The document discusses microbial nutrition and the requirements for various nutrients including carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur. It describes the different types of microorganisms based on their carbon and energy sources, including heterotrophs, autotrophs, phototrophs, and chemotrophs. It also discusses uptake of nutrients via passive diffusion, facilitated diffusion, and active transport mechanisms. Culture media types and compositions are outlined.

1. Microbial
Nutrition
SBT 1313 Fundamental of
Microbiology
1

2. 2
Outlin
e
• Introduction
• Requirements for carbon, hydrogen, oxygen and
electrons
• Nutritional types of microorganisms
• Requirements for nitrogen, phosphorus and sulfur
• Growth factors
• Uptake of nutrients by the cell
• Culture media
• Isolation of pure culture

3. 3
Introductio
n

4. 4
• Microbial cells need supply of raw materials or nutrients
and energy to function.
• Nutrients are substances used in biosynthesis and energy
release – require for microbial growth.
Microbial
nutrition

5. Macroelements/
macronutrients
• Carbon
(C
)
• Oxygen
(O
)
• Hydrogen
(H
)
• Nitrogen
(N
)
• Phosphorus
(P)
• Potassium
(K+
)
• Calcium (Ca2+)
• Magnesium(Mg2+)
• Iron (Fe2+,
Fe3+)
The common nutrient
requirements
Components of carbohydrates,
lipids, proteins, and nucleic acids
As cations
– parts of enzymes and
cofactors
5

6. 6
Microelements/
micronutrients
• Manganese
• Zinc
• Cobalt
• Molybdenu
m
• Nickel
• Copper
(Mn2+
)
(Zn2+)
(Co2+)
(Mo2+
)
(Ni2+)
(Cu2+)
• Normally a part of enzymes and cofactors, and aid in the
catalysis of reactions and maintenance of protein structure.
The common nutrient
requirements

7. • Some microorganism need specific elements base on their
specific morphology or environment.
– E.g. Diatom need silicic acid (H4SiO4) to construct their
cell wall of silica [(SiO2)n].
• Balanced mixture of nutrients is essential.
The common nutrient
requirements
7

8. 8
Requirements for carbon,
hydrogen, oxygen and
electrons

9. 9
Requirements for carbon,
hydrogen, oxygen and
electrons
• C is needed for the skeletons or backbones of all organic
molecules.
• H and O are important elements found in organic molecules.
• Electrons are needed:
– To reduce molecules during biosynthesis (e.g. the
reduction of CO2 to form organic molecules), and
– To provide energy through oxidation-reduction reactions.

10. 10
Heterotroph
s
• Organisms use organic molecules as their carbon source.
• Organic molecules also supply hydrogen, oxygen, and
electrons.
– Electrons from organic C sources can be used in electron
transport and oxidation-reduction reactions  energy source.
– The more reduced the organic C source (i.e. the more
electrons it carries), the higher its energy content.
– Lipids have a higher energy content than carbohydrates.
• Exception: CO2 supplies only carbon and oxygen, cannot be
used as a source of hydrogen, electrons, or energy.
– Lacks hydrogen, and is unable to donate electrons
during oxidation-reduction reactions.

11. 11
Autotroph
s
• Organisms that use CO2 as their sole or principle carbon
source.
• CO2 cannot supply energy – only supply carbon and
oxygen.
• Must obtain energy from other sources such as light and
reduced inorganic molecules.

12. 12
Nutritional types of
microorganisms

13. Table5.1 Sources of Carbon,Energy,and Electrons
Carbon Sources
Autotrophs CO2 sole or principal biosynthetic carbon
source (section 10.3)
Heterotrophs Reduced, preformed, organic molecules from
other organisms (chapters 9 and 10)
Energy Sources
Phototrophs Light (section 9.12)
Chemotrophs Oxidation of organic or inorganic compounds
(chapter 9)
Electron Sources
Lithotrophs Reduced inorganic molecules (section 9.11)
Organotrophs Organic molecules (chapter 9)
13

