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OCN621: Biological Oceanography-Bioenergetics-III

Guangyi WangGua gy a gPOST 103B

guangyi@hawaii.edu

http://www.soest.hawaii.edu/oceanography/zij/education/ocn621/

dC1/dt = growth

StandingStock

C1

Overview

1.Energy “Currency” ATP1) Active transport & ATP2) Mechanical work & ATP 3) Bi th i & ATP3) Biosynthesis & ATP

2.Application of Bioenergetics to Biological Oceanography1) Measurement of standing stocks (methods)2) Measurement of rate processes3) Relationship among cell constituents4) E ti ffi i4) Energetic efficiency5) Energy implications of adaptations

3. Assignment question1) Link bioenergetics to physical and biogeochemical

oceanographic processes????

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Biological Utilization of Chemical Energy1. Energy “Currency” ATP - Economic analogy for the transformation of energy in the cell

- need for a "medium of exchange". Most biochemical reaction series requires elaborate cell machinery and organization, and many specific enzymes. It is not efficient, and not possible, for enzyme complexes to handle all possible combinations of substrates, intermediates, and sources of energy. METABOLIC processes (e.g, respiration) "oxidize" organic molecules, capturing some of their energy in a single molecule which is recognized as the "energy donor" or "medium of exchange" in all subsequent reactions. This molecule, ATP, is special because it carries a fixed amount of energy in an easily released form – high energy phosphate bonds.

2. Active Transport: work of moving molecules &ions against concentration gradientsAccording to the 2nd Law of Thermodynamics - everything in universe tends toward increased entropy (randomness). Therefore, energy must be expended to bring things (e.g, molecules) into a more organized and concentrated state.

Functions of active transport:1. Provides proper chemical environment for cellular processes (e.g., pH).2. Brings needed substrates (glucose, amino acids) &essential minerals (nitrate, phosphate, & important ions K+ and Ca++) where they are needed.3. Gets rid of waste products (H+, Na+ , C02, lactic acid).

Characteristics of active transport:1. A given systems is specific for a particular molecule or ion.2. Transport occurs in a specific direction across membrane. Transport is accomplished by enzymes at "active sites" i e substrate specific with specific orientationby enzymes at active sites - i.e., substrate specific with specific orientation (directionality) in cell membrane matrix.3. Powered by ATP molecules which "fit" into an active sites and donates high energy phosphate bonds to the process.4. Works against continuous back diffusion (which occurs at a slower rate because it is not enzyme aided). Active transport is generally a continuous process in living cells, concentrations on either side of the membrane are maintained in "dynamic equilibrium"

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2. Active Transport (cont.)

3. Mechanical: work associated with contractionsFunctions of contractions:1. Locomotion2. Organs (intestines, heart, liver, brain)3. Cell division4. Subcellular movements in membranes of organelles

Ciliar and flagellar motion results from the coordinated sliding of outer doublet microtubules relative to their neighbors, driven by ATP.

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4. Biosynthesis: creation of complex organic molecules from simple precursors, minerals, and nutrients. Synthesis is a continuous active process because molecules are constantly breaking down and need to be replaced ('Homeostasis" = maintenance). Mechanism:Biosynthetic reactions involve enzymes (proteins) – in which terminal phosphates of ATP are used directly or indirectly to activate building-block molecules so that theyATP are used, directly or indirectly, to activate building block molecules so that they react with other building blocks in an energetically favorable manner.

IMPORTANT MACROMOLECULES - precursors & functions

Precursors Macromolecule Function

Amino Acids Proteins Structure – membranesSynthesis – enzymesActive Transport – enzymesContraction - actomyosin

Monosaccharides (sugars) Polysaccharides Structure membranes cellulose (plant) chitin (animals)Monosaccharides (sugars) PolysaccharidesCarbohydrates

Structure – membranes, cellulose (plant), chitin (animals)Storage - starch (plants), glycogen (animals)

Fatty Acids Lipids Structure – membranesStorage - fats, oils, waxesLight absorption – pigmentsGrowth regulation - hormones

Mononucleotides DNA & RNA Genetic info.- DNAProtein synthesis - RNA

ATP pool (E. coli) = 1,000,000 moleculesATP utilization rate = 2,500,000 molecules/secTurnover rate of ATP pool = 0.4 sec

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Bacteria invest a large fraction of their energetic budget in proteins. Relative to some eukaryotes, E. coli is a simple organism with an extreme life-history strategy - to reproduce as fast as possible. In more complex organisms, we expect more of an energetic investment in "adaptations":

a. differentiation and specialization of tissues and appendagesb. metabolic or reproductive storage products

Greater specialization and complexity brings the need for more information (organization). Biosynthesis is not simply the process of building molecules, but involves the organization of molecules into a hierarchy of increasing complexity:

-ORGANISM-TISSUES & ORGANS-CELLS-ORGANELLES (chloroplasts, mitochondria, nuclei)-SUBCELLULAR COMPLEXES (membranes, enzyme systems)-MACROMOLECULES (proteins, carbohydrates, lipids, nucleic acids)-BUILDING BLOCKS (sugars, amino acids, fatty acids, mononucleotides)-INORGANIC PRECURSORS (CO2, H2O, NH4)

Application of Bioenergetics to Biological Oceanography

Biochemical parameters indicative of stock sizes and process rates

dC1/dt = growthtransfer dC /dt

1. MEASUREMENT OF STANDING STOCKS:Carbon = the "standard unit" of measurement, basic to all organic molecules.Bulk measurements straightforward but difficult to separate community

C2

StandingStock

C1

transferefficiency

dC2/dt

Bulk measurements straightforward, but difficult to separate communitycomponents:

"dead" carbon (detritus) >> living C

[cells] × biovolume / cell × carbon = population carbon

●microscope work is tedious●carbon / biovolume varies with nutritional status, taxa, etc.

