How to Optimize Nutrient Uptake & Increase Production

STUDY NOTES: MICROBIAL PHYSIOLOGY & BIOCHEMISTRY)

(I made these notes after I wrote the essay, while I was preparing for my exams. Interesting how half an hour of thinking can produce such enlightening points J Because Booster’s lecturing style is more of shooting question after question at his students instead of making them copy down notes, all my Physiology and Ecology notes have consequently been influenced to the extent that they are not so much notes as a discourse with myself. Sorry about that. However, I would suggest that you take a look at the questions I have asked myself, and search for your own answers)

(those of you who are interested in Industrial Microbiology, pay close attention)

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• Modes of transportation: passive diffusion, group translocation, active transport, facilitated diffusion, active transport
• Consider:

  i. The Physical Nature of Raw Material Used

  • Cells can only transport solutes (polar-non-polar): fat/water soluble; also consider size of solutes!!!
  • Also: if more than one substrate/nutrient → will they react to form complexes that cannot be utilized? Or change reaction?

  ii. Type of Raw Material Used

  • Aside from carbon source & substrates, might the microbes need other nutrients, microelements for activators etc?
  • Carbon source must be suitable for utilization
  • Energy yield of substrates?
  • Reducing potential of carbon sources: anaerobes require low redox potential sources

  iii. Modes of Transportation

  • Usually small non-polar, fat-soluble molecules: passive diffusion; amino acids and organic acids: active diffusion
  • One transport system serves many substrates, one substrate may enter cell in more than one way
  • Therefore choose substrate that can enter cell in more than one way

  iv. The Quantity of Substrate

• By increasing substrate can production be increased?
• Assuming SUBSTRATE → PRODUCT process involves one enzyme, substrate quantity can only be increased to a certain level because:
  1. production will only increase up to enzyme saturation point; will go no higher after that
  2. besides substrate used for productions, microbes need other nutrients (to increase substrate means to decrease other nutrients. Or would you have the microbes smother to death???)
  3. increasing the substrate makes the liquid more viscous, therefore less oxygen can diffuse in (will affect microbial respiration unless it’s an anaerobe); also viscosity affects bacterial motion/movement. (but more substrate also means that the microbes will not have to travel such great distances to get food! Therefore minimizes distance, maximizes collision and uptake)
  4. water concentration gradient: osmosis will occur → plasmolysis of cell if liquid is too hypertonic
  5. increasing substrates also means decreasing number of microbes. (or have you forgotten that the holding tank has a finite volume??)

v. The Quantity of Microbes

• Will increasing microbial number increase production???
• Rate of production will remain the same, only more microbes to do the work
• In industry, microbial number limited to about 10% because increasing microbe numbers means decreasing quantity of substrate)
• Must also consider the fact that microbes will multiply and increase viscosity of liquid! (pseudoviscosity)

vi. Environmental Parameters

• To optimize or not? That is the question!
• Optimization:
  1. pH of transport system
     • if it is valid that permease enzymes are involved in facilitated diffusion!
     • Increasing nutrient uptake by ensuring that pH of substance is optimum for transport system involved
     • pH will change as microbes metabolize substrate: must regulate (different pH may result in failure of transport system, cell lysis due to collapse of membrane, activation of different enzymes leading to different metabolic processes etc)
  2. temperature
     • think of the kinetic theory & Brownian movement!
     • Temperature increase → Brownian movement increases in velocity à more frequent collisions between microbes and substrate
     • But at high-speed collisions, will cells be damaged?
     • Proteins denature at > 40ºC! (transport system --> enzymes!)
     • Association/dissociation of substrates at higher temperature?
• The question is: is the optimal condition for vegetative growth optimal for product formation??? Eg. In aerobic conditions, yeast will carry out TCA cycle; only in anaerobic conditions will they carry out fermentation of pyruvate!
• What about torturing the microbes? (shut up, you microbial activists. I know what I’m saying) since under stressed conditions, bacterial metabolism is higher (see biofilms)
• But it may be damaging to microbial cells in the long run (may affect physiology: growth may be halted)
• Anyway, will products be the desired ones?
• What if the microbes become dormant? What happens then?

vii. Quality of Microbes

• Strains used must be stable i.e. low mutation frequency; recombination will not result in strains carrying out different metabolism or metabolism of subtrates to unwanted products
• Eg. Usage of petite mutants (yeast): cannot carry out TCA cycle!
• Culture must be free of contaminants (i.e. other undesirable strains)
• Assuming one gene codes for one enzyme, can production be increased by amplifying the gene? This means that the microbial genome will increase à will result in genetic instability and short life-span even though quantity of enzymes might increase production. Therefore, is it economical in the long run?
• Properties of a useful industrial microorganism:
  1. must be available in pure culture
  2. must be genetically stable
  3. must grow in large-scale culture
  4. possible to maintain cultures for a long period of time in the lab and industrial plants
  5. can be easily inoculated (produce spores or other reproduction cell forms)
  6. grow rapidly, produce desired products in relatively short period of time
  7. able to grow in relatively inexpensive culture medial obtainable in bulk quantity
  8. should not be harmful to humans, economically important to animals/plants
  9. possible to remove microbes from culture medium relatively easily
  10. amenable to genetic manipulation

viii. Reactions Involved

• Will microbes produce other metabolites in addition to desired products? Can these two be separated? (eg. Mixed acid fermentations)
• If desired product is produced thus: A → B → C → D → E → F whereby C is the desired product, therefore reaction must be stopped at B à C
• If F is needed for microbial growth, D must be supplied etc
• What if C, E or F are toxic to the microbial cell? There must be a way to remove them or alter their physical properties so that they don’t affect the microbial cell
• What kind of reaction is involved? Aerobic or anaerobic? If aerobic, concentration of substrates and microbes will affect amount of oxygen diffusing into the solution

ix. Others

• Hyperbaric pressure: what effect does it have upon microbes at the bottom of the fermenter? Will it affect their metabolic pathways? Stress?
• To sterilize or not to sterilize substrate? If not, there may be contaminants. But if so, might the procedure (eg. Autoclaving) change the structure of the substrate?