Growth and Kinetics III: microbial growth and nutrition

Question 1

Nutrition in the Bacteria and the Archaea

Answer 1

• we consider the following key nutrients:
  
  • macronutrients: C, H, O, N
  • micronutrients: S, P, K, Cl, Na, Zn, Mg, Fe, Co, Cu…
    [micronutrients vary with the organism and how it is grown e.g. if you grow a methanotroph on methane it will require a lot of Cu, but grown on methanol it will need much less – see L09 Metabolism IV]
• biomass has a formula: C12H24O6N3 which is about 306.34 Da. 1 mol of biomass is 306.34 g dry material.
• sometimes this is shorthanded to [CH2O]n, meaning “carbohydrate of n repeating units” really but it’s shorthand for biomass – sometimes the formula for a hexose is used instead. These methods ignore N which is not correct.
• dead biomass is necromass and sometimes has to be considered.
• C12H24O6N3 is 47 % w/w C [do the maths and check for yourself] so we always consider C as 47 % of the dry biomass.
• only time you see variation in these numbers is 1) if the biomass isn’t properly dried or 2) there is e.g. glycogen granules present, which will raise the C, H and O but not the
  N.

Question 2

Where do they get their nutrients?
In complex media

Answer 2

C - amino acids, sugars, proteins, peptides
H - water, sugars
O - water, sugars, molecular oxygen
N - amino acids, proteins, peptides, maybe amines
S - methionine, cysteine, cystine
P - nucleotides, DNA, RNA
Fe - heme, hemin,
Cl - dissolved inorganics
Mg - dissolved inorganics
Mn - dissolved inorganics

Question 3

Where do they get their nutrients?
In defined media

Answer 3

C -
H - water etc
O - molecular oxygen etc
N - ammonium, nitrate, nitrite, cyanate, thiocyanate, amino acids
S - sulfate, thiosulfate, cysteine, methionine
P - inorganic P
Fe - Fe (II), Fe(III)/Fe EDTAate
Cl - Cl-
Mg - Mg(II)
Mn - Mn(II)/Mn EDTAate

Question 4

Metabolic modes

Answer 4

• primary metabolic modes are in the form “energy, H, carbon”
• ENERGY: photo- (electromagnetic radiation) OR chemo- (from chemical reactions)
• CARBON: auto- (CO2/DIC) OR hetero- (any C compound that is not CO2/DIC)
• e.g. most Viridiplantae are photolithoautotrophs– energy is conserved from visible light, H is from an inorganic electron donor (water) and carbon is from CO2.
• e.g. Homo sapiens subsp. sapiens L is a chemoorganoheterotroph– energy is conserved from chemical redox reactions, H is from an organic electron donor (ultimately D-(+)-glucose) and carbon is from (mostly) D-glucose.
• Thiobacillus thioparus is a chemolithoautotroph– energy is conserved from oxidation of thiosulfate to sulfate, H is from thiosulfate, carbon is from CO2.
• Escherichia coli is a chemoorganoheterotroph- energy is conserved from chemical redox reactions, H is from an organic electron donor (such as sugars
  or amino acids) and carbon is from sugars or amino acids.
  Often we will abbreviate and say “autotrophs” and “heterotrophs” in general terms.

Question 5

Key things needed for growth

Answer 5

1) an energy source – this could be electromagnetic radiation or a chemical electron donor/energy source (duel function).
2) an electron donor (if not the same as the above).
3) a terminal electron acceptor (in respiratory organisms only) e.g. O2 in aerobes, sulfate in some anaerobes…lots of them…
4) a nitrogen source (usually NH4+, NO3- or amino acids – sometimes N2 gas in diazotrophs (L12, Metabolism VII)

