Cultivation of microorganisms

Requirements for growth

Organic matter: C, H, O, N, P, S

Inorganic ions: K+, Na+, Fe2+, Mg2+, Ca2+, and Cl-: required for enzymatic catalysis and maintaining chemical gradients across the cell membrane.

Chemical energy

ATP

Proton motive force

Microbial Physiology

Nutrition

Nutrients: synthetic vs. nonsynthetic media

Carbon source

Autotrophs (lithotrophs): use CO2 as the C source

Photosynthetic autotrophs: use light energy to reduce CO2.

Chemolithotrophs: use inorganics, such as H2 or thiosulfate

to reduce CO2.

Heterotrophs (organotrophs): use organic carbon (eg. glucose) for growth.

Nitrogen source

Ammonium (NH4+) is used as the sole N source by most micro

organisms. Ammonium could be produced from N2 by nitrogen

fixation, or from reduction of nitrate and nitrite.

Sulfur source

A component of several coenzymes and amino acids.

Most microorganisms can use sulfate (SO42-) as the S s

ource.

Phosphorus source

A component of ATP, nucleic acids, coenzymes, lipids, teichoic acid, capsular polysaccharides; also is required for signal transduction.

Phosphate (PO43-) is usually used as the P source.

Mineral source

Required for enzyme function.

For most microorganisms, it is necessary to provide so

urces of K+, Mg2+, Ca2+, Fe2+, Na+ and Cl-. Many other minerals (e.g., Mn2+, Mo2+, Co2+, Cu2+ and Zn2+) can be provided in tap water or as contaminants of other medium ingredients.

Uptake of Fe is facilitated by production of siderophores (hydroxamates and catechol derivatives).

Growth factors: organic compounds (e.g., amino acids, sugars, nucleotides) a cell must contain in order to grow but which it is unable to synthesize.

Fastidious microorganisms

pH value Neutrophils ( pH 6-8) Acidophils ( pH 1-5) Alkalophils ( pH 9-11) Internal pH is regulated by variou

s proton transport systems in the cytoplasmic membrane.

Temperature Psychrophils ( 15-20 oC) Mesophils ( 30-37 oC) Thermophils ( at 50-60 oC)

Heat-shock response is induced to stabilize the heat-sensitive proteins of the cell.

Environmental factors

Aeration

Obligate aerobes

Facultative anaerobes

Microaerophilics

Obligate anaerobes

(Capnophilics: bacteria that do not produce enough CO2

and, therefore, require additional CO2 for growth.)

Ionic strength and osmotic pressure Halophilic

Toxicity of O2 for anaerobes:

1. O2 reduced to H2O2 by enzymes.

2. O2 reduced to O2- by ferrous ion.

In aerobes and aerotolerant anaerobes, O2- i

s removed by superoxide dismutase, while H

2O2 is removed by catalase.

Strict anaerobes lack both catalase and sup

eroxide dismutase.

Excluding oxygen

Reducing agents

Anaerobic jar

Anaerobic glove chamber

Anaerobic cultivation methods

Microbial metabolism

Intermediary metabolism

Assimilation (Anabolism)

Assimilatory pathways for the formation of key intermediates.

Biosynthetic sequences for the conversion of key intermediates to end products.

Dissimilation (Catabolism)

Pathways that yield metabolic energy for growth and maintenance.

Pyruvate: universal intermediate

Aerobic respiration

Fermentation

Glycolysis (EMP pathway)

Substrate-level phosphorylation

Fermentation: metabolic process in which the final electron acceptor is an organic compound.

Sources of metabolic energy Respiration: chemical reduct

ion of an electron acceptor through a specific series of electron carriers in the membrane. The electron acceptor is commonly O2, but CO2, SO4

2-, and NO3- are employed by some microorganisms.

Photosynthesis: similar to respiration except that the reductant and oxidant are created by light energy. Respiration can provide photosynthetic organisms with energy in the absence of light.

Substrate-level phosphorylation

In fermentation, the NADH produced during glycolysis is recycled to NAD.

Many bacteria are identified on the basis of their fermentative end products.

Fermentation of bacteria produces yogurt, sauerkraut, flavors to various cheeses and wines.

Alcoholic fermentation is uncommon in bacteria.

Fermentation

Saccharomycetes

E. coliClostridium

Propionebacterium Enterobacter

StreptococcusLactobacillus

Function of TCA cycle

1. Generation of ATP

2. Supplies key intermediates for amino acids, li

pids, purines, and pyrimidines

3. The final pathway for the complete oxidation of

amino acids, fatty acids, and carbohydrates.

Functions:

1. Provides various suga

rs as precursors of bio

synthesis, and NADP

H for use in biosynthe

sis

2. The various sugars m

ay be shunted back to

the glycolytic pathway.

Pentose phosphate pathway (hexose monophosphate shunt)

1. Ribose-5-P (product of HMP) synthesis of purine r

ing from sugar moiety inosine monophosphate

purine monophosphate

2. Pyrimidine orotate orotidine monophosphate (pyri

midine orotate attaches to ribose phosphate)

cytidine or urine (pyrimidine) monophosphate

3. Reduction of ribonucleotides at the 2’ carbon of the sug

ar portion deoxynucleotides

Nucleic acid synthesis

Cultivation methods

Medium

Basic media

Rich media

Enrichment media

Selective media

Differential media

Agar: an acidic polysaccharide extracted from red algae

For microbiologic examination

Use as many different media and conditions of incubation as is practicable. Solid media are preferred; avoid crowding of colonies.

For isolation of a particular organism

Enrichment cultureDifferential mediumSelective medium

Isolation of microorganisms in pure culture

Pour plate methodStreak method

For growing bacterial cells

Provide nutrients and conditions reproducing the organism's natural environment.

Most bacteria reproduce by binary fission.

Measurement of microbial concentrations:

Cell concentration (no. of cells/unit vol. of culture)

Viable cell count

Turbidimetric measurements

Biomass concentration (dry wt. of cells/unit vol. of culture): can be estimated by measuring the amount of protein or the volume occupied by cells.

Growth, survival and death of microorganisms

0.1 ml

10-1 10-2 10-3 10-4 10-5 10-6 10-7

> 1000 220 18

Bacterial concentration:

220 x 106 x 10 = 2.2 x 109/ml

Lag phase (adaptation)

Exponential phase

Determination of the generation time (doubling time)

The ending of this phase is due to exhaustion of nutrients in the medium and accumulation of toxic metabolic products.

Stationary phase

A balance between slow loss of cells through death and formation of new cells through growth.

Alarmones is induced.

Some bacteria undergo sporulation.

Decline phase (the death phase)

Bacterial growth curve

Death: irreversible loss of the ability to reproduce.

Empirical test of death: culture of cells on solid media. Viable but nonculturable cells

Bacterial growth in nature

Interaction of mixed communities

A natural environment may be similar to a continuous culture.

Bacteria grow in close association with other kinds of organisms.

The conditions in bacterial close association are very difficult to reproduce in the laboratory. This is part of the reason why so few environmental bacteria have been isolated in pure culture.

Biofilms

Polysaccharide encased community of bacteria attached to a surface.

Attachment of bacteria to a surface or to each other is mediated by glycocalyx.

About 65% of human bacterial infection involve biofilms.

Biofilms also causes problems in industry.

Bioremediation is enhanced by biofilms.

Biofilm: a community of microbes embedded in an organic polymeric matrix (glycocalyx, slime), adhering to an inert or living surface.