Processes 2018, 6,38
Biomass to OD 730 correlation for Synechococcus 7002 was determined from biomass combined
from 50-mL shake flask cultures. Cells were harvested by centrifugation (4000 rpm, 20 min,
4 ◦ C), re-suspended in A+ media and centrifuged again, and a series of dilutions was prepared.
Three milliliters of each dilution were aliquoted into pre-dried, pre-weighed aluminum pans, dried at
80 ◦ C for 24 h, and weighed on a microbalance with accuracy to 0.001 mg (Mettler Toledo MT5).
Pans were dried and weighed again to confirm stability. The correlation curve is provided in Appendix A
(Figure A1a).
Biomass to OD 600 correlation for A. acidocaldarius was determined from biomass grown in a batch
fermentor aerated at 1 vessel volume per minute and agitated at 600 rpm. Cells were harvested by
centrifugation (6000 rpm, 5 min, 4 ◦ C), re-suspended in water and centrifuged again, and a series of
dilutions was prepared in pre-weighed 50-mL polypropylene centrifuge tubes, which had been dried
at 100 ◦ C for one week before pre-weighing. Tubes were dried at 100 ◦ C for one week and weighed on
an analytical balance with accuracy to 0.1 mg. Tubes were dried and weighed again to confirm stability.
The correlation curve is provided in Appendix A (Figure A1b).
3. Modeling Methods
A metabolic network model for A. acidocaldarius was constructed in CellNetAnalyzer [26,27]from
the annotated genome [28] with the aid of MetaCyc, KEGG, BRENDA, and NCBI [29–31] databases.
Reversible exchange reactions were defined for protons and water. Irreversible exchange reactions
were defined to permit ammonium, sulfate, oxygen, and glucose or xylose uptake and carbon dioxide
evolution, as well as secretion of possible byproducts, including acetate, lactate, ethanol, and formate.
Macromolecular synthesis reactions were defined for nucleic acids, glycogen, lipid, and protein.
Synthesis reactions utilized two phosphate bonds per nucleic acid monomer, one phosphate bond per
glycogen monomer, and four phosphate bonds per protein monomer [32]. Nucleotide distributions
were set based on percent GC content of the genome for DNA and nucleotide sequence of the rRNA
genes for RNA. Fatty acid distribution was assigned based on literature values [33,34]. The amino
acid distribution was set using the experimentally measured values in the current study. All reactions
were balanced for elements, charge, and electrons. Thermodynamic considerations were built into
the model via reaction reversibilities based on data from BRENDA [31]. Model simulations were
performed with elementary flux mode analysis. Flux vectors v satisfying the stoichiometric matrix S at
steady state (Sv=0) subject to conservation of mass, specified irreversibilities, and indecomposability
constraints were computed, resulting in the collection of minimal pathways through the network,
called elementary flux modes (EFMs) [35]. EFMs were enumerated using EFMtool [36]. Analysis of
resulting EFMs (e.g., biomass yield) was performed with MATLAB. Maintenance energy was fit to
experimental glucose and oxygen yield data for A. acidocaldarius obtained from [37]. Both growth
associated (dominant in fast-growing environmental conditions) and non-growth associated (dominant
in slow-growing environmental conditions) maintenance terms were determined. The metabolic model
with supporting details, CellNetAnalyzer metabolite and reaction input, an SBML file, and maintenance
calculations can be found in the Supplementary Materials (Files S1, S2, and S3).
4. Carbohydrate
4.1. Literature Review
Carbohydrates are common cellular energy storage molecules and constituents of cell walls.
HPLC methods can be used to separate and quantify specific sugars [38,39]; however, methods for
quantifying total carbohydrates were the focus of the current work. Chaplin [40] reviewed many
colorimetric methods for carbohydrate quantification and detailed the advantages and disadvantages
of each. The phenol sulfuric acid method [41,42] is widely used, and the L-cysteine and anthrone
methods [40,43] are also frequently found in the literature. An issue with many methods is interference
from other cellular constituents. For example, protein interferes with measured absorbance in the
157
Biomass to OD 730 correlation for Synechococcus 7002 was determined from biomass combined
from 50-mL shake flask cultures. Cells were harvested by centrifugation (4000 rpm, 20 min,
4 ◦ C), re-suspended in A+ media and centrifuged again, and a series of dilutions was prepared.
