Industrial Microbiology

Lecture Series
Tuesdays 10:15 – 11:45 am

Drs. Dam Thuy Hang, Nguyen Thanh Hoa, Le Thanh Ha, Pham Tuan Anh
Dept. of Bioengineering
School of Chemistry and Life Sciences
 Agriculture Food
N2 fixation Food preservation
Nutrient cycling Fermented food
Animal husbandry Food additives
Disease
Identify new diseases
Treatment
Prevention
Energy/Environment Biotechnology
Biofuels (methane, ethanol) GMO
Bioremediation Produce pharmaceuticals
Microbial mining Gene therapy
Industrial Microbiology – Putting Microbes to Use

• Use and control of microorganisms for the benefit of humankind

use of microorganisms to make product: food, chemical, enzyme, etc.

control of microorganisms because they cause food spoilage, disease or
other biodeterioration

• Bacteria & archaea, yeasts & fungi, viruses, protists

• Modern definition of industrial microbiology:

The use of microbiology, biochemistry and engineering in an
integrated fashion with the goal of using microorganisms or their
parts to manufacture useful products.
 Use - Microbial Applications
1. Food and beverage biotechnology
• fermented foods, alcoholic beverages (beer, wine)
• flavors
2. Enzyme technology
• production and application of enzymes
3. Metabolites from microorganisms
• amino acids
• antibiotics, vaccines, biopharmaceuticals
• bacterial polysaccharides and polyesters
• specialty chemicals for organic synthesis
4. Biological fuel generation
• ethanol or methane from biomass, single cell protein, production of biomass
• microbial recovery of petroleum
5. Environmental biotechnology
• water and wastewater treatment, composting (and landfilling) of solid waste
• biodegradation/bioremediation of toxic chemicals and hazardous waste
6. Agricultural biotechnology
• soil fertility
• microbial insecticides, plant cloning technologies
7. Diagnostic tools
• testing & diagnosis for clinical, food, environmental, agricultural applications
• biosensors
Selman Waksman (~1945):
“There is no field of human endeavor, whether it be in industry or in
agriculture, or in the preparation of food or in connection with problems of
shelter or clothing, or in the conservation of human and animal health and the
combating of disease, where the microbe does not play an important and often
dominant role.”

Selman Waksman
Learning Goals: To gain a fundamental understanding of how microbes are utilized
and controlled for the benefit of humankind and to develop the
skills needed to study these applications.

Reading: Slides and assigned papers

Brock Biology of microorganisms
Industrial Microbiology. An Introduction. M.J. Waites, N.L. Morgan,
J.S. Rockney & G. Highton Blackwell Science Publishers, 2001;
ISBN 0-632-05307-0
[Link] cells or cell products

[Link] biotransformation
products

[Link] products (de

novo synthesis)
Primary vs. Secondary Metabolism
Waites et al.
 Control of Microorganisms
• making sure unwanted microorganisms don’t grow

1. Preventing food spoilage

• sterilization methods, canning
• chemical & physical control of growth
• water activity, acidity, pickling, etc.
• fermented foods

2. Sanitation, prevention of waterborne disease

• disinfectants, antiseptics, etc.
• water treatment (filtration, chlorination…)
• wastewater treatment

3. Prevention of biodeterioration
• water damage to buildings
• preservation of paper, books, artwork, etc.
 History of (Industrial) Microbiology
Biotechnological production of foods and beverages
• alcoholic fermentation, spontaneous fermentation of sugar-containing fruits during storage in almost all natural
populations from pre-historic period
• beer production by Sumerians and Babylonians 6000 B.C.
• wine before 2000 B.C. in Assyria
• fermented beverage of rice, honey and fruit produced in China >9,000 years ago
• leavened bread in Egypt 4000 B.C.
• fermented and curdled milk products, cheese
 Discovery and description of microorganisms

• early microscopy by Robert Hooke (1665) and Antoni van Leeuwenhoek (~1680)

Robert Hooke
• described fruiting structures of molds
in 1664
• first person to describe
microoganisms

Antoni van Leeuwenhoek

• discovery of bacteria in 1676, published
1684, Royal Society of London
• “animacules”
 Discovery and description of microorganisms

• refuting spontaneous generation

• demonstrating relationship between the growth of infusoria (microorganisms in
organic broths) and the onset of chemical changes (fermentation and putrefaction)
• Appert invented canning 1810
• Schwann’s 1837 report on alcohol fermentation and putrefaction
• Pasteur’s swan-neck flask experiments 1860's demonstrating that fermentation
was caused by “infusoria”
• fermentative ability of microorganisms described by Pasteur 1857
• detection of fermentation enzymes in yeast by Buchner 1897

• microbes as causative agents of disease – Koch’s postulates 1884

• enrichment culture method

• Winogradsky and Beijerink 1900s
• activities of microbes in the environment, biogeochemical cycling of C, S and N
 Louis Pasteur
Much of Pasteur’s work arose from the requests to solve
practical problems in their industrial processes:
• why was French beer inferior to German beer?
→ discovered the existence of anaerobic life
• problems with souring of alcohol
• budding yeast cells observed in vats of good wine and
rod-shaped bacteria in bad wine
→ discovery of different fermentations and versatility of
microbial metabolism
role of yeast in alcoholic fermentation
lactic acid fermentation by rod-shaped bacteria
• heat-killing of microorganisms ended controversy of
spontaneous generation (swan-neck flask experiments)
development of sterilization methods
• development of vaccines for the diseases anthrax,
cholera and rabies
 Microbial Diversity:
Physiology, Ecology and Industrial Use

Taxonomy
= systematics: identification + nomenclature

Identification: characterization to determine species

Nomenclature: naming

Classification: ordering into groups based upon common properties

What is a species? - Who cares about names? - Why?

