
!.i'P3.Z'I..
8'.'.
0,00 IJ(90CW Sn)
c
0 tnl
S'I !arpSj1,r:..x.
L0,c5t,2In
000 iiiS0CW mm)
k'c
0.0 nmg Sot
5.Fl,.','00
t't!t.i.JO
307 LJ(9DCW Sn)
5.34 IJ(OOCW Sn)
Si splake. k'o
0,00 (g0CW90
Nonnolond 13. PsI..
S ml,
Floct O,d.c Ronction Co.fllcI.,t
0. opt,kn, k',.
C'Li!.'.Z Is'
..I.
s'S
.
08
.
.Il.
_L:
I.
K .t0'2.3.flt4I ..I
0
J..J.Y13.
0"/'Zm?AL'
i.n
t'l.m20jj.ftfl6.kt.
trtr.w5
1tR'S
..o.
ti'1P&
,.
:.15...l.
.
.
.
.W.
.'.
136
137
Appendix B: Experimental Procedures
138
Culture Maintenance
Materials
Four 500 mL flasks with foam stoppers per combined parent flask (combined parent
flask is comprised of three culture flasks combined into one flask), 400 mL Diatom
Nitrate LDM medium per combined parent flask, 10 mL volumetric pipette, Sterile
laminar flow hood (Edge Gard Hood US pat # 3,318,076), Sterile 100 mL graduated
cy]inder, 70% ethanol solution, Wiping Tissues (Kimberly-Clark Professional
Kimwipes EX-L), Nitrile lab gloves (Kimberly-Clark Safeskin powder-free purple
nitrile exam gloves) and an incubator (Precision Scientific low temperature incubator
815).
Culture
Nitzschia frustulum from UTEX algal collection #2042 ORJG1IN: deposition. 1/76 by
J.C. Lewin as 53-M (Lewin & Lewin 1960) swirled for five seconds once per day.
Incubator Conditions
22 °C, 14:10 light/dark cycle, Light intensity of 55 p.Em2s' at the flask exterior,
illuminated by Feit Electric 9 Watt Compact Flourescent 2700 °K / PL9, air
circulation is provided by a cooling fan (120 VAC 60 Hz 22 W impedance protected
Radio Shack cooling fan No. E89061 cat # 273-241C).
Subculturing Procedure
1.
Repeat subculturing procedure every two weeks.
2.
Autoclave five 500 mL flasks with foam stoppers and a 100 mL graduated
cylinder for 30 minutes at 123 °C and 23 psig. Allow to cool.
3.
While always wearing nitrile lab gloves Spray 70% Ethanol solution onto surfaces
in the laminar flow hood and wipe dry with large Kimwipes.
4. Four hours before subculturing remove three flasks from the incubator and place
in the sterile laminar flow hood. Combine the contents of the three flasks into
one flask and place the full flask back in the incubator.
5. In the laminar hood, using aseptic technique, transfer 80 mL of Diatom Nitrate
LDM Medium to each of the five 500 mL flasks.
6. Bring the flask containing culture into the laminar flow hood.
139
7.
With the sterile volumetric pipette remove 10 mL of culture from no more than
116 of the bottom of the flask (the culture should have settled onto the bottom of
the flask) and transfer to the first flask. Repeat with the remaining four flasks.
8.
Place four of the new cultures into the incubator. Place the fifth flask into the
door of the incubator labeled as a "back-up" culture.
9. Dispose of the oldest back-up culture and the remaining culture in the parent flask
by adding one capful of bleach and allow standing until the culture turns white.
Pour the culture down the lab sink while running high flow tap water.
Diatom Nitrate LDM Medium Preparation
Silicon Stock
Add 8.49 g Na2SiO3*5H20 (sodium metasilicate) to 250 mL flask and Fully dissolve
solute in 150 mL DI H20. Fill to 200 mL with DI H20 (Makes 200 mM Si stock) and
store in sterile polycarbonate.
PIV Metal Solution
Fully dissolve the following to 500 mL DI H20 in a I L bottle: 0.375 g Na2EDTA,
50 mg Fe(SO4)*7H20, 20.5 mg MnCL2*4H20, 2.5 mg ZnCl2, I mg C0C12*6H20,
and 2 mg Na2MoO4*H20.