Three milliliters of each dilution were aliquoted into pre-dried, pre-weighed aluminum pans, dried at
80 ◦ C for 24 h, and weighed on a microbalance with accuracy to 0.001 mg (Mettler Toledo MT5).
Pans were dried and weighed again to confirm stability. The correlation curve is provided in Appendix A
(Figure A1a).
Biomass to OD 600 correlation for A. acidocaldarius was determined from biomass grown in a batch
fermentor aerated at 1 vessel volume per minute and agitated at 600 rpm. Cells were harvested by
centrifugation (6000 rpm, 5 min, 4 ◦ C), re-suspended in water and centrifuged again, and a series of
dilutions was prepared in pre-weighed 50-mL polypropylene centrifuge tubes, which had been dried
at 100 ◦ C for one week before pre-weighing. Tubes were dried at 100 ◦ C for one week and weighed on
an analytical balance with accuracy to 0.1 mg. Tubes were dried and weighed again to confirm stability.
The correlation curve is provided in Appendix A (Figure A1b).
3. Modeling Methods
A metabolic network model for A. acidocaldarius was constructed in CellNetAnalyzer [26,27]from
the annotated genome [28] with the aid of MetaCyc, KEGG, BRENDA, and NCBI [29–31] databases.
Reversible exchange reactions were defined for protons and water. Irreversible exchange reactions
were defined to permit ammonium, sulfate, oxygen, and glucose or xylose uptake and carbon dioxide
evolution, as well as secretion of possible byproducts, including acetate, lactate, ethanol, and formate.
Macromolecular synthesis reactions were defined for nucleic acids, glycogen, lipid, and protein.
Synthesis reactions utilized two phosphate bonds per nucleic acid monomer, one phosphate bond per
glycogen monomer, and four phosphate bonds per protein monomer [32]. Nucleotide distributions
were set based on percent GC content of the genome for DNA and nucleotide sequence of the rRNA
genes for RNA. Fatty acid distribution was assigned based on literature values [33,34]. The amino
acid distribution was set using the experimentally measured values in the current study. All reactions
were balanced for elements, charge, and electrons. Thermodynamic considerations were built into
the model via reaction reversibilities based on data from BRENDA [31]. Model simulations were
performed with elementary flux mode analysis. Flux vectors v satisfying the stoichiometric matrix S at
steady state (Sv=0) subject to conservation of mass, specified irreversibilities, and indecomposability
constraints were computed, resulting in the collection of minimal pathways through the network,
called elementary flux modes (EFMs) [35]. EFMs were enumerated using EFMtool [36]. Analysis of
resulting EFMs (e.g., biomass yield) was performed with MATLAB. Maintenance energy was fit to
experimental glucose and oxygen yield data for A. acidocaldarius obtained from [37]. Both growth
associated (dominant in fast-growing environmental conditions) and non-growth associated (dominant
in slow-growing environmental conditions) maintenance terms were determined. The metabolic model
with supporting details, CellNetAnalyzer metabolite and reaction input, an SBML file, and maintenance
calculations can be found in the Supplementary Materials (Files S1, S2, and S3).
4. Carbohydrate
4.1. Literature Review
Carbohydrates are common cellular energy storage molecules and constituents of cell walls.
HPLC methods can be used to separate and quantify specific sugars [38,39]; however, methods for
quantifying total carbohydrates were the focus of the current work. Chaplin [40] reviewed many
colorimetric methods for carbohydrate quantification and detailed the advantages and disadvantages
of each. The phenol sulfuric acid method [41,42] is widely used, and the L-cysteine and anthrone
methods [40,43] are also frequently found in the literature. An issue with many methods is interference
from other cellular constituents. For example, protein interferes with measured absorbance in the
157