14. Table5.2 Major Nutritional Types of Microorganisms
Nutritional Type Carbon Source Energy Source Electron Source
Representative
Microorganisms
Photolithoautotrophy
(photolithotrophic
autotrophy)
CO2 Light Inorganic e—
donor Purple and green sulfur bacteria,
cyanobacteria
Photoorganoheterotrophy
(photoorganotrophic
heterotrophy)
Organic carbon,
but CO2 may also
be used
Light Organic e—
donor Purple nonsulfur bacteria, green
nonsulfur bacteria
Chemolithoautotrophy
(chemolithotrophic
autotrophy)
CO2 Inorganic chemicals Inorganic e—
donor Sulfur-oxidizing bacteria,
hydrogen-oxidizing bacteria,
methanogens, nitrifying
bacteria, iron-oxidizing
bacteria
Chemolithoheterotrophy
or mixotrophy
(chemolithotrophic
heterotrophy)
Organic carbon,
but CO2 may also
be used
Inorganic chemicals Inorganic e—
donor Some sulfur-oxidizing bacteria
(e.g., Beggiatoa)
Chemoorganoheterotrophy
(chemoorganotrophic
heterotrophy)
Organic carbon Organic chemicals
often same as C
source
Organic e—
donor,
often same as C
source
Most nonphotosynthetic
microbes, including most
pathogens, fungi, many
protists, and many archaea
14
Energy  electron 
carbon

15. 1
(a) Bloom of cyanobacteria (photolithoautotrophic bacteria) (b) Purple sulfur bacteria (photoheterotrophs)
Internal membrane system
used for oxidation of nitrite
0.25 U
(a) Nitrobacter winogradskyi, a chemolithoautotroph
Sulfur granule within filaments
(b) Beggiatoa alba, a chemolithoheterotroph (mixotroph)

16. 16
Nutritional type of
microorganisms
• Although a particular species usually belongs in only one of
the nutritional classes, some show great metabolic flexibility
and alter their metabolic patterns in response to
environmental changes.
• E.g. many purple nonsulfur bacteria
– In the absence of oxygen – photoorganotrophic
heterotrophs.
– At normal oxygen level – oxidize organic molecules and
function chemoorganotrophically.
– When oxygen is low – phototrophic and
chemoorganotrophic metabolism function
simultaneously.

17. 17
Requirements for
nitrogen, phosphorus
and sulfur

18. 18
• N, P and S may be obtained from the same organic molecules
that supply C, from the direct incorporation of NH3 and PO4
3-,
and by the reduction and assimilation of oxidized inorganic
molecules.
• N is needed for the synthesis of amino acids, purines,
pyrimidines, some carbohydrates and lipids, enzyme
cofactors etc.
• Some bacteria (e.g. cyanobacterium and Rhizobium) can
assimilate atmospheric nitrogen (N2) by reducing it to
ammonium (NH4
+)  nitrogen fixation.
Requirements for
nitrogen

19. 19
• P is present in nucleic acids, phospholipids, nucleotides
(ATP), several cofactors, some proteins, and other cell
components.
• Almost all microorganism use inorganic PO4
3- as their P
source.
• Low PO4
3- levels limit microbial growth in many
aquatic environment.
Requirements for
phosphorus

20. 20
• S is needed for synthesis of substances like the amino acids
cysteine and methionine, some carbohydrates, biotin and
thiamine.
• Most microorganism use SO4
2- as a source of S and
reduce it by assimilatory SO4
2- reduction.
• A few microorganisms require an organic form of S
such as cysteine.
Requirements for
sulfur

21. 21
Growth
factors

22. 22
Growth
factors
• Organic compound that are essential cell components or
precursors of such components but cannot be synthesized
by the organism are called growth factors.
There are 3 major classes of growth factors:
• amino acids
• purines and pyrimidines
• vitamin

23. 23
Growth
factors
• Amino acids are needed for protein synthesis.
• Purine and pyrimidines for nucleic acid synthesis.
• Vitamins are small organic molecules that usually make up of
all or part of enzyme cofactors, and are needed in only small
amount to sustain growth.