Vitamin Stock
Place the following in a I L beaker and dilute with DI H20 to 500 mL: 0.0050 g B12,
0.0050 g biotin, 0.5000 g thiamin HC1, 5.000 g meso-inositol, 0.5000 g thymine,
0.5000 g Ca pantothenate, 0.0500 g p-amonobenzoic acid, and 0.5 g nicotinic acid.
Mix and freeze lOmL aliquots.
Bristol Nitrate Salt Solution
1.
1.125 g MgSO4*H20 into 150 mL DI H20 in 250 mL bottle
2.
1.474 g K2HPO4*3H20 into 150 mL DI H20 in 250 mL bottle
140
3. 2.625 g KH2PO4 into 150 mL DI H20 in 250 mL bottle
4. 33.92 g NaNO3 into 500 mL DI H20 in 1 L bottle
5. Autoclave solutions for storage
6. Add 14 mL of solutions from 1) 2) and 3 and 70 mL of solution 4) to 2L flask
7. Fill flask to 1400 mL
8.
Autoclave solution for 30 minutes at 123 °C and 23 psig, allow to cool
Seawater
1.
Receive seawater from the National Oceanic and Atmospheric Administration
(NOAA, Newport, OR, USA). Store and transport in a 55 gallon polyethylene
Russel Stanley West Inc. Poly Drum (Pat # 4022345).
2. Using a peristaltic pump (Cole-Parmer model # 50000-079, serial # FK3I 14, 45
W, 10.6 gpm) filter seawater through 5irn nylon fiber Omnifilter Whole House
Filter Cartridge.
Medium Preparation
1. To each liter of filtered seawater add the following: 2 mL ESS-lO Nutrient Stock,
6 mL Ply Metal Solution, 112 mL Bristol Nitrate Salt Solution, 3 mL 200 mM
silicon
stock
solution.
141
Table Bi Diatom Nitrate LDM Medium.
Diatom Nitrate LDM
Clayton Jeffryes
211612804
Chemical Name
Chemical
Molecular
Medium
Formula
Weight
Component [aj
(g/gmole)
Bristot Nitrate Salt Solution
Solum nitrate
Superstock Concentration
Stock Concentration
Medium Concentration
mL superstock
Liters Dl t-lO
rug solute
per mL stock
(mg/L)
mL stock per
L seawater
(mg/L)
mL Stock per
mL medium
(mg/L)
(emol'L)
112.0
NaNO5
*
85.
Macronutrier
33920
0.50
67940
0.05
3392.0
9.976-02
33629
3979.93
138.4
Macronutrient
1125
015
7500
0.01
75,0
9.97E-02
7.48
54.05
Magneolum sulfate monohydrate
MOSOn
Dibasicpotasium phosphate trirrydrate
KHPO3HO
228.2
Mac,onulrient
1447
0.15
9547
0.01
96_S
9.97E-02
9.62
42.16
Monokaoic pntasiam phosphate
KF-lPO
136.1
Macrunatrient
2625
0.15
17500
0.01
175.0
9.970-02
17.45
128.24
N5)SiO55t4)O
212.1
Macronntrient
8490
0.20
42450
1.00
42450.0
2.67E-03
113.40
534.75
750.0
5.34E-03
4.01
10.76
FIrO
Silicon Stock Solution
Sodium metasilicate pnntahydrale
3.0
PIV Metal Solution
0.50
6.0
Sodium EDTA
NaEDTA
372.3
Micmnutnent
370 1)
750.0
1.00
Iran Sulphate heptahydrale
Fe(SO))*7H)O
277.9
Micronatlent
50.0
100.0
1.00
100.0
5.34E.03
0.53
1.92
Manganese chloride tetrahydradrate
MnCI
197.b
Micronulrient
20.5
41.0
1.01)
41.0
5,340-03
0.22
1,11
Zinc chloride
ZnCl2
136.3
Micronutrient
2.5
5.0
1.00
50
5.34E-03
0.03
0.20
Cokaftchloridehexahydrate
CoClr6HO
237.8
MiCrnnatrient
1.0
2.0
1.00
2.'
