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CELLULAR BIOLOGY
PERIPLASMIC
SPACE
OUTER
3 m MEMBRANE
1 m
SEX PILUS
RIBOSOMES
FLAGELLA PEPTIDOGLYCAN
CYTOPLASMIC
CHROMOSOME
INNER OR CYTOPLASMIC
COMMON PILUS MEMBRANE
10 m 20 m
CELL WALL
STARCH GRANULES
MITOCHONDRIA
PLASMA
MEMBRANE
NUCLEUS
ENDOPLASMIC CHLOROPLAST
RETICULUM
TONOPLAST
CENTRAL
VACUOLE
GOLGI
LYSOSOME COMPLEX
ROUGH
ENDOPLASMIC
RETICULUM
ANIMAL PLANT
Cell
Group structure Properties Constituent groups
Shuler, Michael L., & Fikret Kargi, Bioprocess Engineering Basic Concepts, Prentice Hall PTR, New Jersey, 1992.
Stanier, Roger; Adelberg, Edward A; Wheelis, Mark L; Decastells; Painter, Page R; Ingraham, John L; The Microbial World, 5th ed., 1986. Reprinted by permission of Pearson Education, Inc., Upper Saddle River, NJ.
BIOLOGY 91
Biochemical Catabolic Pathways
Catabolism is the breakdown of nutrients to obtain energy and precursors for biosynthesis. Carbohydrates are the most
important class of carbonaceous nutrients for fermentations, although some microbial species can also utilize amino acids,
hydrocarbons and other compounds. As an illustration of microbial diversity, almost any carbohydrate or related compound can
be fermented by some microbe.
Embden-Meyerhof-Parnas (EMP)
6
CH2 OH
5
O GLYCERALDEHYDE
H H H 3-PHOSPHATE
4
OH H
1 GLUCOSE
GLYCERALDEHYDE NAD +
HO OH 3-PHOSPHATE
DEHYDROGENASE
H
3 2
OH NADH
2
CH 2 OPO 3
ATP
HEXOKINASE HCOH 1,3-DIPHOSPHOGLYCERATE
ADP 2
C OPO 3
CH2 OPO 3 2
O
O
H H 3-PHOSPHOGLYCERATE
ADP
GLUCOSE 6-PHOSPHATE
OH H KINASE
HO OH ATP
H OH CH 2 OPO 3
2
HCOH
TAKEN TWICE FOR EACH GLUCOSE MOLECULE
PHOSPHOHEXOISOMERASE 3-PHOSPHOGLYCERATE
COO
CH2 OPO 3 2
O CH 3OH
PHOSPHOGLYCERAMUTASE
FRUCTOSE 6-PHOSPHATE
H HO
H OH CH 2 OH
OH H HCOPO 3 2 2-PHOSPHOGLYCERATE
ATP
PHOSPHOFRUCTOKINASE COO
ADP
ENOLASE
CH2 OPO 3 2 3OH H2 O
O CH 2 OPO 3
2
CH 2
FRUCTOSE 1,6-DIPHOSPHATE
H HO C O PO 3
2
H OH
OH H COO PHOSPHOENOLPYRUVATE
ADP
PYRUVATE KINASE
ALDOLASE ATP
CH 3
2
H C=O PYRUVATE
CH 2 OPO 3 C=O
TRIOSE ISOMERASE COO
C=O HCOH
2
CH 2 OH CH 2 OPO 3
DIHYDROXYACETONE GLYCERALDEHYDE
PHOSPHATE 3-PHOSPHATE
The Embden-Meyerhof-Parnas (EMP) pathway. Notice that each six-carbon glucose substrate molecule yields two
three-carbon intermediates, each of which passes through the reaction sequence on the right-hand side.
Bailey, J.E. and D.F. Olis. Biochemical Engineering Fundamentals, 2nd ed., McGraw-Hill, New York, 1986.