24. 2
Table 5.3 Functions of Some Common Vitamins in Microorganisms
Vitamin Functions Examples of Microorganisms Requiring Vitamina
Biotin Carboxylation (CO2 fixation) Leuconostoc mesenteroides (B)
One-carbon metabolism Saccharomyces cerevisiae (F)
Ochromonas malhamensis (P)
Acanthamoeba castellanii (P)
Cyanocobalamin (B12) Molecular rearrangements Lactobacillus spp. (B)
One-carbon metabolism—carries methyl groups Euglena gracilis (P)
Diatoms (P)
Acanthamoeba castellanii (P)
Folic acid One-carbon metabolism Enterococcus faecalis (B)
Tetrahymena pyriformis (P)
Lipoic acid Transfer of acyl groups Lactobacillus casei (B)
Tetrahymena spp. (P)
Pantothenic acid Precursor of coenzyme A—carries acyl groups Proteus morganii (B)
(pyruvate oxidation, fatty acid metabolism) Hanseniaspora spp. (F)
Paramecium spp. (P)
Pyridoxine (B6) Amino acid metabolism (e.g., transamination) Lactobacillus spp. (B)
Tetrahymena pyriformis (P)
Niacin (nicotinic acid) Precursor of NAD and NADP—carry electrons Brucella abortus, Haemophilus influenzae (B)
and hydrogen atoms Blastocladia pringsheimii (F)
Crithidia fasciculata (P)
Riboflavin (B2) Precursor of FAD and FMN—carry electrons Caulobacter vibrioides (B)
or hydrogen atoms Dictyostelium spp. (P)
Tetrahymena pyriformis (P)
Thiamine (B1) Aldehyde group transfer
(pyruvate decarboxylation, a-keto acid oxidation)
Bacillus anthracis (B)
Phycomyces blakesleeanus (F)
Ochromonas malhamensis (P)
Colpidium campylum (P)
a
The representative microorganisms are members of the following groups: Bacteria (B), Fungi (F), and protists (P).

25. 25
Growth
factors
• Understanding the growth factor requirements of microbes
has important practical applications.
• Ideally, the amount of growth is directly proportional to
the amount of growth factor – if the growth factor
concentration doubles the amount of microbial growth
doubles.
• Microbes that are able to synthesize large quantities of
vitamins can be used to manufacture these compounds
for human use.
– Riboflavin – Clostridium, Candida, Ashbya, Eremothecium
– Coenzyme A – Brevibacterium
– Vitamin B12 – Streptomyces, Propionibacterium, Pseudomonas
– Vitamin C – Gluconobacter, Erwinia, Corynebacterium

26. 26
Uptake of nutrients by the
cell

27. 27
Passive
diffusion
• Often called diffusion or simple diffusion.
• Molecules move from a region of higher concentration to
one of lower concentration.
• The rate is dependent on the size of the concentration
gradient between the cell exterior and its interior.
• A fairly large concentration gradient is required for
adequate nutrient uptake
– The external nutrient concentration must be high while
the internal concentration is low.
• The rate decreases as more nutrient is acquired unless
is used immediately.
• Very small molecules – H2O, O2, CO2.

28. Facilitated
diffusion
• Diffusion involving carrier proteins.
• The rate of diffusion across selectively permeable membranes
is greatly increased by using carrier proteins, called
permeases, which are embedded in the plasma membrane.
• The rate of facilitated diffusion increases with the
concentration gradient much more rapidly and at lower
concentrations of the diffusing molecule than that of
passive diffusion.
28

29. Facilitated
diffusion
Concentration gradient 29
Passive
diffusion
Rate
of
transport

30. 30
Facilitated
diffusion
• The diffusion rate levels off or reaches a plateau above a
specific gradient value because the carrier is saturated.
– The carrier protein is binding and transporting as many
solute molecules as possible.
• Carrier proteins are selective.
• Facilitated diffusion is truly diffusion, no energy involved.
• A concentration gradient drives the movement of
molecules.

31. 31
Facilitated
diffusion
• Some permeases are related to the major intrinsic protein
(MIP) family of proteins.
– Aquaporins – transport water.
– Glycerols facilitators – aid glycerol diffusion.
• Is not the major uptake mechanism because nutrient
concentration often are lower outside the cell.
• Is much more prominent in eucaryotic cells – transport
sugars and amino acids.

32. Outside
cell
32
Inside
cell
Outside
cell
Inside
cell

33. 33
Active
transport
• Move solutes against a concentration gradient.
• Energy-dependent processes.
• Definition – transport of solute molecules to higher
concentrations or against a concentration gradient with the
input of energy.
• Permeases bind particular solutes with great specificity.
• Example of active transport system: ATP-binding
cassette transporters (ABC transporters).
– Important primary active transporters.
– Observed in bacteria, archea and eucaryotes.
– Uniport transporters.

