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Manipulating Internal Environment of Cyanobacteria for Biofuel Production

This study explores the effects of manipulating the internal environment of cyanobacteria on biofuel production, specifically focusing on nutrient deprivation and hydrogen synthesis. The hypothesis is that altering the internal conditions will change the allocation of photosynthate and the investment in energy stores, ultimately affecting biofuel production. The study aims to test this hypothesis by altering external and internal conditions and assessing the growth, photosynthesis, respiration, protein synthesis, DNA synthesis, lipid synthesis, and hydrogen synthesis of cyanobacteria.

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Manipulating Internal Environment of Cyanobacteria for Biofuel Production

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  1. Common themes • Nutrient deprivation • N and biodiesel • N and H2 production • S and biodiesel • Biotin and biodiesel • 2. Hydrogen production • N deprivation • Knock down hydrogenases • Knock up hydrogen synthases, H+ pumps • Gene knockouts • FFA recycling • H2 metabolism • N metabolism • Cell walls • Abiotic stresses • Salinity • osmotic • Temperature

  2. Common themes • Growth in different media • Differ in [N] or other nutrients • Growth in common medium, then change • Harvest, then resuspend in new media • Add something to medium • Salt • Biotin/avidin • Inducer • Inhibitor

  3. Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production

  4. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate

  5. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth

  6. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions?

  7. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth?

  8. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis?

  9. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis? • Respiration?

  10. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis? • Respiration? • Protein synthesis?

  11. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis? • Respiration? • Protein synthesis? • DNA synthesis?

  12. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis? • Respiration? • Protein synthesis? • DNA synthesis? • Lipid synthesis?

  13. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis? • Respiration? • Protein synthesis? • DNA synthesis? • Lipid synthesis? • Hydrogen synthesis?

  14. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis? • Respiration? • Protein synthesis? • DNA synthesis? • Lipid synthesis? • Hydrogen synthesis? • How to test?

  15. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis? • Respiration? • Protein synthesis? • DNA synthesis? • Lipid synthesis? • Hydrogen synthesis? • How to test? • Alter external conditions

  16. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis? • Respiration? • Protein synthesis? • DNA synthesis? • Lipid synthesis? • Hydrogen synthesis? • How to test? • Alter external conditions • Alter internal conditions by bioengineering

  17. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Will change allocation of photosynthate • Will invest their income in energy stores cf growth • Predictions? • Growth? • Photosynthesis? • Respiration? • Protein synthesis? • DNA synthesis? • Lipid synthesis? • Hydrogen synthesis? • How to test? • Alter external conditions • Alter internal conditions by bioengineering • Then measure growth, physiology and biofuels

  18. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin)

  19. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG

  20. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG • pCO2: pCO2 in air and [HCO3-] in medium

  21. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG • pCO2: pCO2 in air and [HCO3-] in medium • Temperature

  22. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG • pCO2: pCO2 in air and [HCO3-] in medium • Temperature • Light: intensity & duration

  23. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG • pCO2: pCO2 in air and [HCO3-] in medium • Temperature • Light: intensity & duration

  24. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG • pCO2: pCO2 in air and [HCO3-] in medium • Temperature • Light: intensity & duration • Turn down light reactions with atrazine

  25. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG • pCO2: pCO2 in air and [HCO3-] in medium • Temperature • Light: intensity & duration • Turn down light reactions with atrazine • Bioengineer internal changes • Nutrients (including HCO3-) by altering transporters

  26. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG • pCO2: pCO2 in air and [HCO3-] in medium • Temperature • Light: intensity & duration • Turn down light reactions with atrazine • Bioengineer internal changes • Nutrients (including HCO3-) by altering transporters • Light reactions

  27. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG • pCO2: pCO2 in air and [HCO3-] in medium • Temperature • Light: intensity & duration • Turn down light reactions with atrazine • Bioengineer internal changes • Nutrients (including HCO3-) by altering transporters • Light reactions • H2 production via N2ases, H2ases, H+ pumps, etc • Redirect photosynthate • K/O FFA recycling

  28. Hypothesis: manipulating the internal environment of • cyanobacteria will affect biofuel production • Alter external conditions • Nutrients • N, S, P, cofactors (including biotin) • Salinity/ water potential • NaClvsKClvsmannitol/sorbitol/PEG • pCO2: pCO2 in air and [HCO3-] in medium • Temperature • Light: intensity & duration • Turn down light reactions with atrazine • Bioengineer internal changes • Nutrients (including HCO3-) by altering transporters • Light reactions • H2 production via N2ases, H2ases, H+ pumps, etc • Redirect photosynthate • K/O FFA recycling • ????