92 BIOLOGY
Overall Stoichiometry
C6H12O6 + Pi + 2ADP + 2NAD+ " 2C3H4O3 + 2ATP + 2 _ NADH + H+i
where
ADP is Adenosine diphosphate
ATP is Adenosine triphosphate. ADP and ATP are the primary energy carriers in biosynthesis. Each mole of ATP
carries 7.3 kcal free energy in the process of being reduced to ADP.
NAD is Nicotinamide adenine dinucleotide (+ denotes the oxidized form and NADH denotes the reduced form). The
role of this group is to serve as an electron carrier.
C3H4O3 is pyruvate, a compound of central importance in biosynthesis reactions.
The EMP pathway, among several other biochemical pathways, provides carbon skeletons for cellular biosynthesis and provides
energy sources via substrate level phosphorylation. The EMP pathway is perhaps the most common carbohydrate catabolic
pathway. Other catabolic pathways include the pentose phosphate cycle, yielding 1.67 ATP for each mole of glucose and the
Entner-Doudoroff (ED) pathway, yielding 1 ATP per mole of glucose.
Pathways are called upon by the elaborate control mechanisms within the organism depending on needed synthesis materials
and available nutrients.
H2 O2 H 2O HYDROGEN BACTERIA
H2 SO 24 H 2 O + S2 Desulfovibrio
ORGANIC COMPOUNDS O2 CO 2 + H 2 O MANY BACTERIA, ALL PLANTS AND ANIMALS
NH 3 O2 NO 2 + H 2 O NITRIFYING BACTERIA
NO 2 O2 NO 3 + H 2 O NITRIFYING BACTERIA
ORGANIC COMPOUNDS NO 3 N 2 + CO2 DENITRIFYING BACTERIA
Fe 2 + O2 Fe 3 + Ferrobacillus (iron bacteria)
2
S O2 SO 24 + H 2 O Thiobacillus (sulfur bacteria)
From W.R. Sistrom, Microbial Life, 2d ed., table 4-2, p. 53, Holt, Rinehart, and Winston, Inc. New York, 1969.
BIOLOGY 93
The most important of the respiratory cycles is the tricarboxylic acid (denoted as TCA, citric acid cycle or Krebs cycle).
The TCA cycle is shown in the figure below.
O
CH 3 CCOOH
Pyruvic acid
CoASH
2H CO2
(to NAD)
O
CH 3 C CoA
2H COOH
Acetyl-coenzyme A COOH
(to NAD)
C O HCOH
CH 2 CH 2
HSCoA H2 O
COOH COOH
COOH Oxaloacetic acid C 4 Malic acid C 4
HO C CH 2 COOH H2 O
COOH
CH 2
CH
COOH
CH
Citric acid C 6
COOH
COOH Fumaric Acid C 4
HC CH 2 COOH
2H
CHOH (to FAD)
TCA
COOH (tricarboxylic acid, COOH
Isocitric acid C 6 Krebs, citric acid)
CH 2
2H CYCLE
(to NAD) CH 2
CO2 COOH
H 2 C CH 2 COOH Succinic acid C 4
C O CoASH
COOH ATP
COOH
Ketoglutaric acid C 5
CH 2 ATP + P1
CoASH CO 2 H2 O
2H CH 2
(to NAD)
C-SCoA
O
Succinyl
CoA
Bailey, J.E. and D.F. Olis, Biochemical Engineering Fundamentals, 2nd ed., McGraw-Hill, New York, 1986.
94 BIOLOGY
Important associated respiratory reactions is the oxidative Organismal Growth in Batch Culture
phosphorylation in which ATP is regenerated.