34. 1 2
Solute-
binding
protein Transporter
Nucleotide-
binding
domain
Cytoplasmic
matrix
Periplasm
ATP ADP
+
Pi
ATP ADP
+
Pi
34

35. 35

36. 36
Group
translocation
• Another type of energy-dependent transport.
• Chemically modify the molecule as it is brought into
the cell (chemotaxis).
• Example – phosphoenolpyruvate: sugar phosphotransferase
system (PTS).
• PTS transport a variety of sugars while phosphorylating them
using phosphoenolpyruvate (PEP) as the phosphate donor.
• PEP + sugar (outside)  pyruvate + sugar-phosphate (inside)

37. Mannitol-1-P
37
Glucose-6-P
Pyruvate EI~ P HPr
Cytoplasmic
matrix
HPr~ P
Periplasm
Glucose
Mannitol
EI
PEP
IIB
P
IIA IIC
IIA IIC
P
P
IIB
P

38. 38
PT
Ss
• Widely distributed in bacteria: facultative anaerobic bacteria
(e.g. Escherichia, Salmonella, Staphylococcus), obligate
anaerobic bacteria (e.g. Clostridium). Most aerobic bacteria
lack PTSs.
• Transport many carbohydrates.
– E. coli takes up glucose, fructose, mannitol,
sucrose, N- acetylglucosamine, and cellobiose.

39. 39
Culture
media

40. 40
Culture
media
• Growing microorganism is important for further study.
• A solid or liquid preparation used to grow, transport, and
store microorganisms.
• Effective media contains all nutrients needed by the
microorganism for growth.
• Specialized media are essential in the isolation and
identification of microorganisms, the testing of antibiotic
sensitivities, water and food analysis, industrial microbiology,
and other activities.
• The precise composition of a satisfactory medium depend
on the species – nutritional requirement vary.

41. Classification of culture
media
Table5.4 Types of Media
Physica
l
Nature
Chemical
Composition Functional Type
Liquid
Semisolid
Solid
Defined (synthetic)
Complex
Supportive (general purpose)
Enriched
Selective
Differential
41

42. 42
Chemical and physical types of
media
Defined/ synthetic medium
• Media which all chemicals are known.
• Can be in liquid form (broth) or solidified by an agent such as
agar.
•Often used to culture photolithotrophic autotrophs such
as cyanobacteria and photosynthetic protists.
– CO2 as a C source (often added as sodium carbonate or bicarbonate),
nitrate or ammonia as a N source, sulfate, phosphate, and other
minerals.
• Chemoorganoheterotrophs can also be grown.
– Glucose as a C source and an ammonium salt as a N source.
•Widely used in research – desirable to know exactly
what the microorganism is metabolizing.

43. BG–11 Medium for Cyanobacteria Amount (g/liter)
NaNO3 1.5
K2HPO4 · 3H2O 0.04
MgSO4 · 7H2O 0.075
CaCl2 · 2H2O 0.036
Citric acid 0.006
Ferric ammonium citrate 0.006
EDTA (Na2Mg salt) 0.001
Na2CO3 0.02
Trace metal solutiona
1.0 ml/liter
Final pH 7.4
Medium for Escherichia coli Amount (g/liter)
Glucose 1.0
Na2HPO4 16.4
KH2PO4 1.5
(NH4)2SO4 2.0
MgSO4 · 7H2O 200.0 mg
CaCl2 10.0 mg
FeSO4 · 7H2O 0.5 mg
Final pH 6.8–7.0
Sources: Data from Rippka, et al. Journal of General Microbiology, 111:1–61, 1979; and S. S.
Cohen, and R. Arbogast, Journal of Experimental Medicine, 91:619, 1950.
a
The trace metal solution contains H3BO3, MnCl2 · 4H2O, ZnSO4 · 7H2O, Na2Mo4 · 2H2O,
CuSO4 · 5H2O, and Co(NO3)2 · 6H2O. 43

44. 44
Chemical and physical types of
media
Complex media
•Contain unknown/ undefined chemical composition –
peptones, meat extract, and yeast extract.
• For microorganism that the nutritional requirements are
unknown.
•Very useful – a single complex medium may be sufficiently
rich to meet all the nutritional requirements of many different
microorganisms.
•Also used to culture fastidious microbes, microbes with
complex or cultural requirements.
• Examples: nutrient broth, tryptic soy broth, MacConkey agar.