5,34E.03
0.01
0,04
Sodium Molyhdate monnhydrate
NaMoOr*4HrO
277.9
Micmnutrient
2.0
4.0
1.00
4.0
5.34E-03
0.02
0.08
1355.4
Micmnutdent
5
10.0
1.00
10.0
l.78E.03
0.02
0.01
Micmnutrient
5
10.0
1.00
10.0
1.786-03
0.02
0.07
1.76
5.28
*
4H)O
ESS-lo Nutrient Shock
0.50
Vitamin B,
2.0
Biotin
C,0H,5NO,S
244.3
Thiamine HCI
C,1H,,ClNO5HCl
337.3
MicrOnutrient
500
1000.0
1.00
1000.0
1.78E-03
Meuo-lnos'lol
C5H,uOS
180.2
Micronutrient
5000
10000.0
1.00
10000.0
j,78E-03
17.81
98.83
Thymine
C5H5NO,
126.1
Mlcmnutrienl
500
1000.0
1.00
1000.0
1.78E-03
1.78
14.12
Copantathenate
Chl,eNOrCa
238.3
Micmnutrierrt
500
1000.0
1.00
1000.0
l.78E-03
1.78
7,47
P-aminobenzoic acid
C,HNO
137.1
Micmnutrient
50
100.0
1.00
100.0
1.786-03
0.18
1.30
Nicotinic acid
C6H5NO5
123.
500
1000.0
1.00
1000.0
l.78E.03
1.78
14.47
Tntal:
Micronutrient
142
Reactors
Bioreactor Descriptions
All reactors are jacketed and cooled from a reservoir containing water at 22°C. Inlet
air is filtered (0.2 m PTFE Gelman Acro 50 Lot No. 4254) and moistened by
bubbling (500 mL flask with stopper and stainless steel tubing) before entering the
reactor.
Table B2. Bubble column vessel geometry.
Reactor
Volume [mL]
Height [cm]
Diameter I.D. [cm]
Impeller dimensions
[cm] h x w
Be1co1STR
800
15
10
2.2x5.5
Belco2 STR
3L BC #1
800
3000
3000
2000
15
10
48
36
48
9.8
2.2 x 5.5
n/a
n/a
n/a
3LBC#2
2LBC#1
111.4
f
7.9
143
Table B3. Reactor operating parameters.
Reactor
Stir rate
RPM
Light
intensity
Illumination
source
Number of
bulbs
Air flow
Feit Electric
2
400
Air delivery
[mL min']
[tE m2s']
Belcol
150
100
STR
Belco2
STR
150
200
Swagelok-
9Watt
316JWP
Compact
Fluorescent
2700 °K I
PL9
Feit Electric
stainless
steel fit
2
400
Swagelok-
9Watt
316JWP
Compact
Florescent
3tainless
steel fit
2700 °K/
3L BC #1
n/a
60
3L BC #2
n/a
60
PL9
15W
Sylvania
Cool White
F1ST12/CW
15W
4
1500
Glass fit
4
1500
Glass fit
4
1000
Glass fit
Sylvania
Cool White
F15T12/CW
2L BC #1
ISylvania
n/a
60
15W
Cool White
F15T12ICW
Bioreactor Inoculation
1.
Place a cleaned and sterilized bioreactor vessel assembly in its holder or on its stir
plate and attach to the cooling water and air lines.
2. Check the lights, cooling jacket, air and impeller for proper working order.
3.
Select an inoculum flask from the incubator (generally the most dense culture is
selected) and move to the laminar flow hood.
4. Using aseptic technique remove 1 mL of culture and assayed for cell number by
the hemocytometer cell number assay.
5. With lights, air and stirring at operating conditions add Diatom Nitrate LDM to
the vessel's capacity minus the inoculum volume by addition through the top of
the reactor (remove headplate).
144
6. Allow the medium to equilibrate with the sparge gas for four hours and then add
the inoculum culture via the same procedure as medium addition.
Sampling
1. Draw a sampling syringe (2OmL Norm-Ject, Henke Sass Wolf GMBH
DIN/EN/ISO 7886-1) full of sterile air from a laminar flow hood (Edge Gard
Hood US pat # 3,318,076).
2. Insert the tip of the sampling syringe into the reactor sample port and expel the
sterile air into the reactor to clear stagnant medium and debris out of the sampling
tube.
3. Draw the desired sample volume out of the reactor sampling port with the
sampling syringe.
Experimental Design for Bioreactor Experiments
1.