Question 6

Vitamins and other minor growth factors

Answer 6

• many organisms can make their own B-complex vitamins – we say they are “prototrophic for B12”, for example.
  [Gr. pref. πρωτο- (prōto-), primary; Gr. fem. n. τροφή (trophḗ), nourishment]
• if they cannot, we say they are “aux confuse with autotrophic for B12”, for example – don’t confuse with autotrophic!
  [L. ac. perf. indic. v. auxi, I augment; Gr. fem. n. τροφή (trophḗ), nourishment]
  [Gr. masc. pron. αὐτός (autós), oneself; Gr. fem. n. τροφή (trophḗ), nourishment]
• we often provide B-complex vitamins in media even for prototrophs just because it generally speeds up growth or ensures at least that the production of those vitamins doesn’t become limiting to growth.
• we often supply lipoate (cofactor for many enzymes) in vitamin solutions added to media and this does aid growth but we don’t know if it IS being taken up or if it just stabilises the true vitamins as it is an antioxidant.
• some organisms need non-vitamin micronutrients like cholesterol, hemin, menaquinone– often these are strictly anaerobic organisms, for some reason.
• some more vague ones like boiled soil extract…

Question 7

Terminology re: oxygen

Answer 7

• IF an organism uses molecular oxygen (O2) as the terminal electron acceptor for respiration, it is an aerobe/aerobic.
• IF an organism uses anything BUT molecular oxygen as the terminal electron acceptor for respiration, it is an anaerobe/anaerobic.

(even if it uses O2 for benzene oxidation in catabolism but NO3- as terminal electron acceptor, it is still an anaerobe)

• an environment (inc. flask/reactor in the laboratory) is oxic (it it has oxygen) or anoxic (if it has no oxygen). NO SUCH THING
  as “grown under anaerobic
  conditions” or “an anaerobic sediment” – they are anoxic!
• organisms that use oxygen (regardless of what for) can have different relations with it:
• if they need high/ambient pO2, aerophiles.
• if they need a zero pO2, strict anaerobes (as they are always anaerobic respirers!)
• if they need O2 but only grow at low pO2, microaerophiles (in microxic environments)

Question 8

Assimilation and dissimilation

Answer 8

• in heterotrophs, C compounds are partially assimilated and partially dissimilated but not usually directly e.g. glucose is dissimilated partially to 3 phosphoglycerate (3PGA), some of which is then dissimilated to CO2 to generate energy and some is assimilated into biomass. Refer to Krebs’ cycle and you will see where CO2 leaves and where assimilation (from intermediates) can occur.
• in autotrophs, electron donors e.g. thiosulfate are dissimilated by oxidation to e.g. sulfate (in this case), yielding electrons for growth. Electron donors in autotrophs don’t get assimilated.
• other things like N compounds, S compounds etc do get assimilated as sources for biomass production e.g. NH4+ gets assimilated into amino acids, SO42- gets assimilated into methionine and L-cysteine.
• we can also refer to these as catabolism (dissimilation) and anabolism (assimilation) although they are slightly different really.

Question 9

Theoretical yields of biomass

Answer 9

• if we know an organism grows on benzene (C6H6) using molecular oxygen for respiration and we know all other growth factors are in excess, can we predict how much biomass can be growth (as a theoretical typical amount) in a 5 L
  culture on 30 mM benzene?
  Firstly, how much benzene? 5 × 30 = 150 mmol benzene.
  Given 1 mol benzene = 6 mol C, we have 900 mmol C, or 0.9 mol C.
  Given Ar of C = 12.011 Da, we have 10.81 g C in our culture as a whole.
  Typically, 50 % of C is dissimilated to CO2 for energy, leaving 50% for
  assimilation into biomass, i.e. 5.40 g C for assimilation.
  As biomass = 47 % w/w C, 5.40/47*100 = 11.50 g dry biomass, in theory.
• in reality, the amount dissimilated can be 30-80 % normally and can be 100 % if cells are very stressed, but 50 % is a good estimate.
  [look at Boden and Murrell (2011) FEMS Microbiol Lett 324: 106-110 for how much this varies in Methylococcus capsulatus Bath using methane as the carbon
  source with and without Hg(II) ions]