106
Adding the EMP reactions with the TCA and oxidative
phosphorylation reactions (not shown), results in a STATIONARY
stoichiometry giving a net upper bound on ATP yield from PHASE
BACTERIAL NUMBERS
glucose in a respiring cell with glucose as the primary carbon 105 DEATH
source and oxygen as the electron donor. PHASE
The energy capture efficiency of the EMP-TCA sequence Exponential (log) growth with constant specific growth rate,
compared to the inorganic combustion reaction of glucose
(net free energy of 686 kcal/mole glucose with the negative n = b 1x l b dx l, where
dt
sign indicating energy evolved) is about 40%.
x = the cell/organism number or cell/organism concentration
t = time (hr)
Photosynthesis
= the specific growth rate (time1) while in the exponential
Photosynthesis is a most important process form synthesizing
growth phase.
glucose from carbon dioxide. It also produces oxygen. The
most important photosynthesis reaction is summarized as Logistic GrowthBatch Growth including initial into
follows. stationary phase
6CO2 + 6H2O + light " C6H12O6 + 6O2 dx = kx c1 - x m
dt x3
The light is required to be in the 400- to 700-nm range
x = x 0 _1 - e kt i, where
x
(visible light). Chlorophyll is the primary photosynthesis
3
compound and it is found in organisms ranging from tree
and plant leaves to single celled algae. The reaction above where,
requires an energy equivalent of roughly 1,968 kcal per mole k = logistic growth constant (h1),
of glucose produced. The inorganic synthesis of glucose x0 = initial concentration (g/l)
requires +686 kcal/mole glucose produced. Hence the x = carrying capacity (g/l).
efficiency of photosynthesis is roughly (686 kcal/mole glucose
inorganic)/(1,968 kcal/mole glucose photosynthesis)
686 kcal/mole or 35%.
1968 kcal/mole
Other bacterial photosynthesis reactions may occur where the
carbon source is other organic or inorganic substrates.
BIOLOGY 95
Characteristics of Selected Microbial Cells
Gram Morphological
Organism genus or type Type Metabolism1
reaction2 characteristics3
Rodmay or may not be
Escherichia Bacteria Chemoorganotroph-facultative Negative motile, variable extracellular
material
Rodmotile; significant
En t e ro ba c t e r Bacteria Chemoorganotroph-facultative Negative
extracellular material
Rodusually motile; spore;
Bacillus Bacteria Chemoorganotroph-aerobic Positive can be significant extracellular
material
Rodchainsusually
Lactobacillus Bacteria Chemoorganotroph-facultative Variable nonmotile; little extracellular
material
Coccinonmotile; moderate
Staphylococcus Bacteria Chemoorganotroph-facultative Positive
extracellular material
Chemoautotroph-aerobic; can use Short rodusually nonmotile;
Nitrobacter Bacteria Negative
nitrite as electron donor little extracellular material
Chemoorganotroph-aerobic; nitrogen Rodsmotile; copious
R hiz obium Bacteria Negative
fixing extracellular slime
Chemoorganotroph-aerobic and some
Rodsmotile; little
Pseudomonas Bacteria chemolithotroph facultative (using Negative
extracellular slime
NO3 as electron acceptor)
Rodsmotile; little
Thiobacillus Bacteria Chemoautotroph-facultative Negative
extracellular slime
Rodsusually motile; spore;
Clostridium Bacteria Chemoorganotroph-anaerobic Positive
some extracellular slime
Rods or coccimotility
Methanobacterium Bacteria Chemoautotroph-anaerobic Unknown unknown; some extracellular
slime
Rodsmotile; some
Chromatium Bacteria Photoautotroph-anaerobic N/A
extracellular material
Rod/filaments; little
Spirogyra Alga Photoautotroph-aerobic N/A
extracellular material
Filamentous fanlike or
Chemoorganotroph-aerobic and
Aspergillus Mold -- cylindrical conidia and various
facultative
spores
Usually oval but can form
Chemoorganotroph-aerobic and
Candida Ye ast -- elongated cells, mycelia, and
facultative
various spores
Spherical or ellipsoidal;
Saccharomyces Yeast Chemoorganotroph-facultative -- reproduced by budding; can
form various spores
1
Aerobic requires or can use oxygen as an electron receptor.