After inoculation use the sampling protocol take initial measurements of cell
number density, cell mass density, soluble silicon concentration and pH.
2. The pH, cell density and soluble silicon concentration are monitored throughout
Phase One, the growth stage leading up to silicon starvation.
3. When soluble Si is below 50 jiM for 24 hours or the level of Si is stable for 48
hours pulse silicon starvation has been achieved. Silicon starvation prepares the
cell culture for Phase Two, pulse addition of Ge/Si.
4. The Phase One specific growth rate (ji) is determined from the least-squares slope
of the cell mass density versus time data.
5. A one time addition of Ge/Si is added to the cell culture. Assay Ge and Si
concentrations, at least six data points within the first three hours. Stirred tanks
were only observed for the initial uptake phase. Uptake rates are determined from
least-squares slopes of the natural log of concentration versus time data.
6. After initial uptake monitor Si/Ge levels every four hours until Si/Ge efflux or a
steady-state concentration of soluble Si/Ge is observed.
7.
After efflux or steady-state is observed, measure dry cell mass density, cell
number density and Si/Ge levels daily until shutdown.
145
Bioreactor Shutdown and Reactor Cleaning
Materials
Reactor shutdown and cleaning requires bleach, nitric acid, a long handled bottle
brush, Rain-X, sodium bicarbonate, a DI water source, tin foil, autoclave tape, and an
autoclave.
Procedure
1.
Remove bioreactor assembly from air and cooling water source.
2.
Remove head plate.
3.
Clean the head plate, hose connections, and interior of sample tubes thoroughly
with soap and water and rise with DI H20. Allow to dry.
4.
Add to any remaining reactor medium two capfuls of household bleach per liter
of reactor contents. Allow time for the reactor contents to turn white, indicating
culture death.
5.
Slowly pour dead reactor contents down the lab sink along with tap water.
6.
Rinse reactors thoroughly with soap and water while scrubbing with the bottle
brush.
7.
If any biomass remains after washing with soap and water rinse the reactor with
undiluted bleach.
8.
If any biomass remains after rinsing with undiluted bleach allow the reactor to
stand overnight in 10% vol/vol nitric acid.
9.
Neutralize the nitric acid in the reactor by slowly adding lOg sodium
bicarbonate. After addition of 1 Og allow the evolution of CO2 to cease before
adding another lOg of sodium bicarbonate. Continue until the evolution ofgas is
negligible.
10.
Allow the neutralized contents to slowly flow down the lab
sink
in with tap
water.
11.
Rinse the reactors with tap water and then DI water
12.
If the cooling jackets appear cloudy rinse the inside of the cooling jacket with
10% vol/vol nitric acid to remove the film. Neutralize the acid as previously
described and rinse the inside of the cooling jacket with DI H20.
146
13.
Allow the reactors to dry
14.
For fritted bubble columns, evenly coat the sparge fit with Rain-X. Attach the
reactor to an air source and allow the inflowing air to dry the fl-it. Rinse off the
fit with DI water. Allow reactor to dry.
15.
Reattach head plates
16.
Cover all reactor openings with tin foil and secure with autoclavable tape.
17.
Autoclave reactors at 123 °C for 30 minutes at a steam jacket pressure of 23
psig.
Sample Preparation for Scanning Electron Microscopy
Materials
A culture volume equivalent containing at least 20 mg dry cell mass, 30 mL 30%
H202 (hydrogen peroxide), Centrifuge (International Equipment Company Centra-4B
1M219 Bench Top Centrifuge) and centrifuge tubes (50 mL), 125 mL flask, Magnetic
stir plate (VWR Model 320) and magnetic stir bar, a Drying oven and drying dish
and a glass sample vial (1 mL < volume >5 mL).
Procedure
1.
Centrifuge culture at 500 x g for 10 minutes.
2. decant supernatant and suspend pellet(s) in DI H20.
3. repeat centrifuge procedure until the entire sample can be suspended in less than 5
mL of sterile seawater.
4. Add 30 mL 30% H202 to a 125 mL flask and stir over heat to 80°C.
5. Add the suspended culture to the heated H202.
6.
Allow stirring for four hours or until the liquid sample becomes clear, whichever
is longer.
7. Allow the sample to cool and add 50 mL cool DI H20.
147
8.