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[Chl a] • C:Chl a = phytoplankton carbon• C:Chl varies with nutritional state, light• range 25:1 (high nutrient, low light) - 200:1• more typical 50 -100:1

[ATP] • C:ATP = living carbon• C:ATP ≈ 250:1, varies with nutritional state, taxa, etc.C:ATP 250:1, varies with nutritional state, taxa, etc.

Other bulk measurable possible:lipopolysaccharide (LPS) – bacterial cell wall componenttaxa-specific photosynthetic accessory pigmentsDNA (specific)

Holm-Hansen 1969L/O 14:740-747

Holm-Hansen and Paerl 1972Mem. Ist. Ital. Idrobiol. 29:

Flow Cytometry: another approach to quantify biomass is to count cells (and biomass)

(A) Measurement Parameters1. LFALS: Log Forward Angle Light Scatter - cell size proxy2. L90LS: Log 90° Light Scatter - cell size proxyg g p y3. LIBFL: Log Integrated Blue Fluorescence - DNA content (Hoechst stain)4. LIOFL: Log Integrated Orange Fluorescence - phycoerythrin content (cyanobacteria)5. LIRFL: Log Integrated Red Fluorescence - chlorophyll content (photoautotrophs)

(B) Applications1. Cell enumeration

heterotrophic bacteriacyanobacteriaprochlorophytes

h t th ti i k t ( 3 )photosynthetic picoeukaryotes (< 3um)photosynthetic nanoeukaryotes (3-20 um)

2. Cell sorting: group-specific 1° production3. Prochlorococcus growth rates (cell cycle)4. Cell Abundance (cell-specific probes)

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Flow Cytometry Principles of Operation:

2. Measurement of Rate Processes:Isotope (Tracer) Methods:

14C-CO218O-O2

Primary ProductionRespiration

32P-PO43-

15N-NH4+ (NO3

-)Phosphorous uptake & cyclingNitrogen uptake & cycling

3H-thymidine3H adenine

Bacterial growth3H-adenine3H-amino acids14C-amino acids

Protein synthesis

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2. Measurement of Rate Processes:other approaches

primary production: oxygen evolution, CO2 consumption, ΔpH, heat production, fluorescence, change in abundance

secondary production: oxygen consumption, CO2 production, ΔpH, change in abundance

PP: light bottle/dark bottle O2 evolution grazing -“dilution experiments”

h ra

tedilution

grow

th

Biochemical Indices:enzymes can be induced by need (substrate availability)assumes “energy economy” in production of cell componentsex. ●RUBISCO – enzyme in photosynthetic dark reaction

●ETS activity:

King and Packard 1975L/O 20: 849-854

●RNA:DNA ratio – protein synthesis/biomass ~ growth●digestive enzymes – substrate specific

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3. Relationship among cell constituents:Carbon ≠ energy content - Lipid (fat) has greater than twice the energy content per carbon molecule (or per unit mass) than protein or carbohydrate.ATP ≠ energy content - ATP content is the pool of immediately available energy. The process of energy utilization can be assessed by the turnover rate of the ATP pool.Respiratory Quotient = RQ = CO evolved/O usedRespiratory Quotient = RQ = CO2 evolved/O2 used

aRQ bcalories/g

Carbohydrate 1.0 4.1

Protein 0.8 4.1

Lipid (Fatty Acid) 0.7 9.3arelevant to carbon budgets, given respiration measured as O2 utilization. Conversion from stoichiometrybrelveant to energy budgets, derived from chemistry.

Elemental composition of organic molecules:Carbon Nitrogen PhosphorousCarbon Nitrogen Phosphorous

Carbohydrates 100 0 Trace

Proteins 100 33 0

Lipids 100 3 3

Nucleic Acids 100 40 1

Redfield Ratio (C:N:P) (molar ratio) = 106:16:1 average of all living material

4. Energetic Efficiencyno process can occur with 100% conservation of energy

RespirationGiven: Glucose yields 686 Kcal/mole

38 moles of ATP/mole glucose respiredATP-phosphate bond liberates ≈ 10 kcal/mole

38 ATP • 10 kcal/ATP = 55% efficiency38 ATP • 10 kcal/ATP = 55% efficiency686 Kcal/mole glucose

BiosynthesisGiven: Heterotroph starts with amino acidsGlycine (AA) = 234 Kcal/molePeptide linkage: cost = 3 ATP = 30 Kcal/mole

bond energy = 5.5 Kcal/moleNeeded ATP is generated with ≈55% efficiencyNeeded ATP is generated with 55% efficiency

234 Kcal/mole glycine • 0.55 =4.330 Kcal/mole peptide bond

About one out of every 5 assimilated glycine molecules must be fully oxidized in respiration to provide enough energy to link the other 4 molecules with peptide bonds.

THEORETICAL MAXIMUM EFFICIENCY: All life processes ≈70%

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5. Energetic Implications of Adaptations

Short-term - Within physiological limits, organisms adjust biochemical systems to the external environment in a manner that promotes energetic efficiency (e.g., enzymes, C:Chl).Long-term (evolutionary) - (morphological specializations, physiological tolerances). Additional capabilities and structures have energetic costs that must be offset, in the long term, by energetic advantages (e.g., homeotherm metabolism).

EXAMPLE: TEMPERATURE TOLERANCE Temperature enhances all enzyme-catalyzed biochemical reactions. The enzyme systems for different species are adapted to specific temperature ranges.

Growth vs. temperature for 5 unicellular algae (Eppley 1972 Fish. Bull. 70)