45. Nutrient Broth Amount (g/liter)
Peptone (gelatin hydrolysate)
Beef extract
5
3
Tryptic SoyBroth
Tryptone (pancreatic digest of casein) 17
Peptone (soybean digest) 3
Glucose 2.5
Sodium chloride 5
Dipotassium phosphate 2.5
MacConkey Agar
Pancreatic digest of gelatin 17.0
Pancreatic digest of casein 1.5
Peptic digest of animal tissue 1.5
Lactose 10.0
Bile salts 1.5
Sodium chloride 5.0
Neutral red 0.03
Crystal violet 0.001
Agar 13.5
45

46. 46
• Most commonly used solidifying agent.
– Media can be solidified with the addition of 1-2% agar; most
commonly 1.5%.
• It is a sulfated polymer, composed mainly of D-
galactose, 3,6- anhydro-L-galactose, and D-glucuronic
acid.
• Extracted from red algae.
• Suitable because:
– Melt at 90°C, once melt does not harden until reach
45°C.
• After melted in boiling water, can be cooled to a temperature
that is tolerated by human hands and microbes.
• Microbes growing on media can be incubated at a wide
range of temperature.
Aga
r

47. 47
Functional types of
media
Supportive media/ general purpose
media
• Sustain the growth of many
microorganism.
• E.g. tryptic soy broth and tryptic soy
agar.

48. Functional types of
media
Enriched media
•To encourage the growth of fastidious microbes – add blood or
other special nutrients to general purpose media.
• E.g. blood agar.
48

49. 49
Functional types of
media
Selective media
• Favor the growth of particular microorganisms.
• Media containing bile salts or dyes such as basic fuchsin and
crystal violet favor the growth of gram-negative bacteria and
suppress gram-positive bacteria.
• Endo agar, eosin methylene blue agar, and MacConkey agar:
– 3 media widely used for the detection of E. coli and related bacteria in
water supplies and elsewhere.
– Contain dyes that suppress the growth of gram-positive bacteria.
• Media containing only cellulose as C and energy source
is quite effective in the isolation of cellulose-digesting
bacteria from samples such as soil.

50. 50
Functional types of
media
Differential media
• Distinguish among different groups of microbes and
permit identification of microorganism based on their
biological characteristics.
• Blood agar is both a differential medium and enriched
one.
• It distinguishes between hemolytic and non-hemolytic
bacteria.
• Hemolytic bacteria (e.g. streptococci and staphylococci
isolated from throat) produce clear zones around their
colonies.

51. Table5.7 Mechanisms of Action of Selective and Differential Media
Medium FunctionalType Mechanism of Action
Blood agar Enriched and differential Blood agar supports the growth of many fastidious bacteria. These can be
differentiated based on their ability to produce hemolysins—proteins that
lyse red blood cells. Hemolysis appears as a clear zone around the colony
(þ-hemolysis) or as a greenish halo around the colony (a-hemolysis) (e.g.,
Streptococcus pyogenes, a þ-hemolytic streptococcus).
Eosin methylene blue
(EMB) agar
Selective and differential Two dyes, eosin Y and methylene blue, inhibit the growth of gram-positive
bacteria. They also react with acidic products released by certain gram-negative
bacteria when they use lactose or sucrose as carbon and energy sources.
Colonies of gram-negative bacteria that produce large amounts of acidic
products have a green, metallic sheen (e.g., fecal bacteria such as E. coli).
MacConkey (MAC) agar Selective and differential The selective components in MAC are bile salts and crystal violet, which inhibit
the growth of gram-positive bacteria. The presence of lactose and neutral red, a
pH indicator, allows the differentiation of gram-negative bacteria based on the
products released when they use lactose as a carbon and energy source. The
colonies of those that release acidic products are red (e.g., E. coli).
Mannitol salt agar Selective and differential A concentration of 7.5% NaCl selects for the growth of staphylococci. Pathogenic
staphylococci can be differentiated based on the release of acidic products
when they use mannitol as a carbon and energy source. The acidic products
cause a pH indicator (phenol red) to turn yellow (e.g., Staphylococcus aureus).
51

52. 52
Isolation of pure
cultures

53. 53
Isolation of pure
cultures
• Naturally microbes grow in complex, mixed population with
many species.
• A problem – a single type of microorganism cannot be
studied adequately in a mixed culture – need pure
culture.
• Pure cultures – a population of cells arising from a single
cell, to characterize an individual species.
• Robert Koch developed pure culture techniques 
transformed microbiology.
• Several ways to prepare pure cultures.