Repeat centrifuge procedure until the white (frustules) precipitate is collected and
suspended in less than 5 mL of DI H20.
9. Pour or pipette the sample into the drying dish and place in drying oven overnight
at 80 °C or until all liquid is gone.
10. Scrape white powder out of drying dish into sample vial, sample is placed directly
into the scanning electron microscope.
Analytical Techniques
Soluble Silicon Concentration Determination
Materials
Two 10 mL sample vials per assay, a spectrophotometer and cuvettes, syringe filters
(Pall Life Sciences Versapor membrane disc filter 3 p.m pore, 25 mm diameter cat #
28149-612), or (Cole-Parmer MFS mixed cellulose ester membrane filters, 3.0 p.m
pore size, 25 mm diameter cat # A300 A025A Lot#41ALBA), Volumetric pipette,
Sterile laminar flow hood or other source of sterile air, and a 250 mL polyethylene
bottle and 250 mL glass or Pyrex bottle.
Reagents
Hydrochloric acid solution, 50% by volume.
Ammonium molybdate reagent:
Dissolve 10 g (NIH4)6Mo7O24*4H20 in 75 mL distilled water (under warning/stirring).
Dilute to 100 mL with distilled water. Adjust pH to 7-8 with
NaOH(aq)
store in a
polyethylene bottle.
Procedure
1. Remove a 10 mL sample from the reactor according to the sampling protocol
2. Remove biomass from sample by membrane filtration via membrane filter and
membrane filter holder assembly.
3.
Collect liquid into sample vial #1 and check for complete removal of biomass. If
biomass is present (sample has turbidity) repeat step 2. Otherwise proceed.
4. Pipette 5.00 mL from sample vial #1 into sample vial #2.
148
5. To 5.00 mL sample add in rapid succession 0.100 mL 50% HC1 and 0.200 mL
ammonium molybdate reagent.
6. Mix by inversions
7. Let stand 10 minutes
8.
Place liquid sample into spectrophotometer cuvette
9. Have a calibration curve prepared by measuring the absorbance at 410 nm of
1.00, 2.00, 3.00, 4.00, 5.00, 7.00 and 10.00mg U1 soluble silicon solutions.
10. Fit the absorbance of the stock solutions to the following empirical relation,
where absorbance is measured in mAU and as,i, as1,2, and aS,3 are empirically
determined constants.
[Si]
0]2
a11 [A4 1
+ a2 [A4 1 o] +
a513
(B 1)
11. Measure the absorbance of the reactor sample and determine the concentration of
silicon in the sample by substation into equation B 1.
149
Soluble Germanium Concentration Determination
Miterici1s
Two 10 mL sample vials per assaya spectrophotometer and cuvettes, syringe filter
(Pall Life Sciences Versapor membrane disc filter 3 tm pore, 25 mm diameter cat #
28149-612), or (Cole-Panner MFS mixed cellulose ester membrane filters, 3.0 pm
pore size, 25 mm diameter cat # A300 A025A Lot#41ALBA), syringe filter holder
(VWR 25 mm cat # 28144-104), a volumetric pipette, three 500 mL glass or Pyrex
bottles, a 500 mL polyethylene bottle, a 100 mL beaker, a 500 mL flask, a 2 L beaker,
and a 2 L flask.
Reagents
Hydrochloric acid 10% (vlv) and H2SO4 25% (vlv), both stored in glass or Pyrex.
Phenylfiourone Reagent: 0.0500 g phenylfourone (2,3 ,7-trihydroxy-9-phenyl-6flourone) to 100 ml beaker, add 50 ml methanol, 1 ml HC1 (12N) stir until well
mixed. Transfer to 500 ml flask, dilute to 500 mL with methanol and thoroughly mix.
Stable for one month, store in glass or Pyrex bottle. Buffer solution (pH 5): sodium
acetate (900 g NaC2H3O2*3H20 or 540 g NaC2H3O2) into 700 ml H20 under heat.
Add to 2 L flask containing 480 mL acetic acid (12N). Cool, dilute to 2 L with DI
water, mix. Store in polyethylene bottle.
Procedure
1. A 5 mL sample is removed according to the sampling protocol.
2. Remove biomass from sample by membrane filtration via membrane filter and
membrane filter holder assembly.
3. Collect liquid into sample vial #1 and check for complete removal of biomass. If
biomass is present (sample has turbidity) repeat step 2. Otherwise proceed.