Facultative can vary the electron receptor from oxygen to organic materials.
Anaerobic organic or inorganics other than oxygen serve as electron acceptor.
Chemoorganotrophs derive energy and carbon from organic materials.
Chemoautotrophs derive energy from organic carbons and carbon from carbon dioxide. Some species can also derive energy from inorganic sources.
Photolithotrophs derive energy from light and carbon from CO2. May be aerobic or anaerobic.
2
Gram negative indicates a complex cell wall with a lipopolysaccharide outer layer. Gram positive indicates a less complicated cell wall with a
peptide-based outer layer.
3
Extracellular material production usually increases with reduced oxygen levels (e.g., facultative). Carbon source also affects production; extracellular
material may be polysaccharides and/or proteins; statements above are to be understood as general in nature.
Adapted from Pelczar, M.J., R.D. Reid, and E.C.S. Chan, Microbiology: Concepts and Applications, McGraw-Hill, New York, 1993.
96 BIOLOGY
Transfer Across Membrane Barriers
Mechanisms
Passive diffusion affected by lipid solubility (high solubility increases transport), molecular size (decreased with molecular
size), and ionization (decreased with ionization).
Passive diffusion is influenced by:
1. Partition coefficient (indicates lipid solubility; high lipid solubility characterizes materials that easily penetrate skin and other
membranes).
2. Molecular size is important in that small molecules tend to transport much easier than do large molecules.
3. Degree of ionization is important because, in most cases, only unionized forms of materials transport easily through
membranes. Ionization is described by the following relationships:
Acids
pKa - pH = log10 ; nonionized form E = log10 HA
ionized form A
Base
nonionized form B
Facilitated diffusion requires participation of a protein carrier molecule. This mode of transport is highly compound
dependent.
Active diffusion requires protein carrier and energy and is similarly affected by ionization and is highly compound dependent.
Other includes the specialized mechanisms occurring in lungs, liver, and spleen.
BIOLOGY 97
BIOPROCESSING
Stoichiometry of Selected Biological Systems
Aerobic Production of Biomass and a Single Extracellular Product
CNcsHm*NcsOn*Ncs + aO2 + bNH3 cCNccH*NccO*NccN*Ncc + dCNcpHx*NcpOy*NcpNz*Ncp + eH2O + fCO2
Substrate Biomass Bioproduct
where
Ncs = the number of carbons in the substrate equivalent molecule
Ncc = the number of carbons in the biomass equivalent molecule
Ncp = the number of carbons in the bioproduct equivalent molecule
Coefficients m, n, , , x, y, z as shown above are multipliers useful in energy balances. The coefficient times the respective
number of carbons is the equivalent number of constituent atoms in the representative molecule as shown in the following table.
Satisfying the carbon, nitrogen, and electron balances plus knowledge of the respiratory coefficient and a yield coefficient is
sufficient to solve for a, b, c, d, and f coefficients.
98 BIOLOGY
Composition Data for Biomass and Selected Organic Energy balance
Compounds QocbNcc = QosNcs Qo4a
Nitrogen Source
Theoretical yield
NH3
coefficientsa HNO3 N2
(shown in the
above equation)
a 2.909 3.569 3.929
b 0.48 0.288 0.144
c 0.6818 0.409 0.409
d 0.00167 0.00167 0.00167
e 4.258 4.66 4.522
f 2.997 4.197 4.917
Substrate to cells
0.5 0.3 0.3
conversion ratio
Substrate to
bioproduct 0.1 0.1 0.1
conversion ratio
Respiration
1.03 1.17 1.06
Quotient (RQ)
a
The stoichiometric coefficients represent theoretical balance conditions. B. Atkinson and F. Mavitona, Biochemical Engineering and Biotechnology Handbook, Macmillan, Inc.,
Product yields in practice are frequently lower and may be adjusted by the
1983. Used with permission of Nature Publishing Group (www.nature.com).
application of yield coefficients.
BIOLOGY 99