Zackary Johnson Department of Oceanography http://www.soest.hawaii.edu/oceanography/zij/education/ocn621 Application of Bioenergetics to Biological Oceanography Biochemical parameters indicative of stock sizes and process rates dc 1 /dt = growth Standing Stock C 1 transfer efficiency dc 2 /dt C 2 1. MEASUREMENT OF STANDING STOCKS: Carbon = the "standard unit" of measurement, basic to all organic molecules. Bulk measurements straightforward, but difficult to separate community components: "dead" carbon (detritus) >> living C [cells] biovolume / cell carbon = population carbon microscope work is tedious carbon / biovolume varies with nutritional status, taxa, etc. 1
[Chl a] C:Chl a = phytoplankton carbon C:Chl varies with nutritional state, light range 25:1 (high nutrient, low light) 200:1 moretypical50 100:1 [ATP] C:ATP = living carbon C:ATP 250:1, varies with nutritional state, taxa, etc. Other bulk measurable possible: lipopolysaccharide (LPS) bacterial cell wall component taxa specific photosynthetic accessory pigments DNA (specific) Holm Hansen 1969 L/O 14:740 747 Holm Hansen and Paerl 1972 Mem. Ist. Ital. Idrobiol. 29: 2. Measurement of Rate Processes: Isotope (Tracer) Methods: 14 C-CO 2 Primary Production 18 O-O 2 Respiration 2 P-PO 4-15 N-NH 4 + (NO - ) H-thymidine H-adenine Phosphorous uptake & cycling Nitrogen uptake & cycling Bacterial growth H-amino acids 14 C-amino acids Protein synthesis 2
2. Measurement of Rate Processes: other approaches primary production: oxygen evolution, CO 2 consumption, ΔpH, heat production, fluorescence, change in abundance secondary production: oxygen consumption, CO 2 production, ΔpH, change in abundance PP: light bottle/dark bottle O 2 evolution grazing dilution experiments growth rate dilution Biochemical Indices: enzymes can be induced by need (substrate availability) assumes energy economy in production of cell components ex. RUBISCO enzyme in photosynthetic dark reaction ETS activity: King and Packard 1975 L/O 20: 849 854 RNA:DNA ratio protein synthesis/biomass ~ growth digestive enzymes substrate specific
. 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 2 evolved/o 2 used a RQ b calories/g Carbohydrate 1.0 4.1 Protein 0.8 4.1 Lipid (Fatty Acid) 0.7 9. a relevant to carbon budgets, given respiration measured as O 2 utilization. Conversion from stoichiometry b relveant to energy budgets, derived from chemistry. Elemental composition of organic molecules: Carbon Nitrogen Phosphorous Carbohydrates 100 0 Trace Proteins 100 0 Lipids 100 Nucleic Acids 100 40 1 Redfield Ratio (C:N:P) (molar ratio) = 106:16:1 average of all living material 4. Energetic Efficiency no process can occur with 100% conservation of energy Respiration Given: Glucose yields 686 Kcal/mole 8 moles of ATP/mole glucose respired ATP phosphate bond liberates 10 kcal/mole 8 ATP 10 kcal/atp = 55% efficiency 686 Kcal/mole glucose Biosynthesis Given: Heterotroph starts with amino acids Glycine (AA) = 24 Kcal/mole Peptide linkage: cost = ATP = 0 Kcal/mole bond energy = 5.5 Kcal/mole Needed ATP is generated with 55% efficiency 24 Kcal/mole glycine 0.55 =4. 0 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% 4
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 enzymecatalyzed 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) Phytoplankton Biomass 1: Quantifying Phytoplankton Biomass a. carbon (nitrogen, phosphorous, etc.) dead carbon > live carbon what fraction is phytoplankton specific? b. counting cells (microscope, flow cytometry) 5
1/25/2008 Phytoplankton Biomass c. chlorophyll (pigments) most abundant phytoplankton pigment present in all oxygenic phytoplankton two strong absorption bands (Soret blue & Qy red band), reflected light is green strong fluorescence at ~680nm 680nm (red) strong can be measured remotely, in situ, or extracted absorption sensitive, simple, rapid fluorescence Phytoplankton Biomass c. chlorophyll pigments (cont): extracted in situ remotely sensed 6
c. chlorophyll pigments (cont): Beer Lambert law (absorption): a Phytoplankton Biomass = clε a is the absorption, c is the concentration, l is the path length, and ε is extinction coefficient rearranging: c = a lε similarly for fluorescence: ( mgchla / m = 11.85E 1.54E 0.08E ) c = fls φ 664 647 60 where fluorescence is directly proportional to c by fluorescence yield, φ for both techniques, extracted pigments (ex. acetone, methanol) work best, because they can be dissociated from contaminants or other sources of intereference and 100% of absorbed energy leads to fluorescence (vs. ~% in intact cells) in situ and remotely sensed values can be compromised from non chlorophyll contamination of the absorption or fluorescence wavebands. Phytoplankton Production 2: Quantifying Primary Production bulk properties photosynthesis: CO 2 + H 2 O CH 2 O + O 2 natural concentrations of CO 2 and O 2 in seawater: carbonate buffer system: CO 2 HCO CO 2 oxygen saturation: 0.21mM (25 o C), 0.6mM (0 o C) [TCO 2 ] 2.0mM typical range primary production values: 0.2 5 μmol CO 2 L 1 d 1 0. 6 μmol O 2 L 1 d 1 sensitivity of methods: CO 2 1μM (Coulometry) O 2 0.02μM (Microwinkler) so, direct measurement of CO 2 not sensitive enough O 2 possible, but only ~10% resolution (not good for precise comparison) 7
1/25/2008 Phytoplankton Production 2: Quantifying Primary Production: incubations with tracers photosynthesis: CO2 + H2O Æ CH2O + O2 carbon incorporation p g bottle technique q dark bottle / light 14CO2 uptake: Phytoplankton Production 2: Quantifying Primary Production (cont). carbon uptake 14CO 2 + H2O Æ 14CH2O + O2 measure radioactive decay using liquid scintillation counter counts per minute (CPM) Æ disintegrations per minute (DPM) photosynthetic rate = 1.05 (DPM sample DPM dark ) [CO2 ] DPM added time notes: 1.05 for preferential uptake of C 12 over C 14 (heavier) DPM always positive, very sensitive DPMdark measures non specific incorporation (including adsorption) incubation time period shapes whether net or gross measurement can be b d done in i situ, it simulated i l t d light li ht incubators, i b t or short h t term t photosynthetrons h t th t 8
Phytoplankton Production 2: Quantifying Primary Production (cont). oxygen evolution CO 2 + H 2 O CH 2 O + O 2 measure production of oxygen in light bottle relative to dark bottle difference is net primary production ([ O ] [ O ]) 2 ([ O ] [ O ]) net production = respiration = light 2initial time notes: gross=net+respiration can be negative low sensitivity can be done in situ incubations, simulated light incubators derivatives include stable isotopes: 2 dark 2initial time CO 2 + H 2 18 O CH 2 O + 18 O 2 used to differentiate net and gross photosynthesis 9