4. Pipette 1.000 mL from sample vial # 1 into sample vial # 2. The final solution
volume will be 5.000 mL.
5. To sample vial # 2 add 0.3 ml sulfuric acid solution and mix.
6. Add 0.300 ml sulfuric acid solution and mix.
7. Add 1.000 ml buffer solution.
8. Add 1.000 ml phenylfiourone reagent.
150
9. Mix and let stand for four minutes.
10. Add 1.700 ml HC1 solution and mix by inversions.
11. Place liquid sample into spectrophotometer cuvette
12. Have a calibration curve prepared by measuring the absorbance at 525 nm of
1.00, 2.00, 3.00, 4.00, 5.00, 7.00 and 10.00 mg U' soluble germanium solutions.
13. Fit the absorbance of the stock solutions to the following empirical relation where
absorbance at 525 nm is measured in mAU and aQe,1, aQe,2, and aGe,3 are
empirically determined constants.
[Ge] = aGeI [A525]2
+
aGe7
[A525]+ aGe3
(B2)
14. Measure the absorbance of the reactor sample and determine the concentration of
silicon in the sample by substation into equation B2.
151
Dry Cell Weight Determination, samples under 30 mL
Materials
Filter paper (Cole-Parmer MFS mixed cellulose ester membrane filters, 3.0tm pore
size, 25mm diameter cat # A300 A025A Lot#41ALBA) or (Pall Life Sciences
Versapor membrane disc filter 3im pore, 25mm diameter cat # 28149-612), syringe
filter holder (VWR 25mm cat # 28 144-104), 30 mL transparent sample vial, tweezers.
Procedure
1.
Weigh and record the mass of the filter to be used in the dry cell mass density
measurement.
2. Remove a 20 mL sample from the bioreactor using the sampling protocol.
3. Remove biomass from sample by membrane filtration via membrane filter and
membrane filter holder.
4. Collect liquid into sample vial and check for complete removal of biomass.
If
biomass is present (sample has turbidity) start over at step 1.
5. Remove sampling syringe from syringe filter holder and draw full of air. Reinsert
sampling syringe into syringe filter holder and press air through membrane filter
to remove any excess liquid.
6. Remove the filter from the filter holder with tweezers.
7. Place membrane filter in a drying dish and allow to dry 24 hours at 23°C.
8.
Record the dry mass of the filter.
9.
Prepare a calibration curve by weighting five dry membranes and separately
process 20 mL of pre-filtered seawater through the filter in the membrane filter
and filter holder.
10. Allow the calibration filters to dry 24 hours at 23°C in air and record the mass.
11. Record the masses and plot initial mass vs. processed and dried filter mass, Mf vs.
(Mf+ Ms). The slope is (Mf + M)IM. The salt correction factor defined as
=M1+M_1
M1
(B3)
152
was determined from the least-squares slope of the data.
12. The dry cell mass density is then determined by the equation
MDW+f+s
ADW
where
XDW
M1(1S)
(B4)
vc
is the dry cell mass density, MDW-1-fs is the mass of the filter with cell
mass cake and salts, and Vc is the culture volume used in the measurement.
153
Dry Cell Weight Determination, samples over 30 mL
Materials
Filter paper (Whatman 42 Ashless 110mm cat# 1442 110), Buchner funnel with 3 L
vacuum flask.
Procedure
13. Weigh and record the mass of the filter to be used in the dry cell mass density
measurement.
14. Remove a 200 mL reactor sample.
15. Remove biomass from sample by using the Buchner funnel and vacuum flask.
16. Inspect the filtrate to make sure no biomass is passing through the filter.
17. After all liquid is filtered through, turn off the aspirator and allow the vacuum to
reside in the vacuum flask. Remove the filter from the funnel with tweezers.
18. Place membrane filter in a drying dish and allow to dry 24 hours at 23°C in air.
19. Record the dry mass of the filter.
20. Prepare a calibration curve by weighting five dry membranes and separately
process 200 mL of pre-filtered seawater through each filter in the Buchner funnel.
21. Allow the calibration filters to dry 24 hours at 23°C in air and record the mass
22. Record the masses and plot initial mass vs. processed and dried filter mass, Mf vs.
(Mf+ Ms). The slope is (Mf + M)/Mf. The salt correction factor defined as
M +M
Sc
1
was determined from the least-squares slope of the data.