  29. Suggested Game Plan • Run everything in parallel in Synechococcuselongatusand Anabaena • We have experience growing S. elongatus + expertise & materials to engineeritsgenome • Anabaena will be the exptl organism

  30. Suggested Game Plan • Run everything in parallel in Synechococcuselongatusand Anabaena • We have experience growing S. elongatus + expertise & materials to engineeritsgenome • Anabaena will be the exptl organism • 2. For environmental folks: • Grow large batches of S. elongatusand Anabaena, then subdivide into different media/ conditions

  31. Suggested Game Plan • Run everything in parallel in Synechococcuselongatusand Anabaena • We have experience growing S. elongatus + expertise & materials to engineeritsgenome • Anabaena will be the exptl organism • 2. For environmental folks: • Grow large batches of S. elongatusand Anabaena, then subdivide into different media/ conditions • At suitable intervals measure • Growth • Heterocysts • H2 production • Photosynthesis • Respiration • DNA, RNA, gene expression in general • Lipids

  32. Suggested Game Plan • 2. For environmental folks: • Grow large batches of S. elongatusand Anabaena, then subdivide into different media/ conditions • At suitable intervals measure • Growth • Heterocysts • H2 production • Photosynthesis • Respiration • DNA, RNA, gene expression in general • Lipids • 3. For gene folks • Identify genes predicted to affect biofuel production

  33. Suggested Game Plan • 2. For environmental folks: • Grow large batches of S. elongatusand Anabaena, then subdivide into different media/ conditions • At suitable intervals measure • Growth • Heterocysts • H2 production • Photosynthesis • Respiration • DNA, RNA, gene expression in general • Lipids • 3. For gene folks • Identify genes predicted to affect biofuel production • Nutrient uptake or metabolism

  34. Suggested Game Plan • 2. For environmental folks: • Grow large batches of S. elongatusand Anabaena, then subdivide into different media/ conditions • At suitable intervals measure • Growth • Heterocysts • H2 production • Photosynthesis • Respiration • DNA, RNA, gene expression in general • Lipids • 3. For gene folks • Identify genes predicted to affect biofuel production • Nutrient uptake or metabolism • Lipid unsaturation

  35. Suggested Game Plan • 2. For environmental folks: • Grow large batches of S. elongatusand Anabaena, then subdivide into different media/ conditions • At suitable intervals measure • Growth • Heterocysts • H2 production • Photosynthesis • Respiration • DNA, RNA, gene expression in general • Lipids • 3. For gene folks • Identify genes predicted to affect biofuel production • Nutrient uptake or metabolism • Lipid unsaturation • Alternate biofuels

  36. Suggested Game Plan • 3. For gene folks • Identify genes predicted to affect biofuel production • Nutrient uptake or metabolism • Lipid unsaturation • Alternate biofuels • 4. Clone and transform genes into host

  37. Suggested Game Plan • 3. For gene folks • Identify genes predicted to affect biofuel production • Nutrient uptake or metabolism • Lipid unsaturation • Alternate biofuels • 4. Clone and transform genes into host • 5. Measure effects of transgenes on physiology and biofuel production

  38. Suggested Game Plan • 3. For gene folks • Identify genes predicted to affect biofuel production • Nutrient uptake or metabolism • Lipid unsaturation • Alternate biofuels • 4. Clone and transform genes into host • 5. Measure effects of transgenes on physiology and biofuel production • Monday • Environmental folks make the various media and start growing cells

  39. Suggested Game Plan • 3. For gene folks • Identify genes predicted to affect biofuel production • Nutrient uptake or metabolism • Lipid unsaturation • Alternate biofuels • 4. Clone and transform genes into host • 5. Measure effects of transgenes on physiology and biofuel production • Monday • Environmental folks make the various media and start growing cells • Gene folks work on identifying suitable targets and devising strategies to clone them.

  40. Mineral Nutrition • Soil nutrients • Amounts & availabilityvary • Many are immobile, eg P, Fe

  41. Mineral Nutrition • Immobile nutrients must be mined • Root hairs get close • Mycorrhizae get closer

  42. Rhizosphere • Endomycorrhizae invade root cells: Vesicular/Arbuscular • Most angiosperms, especially in nutrient-poor soils • Deliver nutrients into symplast or release them when arbuscule dies • Also find bacteria, actinomycetes, protozoa associated with root surface = rhizosphere

  43. Rhizosphere • Also find bacteria, actinomycetes, protozoa associated with root surface = rhizosphere • Plants feed them lots of C! • They help make nutrients available • N-fixing bacteria supply N to many plant spp

  44. Nutrient uptake • Most nutrients are dissolved in water

  45. Nutrient uptake • Most nutrients are dissolved in water • Enter root through apoplast until hit endodermis

  46. Nutrient uptake • Most nutrients are dissolved in water • Enter root through apoplast until hit endodermis • Then must cross plasma membrane

  47. Crossing membranes • A) Diffusion through bilayer • B) Difusion through protein pore • C) Facilitated diffusion • D) Active transport • E) Bulk transport • 1) Exocytosis • 2) Endocytosis Selective Active

  48. Nutrient uptake • Then must cross plasma membrane • Gases, small uncharged & non-polar molecules diffuse

  49. Nutrient uptake • Then must cross plasma membrane • Gases, small uncharged & non-polar molecules diffuse • down their ∆ [ ] • Important for CO2, auxin & NH3 transport

  50. Nutrient uptake • Then must cross plasma membrane • Gases, small uncharged & non-polar molecules diffuse • down their ∆ [ ] • Polar chems must go through proteins!

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