54. 54
• If a mixture of cells is spread out on an agar surface at a
relatively low density, every cell grows into a completely
separate colony.
• Each colony arises from a single cell – each colony
represents a pure culture.
• Successful isolation depends on spatial separation of single
cells.
Spread plate and streak
plate

55. 55
Streak
plate
• To separate cells and obtain pure culture.
• Microbial mixture is transferred to the edge of an agar plate
with an inoculating loop or swab, and then streaked out over
the surface in one of several patterns.
• After the first sector is streaked, the inoculating loop is
sterilized and an inoculum for the second sector is
obtained from the first sector.
• A similar process is followed for streaking in the third sector,
except that the inoculum is from the second sector  dilution
process.
• Eventually, very few cells will be on the loop, and single
cells will drop from it as it is rubbed along the agar surface
 develop into separate colonies.

56. Streak-plate
technique
1 2 3 4 5
56

57. Spread
plate
• An easy, direct way of separating cells.
• A small volume of dilute microbial mixture containing about
30 to 300 cells is transferred to the center of an agar plate
and spread evenly over the surface with a sterile bent-glass
rod.
• The dispersed cells develop into isolated colonies.
• Because the number of colonies should equal the number of
viable organisms in the sample, spread plates can be used
to count the microbial population.
5

58. Spread-plate
technique
1. Pipette a small sample onto cell center of an agar
medium.
2. Dip a glass spreader into ethanol.
3. Briefly flame the ethanol-soaked spreader and allow it to
cool.
4. Spread the sample evenly. Incubate.
58

59. 59
Pour
plate
• Extensively used with bacteria, archaea, and fungi.
• The original sample is diluted several times to reduce the
microbial population sufficiently to obtain separate colonies
when plating.

60. Original
sample
9 ml H2O
(10–1 dilution)
9 ml H2O
(10–2 dilution)
9 ml H2O
(10–3 dilution)
9 ml H2O
(10–4 dilution)
1.0 ml
1.0 ml
1.0 ml
1.0 ml
1.0 ml 1.0 ml
Mix with warm
agar and pour.
60

61. 61
Pour
plate
• The small volumes of several diluted samples are mixed with
liquid agar that has been cooled to about 45°C, and the
mixtures are poured immediately into sterile culture dishes.
• After the agar is hardened, each cell is fixed in place and
forms an individual colony.
• Plates containing between 30 – 300 colonies are counted.
• The total number of colonies equals the number of viable
microorganisms in the sample that are capable of growing
in the medium used.
• Colonies growing on the surface also can be used to
inoculate fresh medium and prepare pure cultures.

62. Petri
dishes
• Special culture dishes for plating techniques.
• Inventor: Julius Richard Petri, a member of Robert Koch’s
lab.
• Petri developed these dishes around 1887 and they
immediately replaced agar-coated glass plates.
• Consist of two round halves, the top half overlaps the bottom.
• Very easy to use, may be stacked on each other to save
space, and are one of the most common items in
microbiology lab.
62

63. Microbial growth on solid
surfaces
• Colony development on agar surface aids in identifying
microbes because individual species often form colonies of
characteristic size and appearance.
6
Spindle
Umbonate
Rhizoid
Irregular
Filamentous
Pulvinate
Convex
Raised
Curled
Filamentous
Erose
Lobate
Undulate
Entire
Flat
Circular
Punctiform
Form
Margin
Elevation

64. Microbial growth on solid
surfaces
• The most rapid cell growth occurs at the colony edge; much
slower in the center, and cell autolysis takes place in the
older central portion of some colonies.
– Gradients of oxygen, nutrients, and toxic products
within the colony.
64

65. 65
Source
s
• Prescott, L.M., Harley, J.P., & Klein, D.A. (2008).
Microbiology, 7th Ed., McGraw-Hill.