(B3)
154
23. The dry cell mass density is then determined by the equation
DW =
Mf (1
S)
(B4)
vc
where XDW is the dry cell mass density, MDW+f+s is the mass of the filter with cell
mass cake and salts, and Vc is the culture volume used in the measurement.
155
Cell Count via Hemocytometer
Materials
Para film (linilin square), a Pasteur pipette, Pasteur pipette bulb, phenosafranin
(0.33% wt.), Hemocytometer cat no. 3720, Hauser Scientific Partnership Cover slip,
bOX microscope Hand tally counter (VWR cat# 23609-102).
Procedure
1. Remove a 1 mL sample from the reactor using the sampling protocol.
2. Squeeze one drop from the sample syringe onto the Para film.
3. Add one small drop phenosafranin to Para film.
4. Touch the phenosafranin with the tip of the Pasteur pipette, allowing the
phenosafranin to enter the pipette through capillary action
5.
Inject the phenosafranin into the drop of culture, sucking it up and down until
well mixed.
6. Place two drops of culture into the central chamber of the hemocytometer and
cover the sample with a cover slip. The sample should travel out of the chamber
and under the cover slip covering the grid.
7.
Place the hemocytometer under the microscope (magnification = 430X)
8.
Each small square represents 1 .25x105 mL.
9. If the sample is particularly dense dilute the sample 2:1 or 4:1 v/v in a beaker with
5 jm filtered sterile seawater. Record the dilution rate.
10. Count the number of viable (whole green) cells in 5-10 randomly picked squares
or until a sum of 150-200 cells have been counted.
156
11. To calculate the cell densities multiply the numbers of cells counted by the
dilution factor and then divide by the number of squares times the volume
represented by each square
ND
N
HN
(B5)
where XN is the cell number density, Ds is the sample dilution, Hv, is the
hemocytorneter chamber square volume and Ns is the number of squares used in
counting the sample.
157
Appendix C: Calculations and Calibrations
158
Azam and Volcani (1974) Units Conversion
tmol Ge 108 cells' min1 to mg Ge g DCW' hr'
from figure 1 and figure 3
5nnol
108 cells
108
108
7.Spmol
mL cell Vol mm
mm
1
1.5
108 cells
mL cell Vol
cells = (0.667mL cell
cells = 0.733gFW[
1.1gFW
mL
0.38 ± 0.025gDCW
gFW
= 0.733gFW
= (0.28 ± 0.O2gDCW)
j
the conversion factor for cells to mg DCW for Azam is therefore
1fl8cp11c
O.2gDC,W
Converting Azam's
R Gc.O
(6.2,wnolGe ')( lO8cells (60min(72.64pgGe')( lmgGe_
108cellsmmn)0.28gDCW)(.\ hr ) pnolGe )1000pgGe)
96.SlmgGe
gDCW
159
Brzezinski (1985) cell silica content calculation
For Nitzschia spi
_
(0.39 pmolSi ')( icell
( lxi o nn ( 1cm3 "( lxi 0_6 janol
cell
cm
pmol )
)16801wn )
) 1,1.1gXDW )1
21 ljunol
gXFW
(
For Nitzschia sp2
(0.O6pmolSi')( lcell
cell
)2l5pn
lxl04jan")3(
)
cm
1cm3
")(1x106/nnol")
) 1.1gXDW)
pmol
2544umol
gXFW
)
Cell mass silicon concentration from data calculation:
L1
"
gSi
YXJ1JgX
imolSi
ISi
)Li09gSi)t\
a
gX
