Microbial nutrition and growth

Chemical composition of microbial cells

 Nutrients required for the growth of a
chemioetherotrophic microorganism
Nutrients required for the growth of etherotrophic microbes:
a) water (composing 70% w/w cell weight);
b) Carbon source, which can be exclusively the source of C
(chemiolytotrophy eterotrophy; in this case energy is produced from the
oxydation of inorganic compounds) or can be also energy source (electron
donor) (chemo-organotrophy and heterotrophy). They can be organic
compounds, like sugars, proteins and lipids, organic acids, hydrocarbons,
xenobiotic compounds, etc.;
c) Nitrogen source, that might be either organic (proteins, aminoacids,
urea, amines, etc.), or inorganic, like NH4+, NO3- and N2;
d) Phosphorous source, which might be organic (phosphorganics) or
inorganic like HPO4=, H2PO4-, PO43-;
e) Sulfur source, which might be organic (sulforated organics) or
inorganic like SO4= ;
f) macro and microelements, such as vitamins, metals and mineral salts
Nutrients available in the environment
Catabolism of organic matter: aerobic respiration (a)

Cell constituents

Energy
Catabolism of organic matter: aerobic respiration (b)
GROWTH WITHOUT INHIBITION BY [S]
m
mmax
MONOD MODEL
mmax * [ S ]
mmax m=
2 Ks + [ S ]
m = specific growth rate (h-1)
mmax = max specific growth rate (h-1)
ks = k of Monod (g/l; M)
KS [S] [S] = concentration of limiting substrate (g/l;M)
GROWTH INHIBITED BY [S]

m mmax
ANDREWS –
HALDANE MODELS
mmax
2 m* * [ S ]
m=
KS + [ S ] + [ S ]2
K1
KS Smax [S]
Catabolism of xenobiotic compounds: aerobic respiration

a) MINERALIZATION (complete oxidation of carbon into CO2)

Xenobiotic compounds Intermediates CO2 + H2O
+ Cl-, N2, etc)
+ Energy

b) BIOCONVERSION (Partial oxidation of carbon)

Xenobiotic compounds Intermediates

Growth dependence on Oxygen

a) (Obligate) aerobic microbes

b) Obligate anaerobic microbes
c) Facultative aerobic microbes
d) Microaerofilic microbes
e) O2 tolerating anaerobic
microbes
 Toxicity due to Oxygen
In theory: 2e-

2H+ +1/2 O2 H2O

Under real conditions:

2e- 2e-
-

4H+ + O2 2H2O
Secondary reactions:
2e- 1e-

2H+ + O2 H2O2 O2 O2-

In aerobes and facultative anaerobes:

Superoxide
2O2 - + 2H+ dismutase H2O2 + O2
catalyse
2H2O2 2H2O + O2
Reduced peroxidase Oxidized
Organic + H2O2 organic + H2O
Compound compounds
The growth of anaerobic microbes
 Media used for growing microbes in the lab (a)
natural substrates from
animal origin BACTERIA
(meat, milk)

natural

natural substrates form vegetable

Sources YEASTS
MUDS,FUNGI
(malto, patatoes, vegetals)

Cultural Media

rich or complex BACTERIA

synthetic MUDS
YEASTS
minimum or mineral
 Media used for growing microbes in the lab (b)
growth and production of biomass

liquid

metabolic and kinetics studies

Cultural media

Isolation of pure cultures or cell

counting on plates

solid (+AGAR)
(plates,
slands) Mantainement of strains
 Pure cultures, mixed cultures and co-cultures

Pure Cultures: they are consisting of cells of the same microorganism.

They have a well defines, reproducible and predictable metabolisms and
activities, even if restricted to a limited number of substrates. They can be
easily preserved and stored.
Mixed cultures: they are consisting of cells of different microorganisms
cooperating/competing via different modes. They are not completely
defined in composition and do not have a reproducible and predictable
behavior. They however use a large number of substrates. They are not
easily preserved and stored.

Co-cultures: they are consisting of a mix of defined pure cultures. They

are completely defined in composition and might have a reproducible and
predictable behavior. They however use a number of substrates larger than
those used by the single pure cultures.
Obtainment of pure cultures from mixed ones (a)
Obtainment of pure cultures from mixed ones (b)
Obtainment of pure cultures from mixed ones (c)
Obtainment of pure cultures from mixed ones (d)
Pure culture characterization
Pure and mixed culture characterization
DNA extraction 16S rDNA amplification
and purification with labelled and non
(CsCl gradient) labelled primers (via
PCR)

Digestion of Cloning of the

the fluorescent non fluorecent
PCR product PCR product
and screening
by RFLP

Elettrophoretic
Sequencing of unique clones
separation
cgg tag cga ccg tcg agc acg tca ctg aca gct cga

T-RFLP profile

Phylogenetic
analysis
Biomass concentration measurements (a)

(Living and died

cells + particles)
Biomass concentration measurements (b)
Growth rate Parameters affecting the microbial growth: pH

pH
Parameters affecting the microbial growth:
Temperature
Parameters affecting the microbial growth: the
osmotic pressure

3 (marine 33
environment) (saturation)
(% w/v)
Chemicals affecting the microbial growth
Sterilization with heat (saturated water vapor)
Sterilization/disinfection with UV
Sterilization/disinfection with filtration
Cultivation and exploitation of microbes in
liquid cultures/bioreactors

1. BATCH CULTIVATION/BIOPROCESSING

2. CONTINUOUS CULTIVATION/BIOPROCESSING
(WITH THE RECYCLE OF BIOMASS)

3. FED-BACTH CULTIVATION//BIOPROCESSING
(REPEATED FEEDING, SBR)
Batch cultivation/processing (a)
medium
(influent)

broth
(effluent)
c
o
n
c
e
n
t
r
a
t
i
o
n
time
= [ biomass] (g/L o cells/mL)
= [substrate] (g/L)
 Batch cultivation/processing (b)
lnX

a b c d time
a lag phase
b exponential growth/log phase
c decelerating phase
d stationary phase
e died cell phase
Exponential growth phase :

dX m= specific growth rate (h-1); X= biomass conc (mg/l or CFU/ml)

=m*X
dt dX = m dt lnX = lnX0 + mt
X
 Batch cultivation/processing (c)

mmax

Ks [S]

m = mmax [S] m = specific growth rate (h-1)

ks + [S] mmax = max specific growth rate (h-1)
ks = k of Monod (g/l; M)
[S] = concentration of limiting substrate (g/l;M)
Batch cultivation/processing (d)

P
r
MAX PRODUCTIVITY
o
d
u
TOTAL PRODUCTIVITY
c
t
i
v
i
t
y
Bioreactor lag Log phase
cleaning phase
Time
Feeding,
sterilization and
inoculation
 Continuous cultivation/processing (a)

MEDIUM/influent
exausted broth/treated effluents

BATCH CULTIVATION/PROCESSING

MEDIUM/influent
exausted broth/treated effluents

CONTINUOUS CULTIVATION/PROCESSING
Continuous cultivation/processing (b)
 Continuous cultivation/processing (c)

Ks [S]
Fed-batch cultivation/processing: SBR