gX
1(1x1O6/nnol'\
mol
J
conversion
O.38gX
i3528pmolSi gX
gX,
gSi
1
Y,,1
160
Fresh Cell Mass to Dry Cell Mass Conversion
Fresh Cell Weight To Dry Cell Weight Conversion 5/14/2004
settled
dish plus
fresh cells
Md
(9)
Dish Mass
sample#
Md (g)
biomass mL
1
10
2
20
10
20
20
20
20
20
30
3
4
5
6
7
8
9
1.9106
1.8992
1.8724
1.8853
1.8988
1.9147
1.8842
1.9454
dish plus dry
cells Md,DW
(9)
1.9509
2.0447
2.0181
2.2314
2.0398
2.0778
2.0613
2.1667
2.1715
2.0121
2.2836
2.2266
2.9389
2.1542
2.2597
2.2731
2.4225
2.3241
1.8931
mass fresh
mass dry
)WW
cells M (g) cells M
(g) (gDw/gFw)
0.1015
0.3844
0.3542
1.0536
0.2554
0.3450
0.3889
0.4771
0.4310
0.0403
0.1455
0.1457
0.3461
0141
0.1631
0.1771
0.2213
0.2784
DW/EW 1 SE
egreon
0.3970
0.3785
0.4113
0.3285
0.5521
0.4728
0.4554
0.4638
0.6459
Fresh Cell Mass To Dry Cell Mass
0.45
0.4
0
0.35
0.3
0.25
'S
C.;
'
0.2
0.15
0.1
0.05
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Fresh Cell Mass (g FCW)
SUMMARY OUTPUT
Regression Statistics
Multiple R
0.916
R Square
0.838
Adjusted R
0.696
Standard E
0.035
Observatio
8.000
ANOVA
df
Regressior
Residual
Total
1.000
7.000
8.000
SS
0.044
0.008
0.053
Coefficientsandard Em
#N/A
0.000
XVariable
0.380
0.025
Intercept
MS
0.044
F
36.340
ignificance F
0.001
0.001
t Stat
#N/A
15.255
P-value Lower 95% Upper 95%
#N/A
#N/A
#N/A
0.000
0.321
0.439
0.38
0.025
161
filter mass calibratiou 11/1/2003
pall life sciences versapor 3000
filter
filt+salt
sample mL
0.0208
0.0207
0.0196
0.0206
0.0200
0.0235
0.0233
0.0220
0.0233
0.0228
30
30
30
30
30
0.0236
0.0234
0.0232
0.0230
0.0228
0.0226
0.0224
0.0222
0.0220
0.0218
0.0195
0.0200
0.0205
Initial Mass (g)
0.0210
162
Whatman 42 Ashless
110 mm
cat# 1442 110
initial filter mass g
0.8743
0.9184
0.8787
0.8874
0.9363
seawater sample vol mL
200
200
200
200
200
dried filter mass g.
0.91 76
0.9563
0.91 97
0.92 74
0.9891
I
0.99
0.98
0.97
0.96
0.95
U0)
0.93
0.92
0.91
0.86
0.88
0.9
Initial Mass (g)
0.92
0.94
163
7/26/2003 Si Calibration
A410
mgSi/L
1
82
89
147
1
141
2
185
193
238
0
0
2
3
240
284
297
348
353
3
4
4
5
5
7
441
436
564
563
746
740
864
869
7
10
10
15
15
20
20
25
a
20
y= 1E-05x2
0.012x-O.8681
R2 = 0.9983
15
U)
a,
.alO
0
U)
5
0
0
200
400
600
A410 (mAU)
800
1000
164
Si Assay
2/16/2004
A410
mgSi/L
6
79
77
247
246
428
428
673
6
671
o
o
2
2
4
4
6
5
-J
0)
E4
Cl)
w3
0
0
0
200
400
A410 (mAU)
600
800
165
8/17/03 Ge Assay
A525
mg GeIL
13
o
o
17
154
156
293
303
451
1
1
2
2
3
447
574
566
3
4
4
5
5
6
6
671
662
743
729
7
y = 4E-06x2 + O.005x + 0.0248
R2 = 0.9959
-J
C)
0
Q
0
C,)
I
0
200
400
A525 (mAU)
600
800
166
1/12/2004 Ge Assay
A525
mg/L
1
33
37
33
306
1
311
o
o
o
302
620
1
2
2
2
611
597
1115
3
3
3
1171
1194
3.5
y = -IE-06x2 + 0.0044x 0.1657
3
R2 = 0.999
2.5
0)
E
'
15
11
0.5
-0.5
0
1000
500
A525 (mAU)
1500
167
2/2/2004 Low Ge Assay
A525
rng Ge/L
42
42
128
o
o
0.125
0.125
122
0.25
189
162
262
259
478
481
0.25
0.5
0.5
1
1
1.2
1
0.8
-J
0)
E
0.6
I
0
200
400
A525 (mAU)
600