 Prokaryotes: The unseen majority
W.B. Whitman, D.C. Coleman & W.J. Wiebe

Proc. Natl. Acad. Sci. 95:6578-6583 (1998)

doi:10.1073/pnas.95.12.6578

The number of prokaryotes and the total amount of their cellular carbon on earth
are estimated to be 4-6 × 1030 cells and 350-550 Pg of C (1 Pg = 1015 g),
respectively. Thus, the total amount of prokaryotic carbon is 60-100% of the
estimated total carbon in plants, and inclusion of prokaryotic carbon in global
models will almost double estimates of the amount of carbon stored in living
organisms. In addition, the earth's prokaryotes contain 85-130 Pg of N and 9-14 Pg
of P, or about 10-fold more of these nutrients than do plants, and represent the
largest pool of these nutrients in living organisms.
 Number and biomass of prokaryotes in the world

Environment No. of prokaryotic Pg of C

cells in prokaryotes
× 1028

Aquatic habitats 12 2.2

Oceanic subsurface 355 303
Soil 26 26
Terrestrial subsurface 25-250 22-215

Total 415-640 353-546

From Whitman et al 1998

 Prokaryotes: The unseen majority
W.B. Whittman, D.C. Coleman & W.J. Wiebe (1998) PNAS 95:6578-6583

• The number of prokaryotes estimated at 4-6 × 1030 cells

• Total amount of prokaryotic cellular carbon on earth 350-550 Pg of C

(1 Pg = 1015 g)

• Thus, the total amount of prokaryotic carbon is 60-100% of the estimated total
carbon in plants

• In addition, the earth's prokaryotes contain

85-130 Pg of N and 9-14 Pg of P
~10-fold more of N&P than in plants
largest pool of these nutrients in living organisms
 What is a Species?

Biological Species

as applied to plants/animals:

A population whose members are able to interbreed freely under natural conditions
(Wilson 1992)
 What is a Bacterial / Archaeal Species?

A collection of similar strains that differ sufficiently from other groups of strains and
shares common ancestry

How do you define sufficiently?

Species concept vs. species definition:

• Species concept is the theoretical framework that attempts to describe what
bacterial species are and explain how they are formed
• Species definition is how this concept is exercised in practice
 Classification in Bacteriology
• based on the principle of degree of relatedness

• the grouping of organisms, whether or not they are formally designated taxa
(e.g., species, genera, domains, or groups, clusters, etc.) generally brings together
organisms on the basis of shared properties

• classification is done by generating as much data on the properties of novel

isolates as possible. Includes the theory and process of ordering the characterized
organisms into one or more systems

• the process of identification, e.g., comparing the data with the database of
previously classified organisms

• nomenclature is the naming of the appropriate taxon within a classification

system
Stackebrandt E (2000) Defining Taxonomic Ranks, In: The Prokaryotes, Vol 1.
 Three Domains:
Universal phylogenetic “tree of life” based on rRNA gene sequences
 Three domain system (Woese)
based on phylogeny of ribosomal RNA sequences:

Bacteria, Archaea, Eukarya

Major differential characteristics of the three domains of life:

Characteristic Bacteria Archaea Eukarya

Nuclear membrane No No Yes
Mitochondria and chloroplasts No No Yes
Peptidoglycan cell walls Yes * No No
Ribosome size 70 S 70 S 80 S
Membrane lipids ester-linked ether-linked ester-linked
 Classification of Microorganisms

A. Subcellular
Viruses (non-living)
B. Prokaryotes (no nuclear membrane)
Bacteria
Archaea
C. Eukarya (membrane bound nuclei)
Fungi cell wall, no chlorophyll, organotrophy
Algae cell wall, contain chlorophyll, phototrophy
Protozoa colorless, motile, lack cell wall, ingest organisms or organic particles

Characteristics (structural differentiation) of prokaryotes vs. eukaryotes:

rRNA Gene Sequence Analysis - Phylogeny
 Polyphasic Taxonomic Description of a Species
Morphological and cultural observation
• cell shape, size, arrangement, flagellation
• staining reactions
• colony morphology
Physiological and metabolic properties
• carbon and nitrogen utilization, energy sources, fermentation products
• temperature range and optimum for growth
• oxygen tolerance and pH optimum, osmotic tolerance
• pigments, storage products
• antibiotic sensitivity
Chemical markers
• cell wall constituents, peptidoglycan, etc.
• complex lipids, fatty acids of membrane phospholipids
• whole organism proteins
 Morphological and cultural observation

• cell shape, size, arrangement, flagellation

• staining reactions
• colony morphology
 Physiological and metabolic properties

• carbon and nitrogen utilization

• energy sources
• fermentation products
• growth and temperature range
• oxygen tolerance and pH optimum, etc.
• pigments, storage products
• antibiotic sensitivity
 Chemical markers = Chemotaxonomy

• cell wall constituents, peptidoglycan

• complex lipids
• fatty acids of membrane phospholipids
• whole organism proteins
Gram-Negative cell wall
Gram-Positive cell wall
 Lipids
1. Long-chain fatty acids
- polar lipids of the plasma membrane
- lipopolysaccharide of Gram-negative bacteria
2. Mycolic acids (2-alkyl-3-hydroxy-branched fatty acids)
- nocardioform bacteria
3. Polar lipids
- “free lipids”
- phospholipids, glycolipids, amino acid amines
4. Isoprenoid quinones
- respiratory quinones in plasma membrane
5. Sterols
- e.g. ergosterol in fungi

Long-chain fatty acids

• cellular fatty acids, CFAs; FAME, fatty acid methyl esters
• most microorganisms synthesize fatty acids with chain lengths of 10 to 20 carbon atoms. The most prevalent are
those with 16-18 carbons
• the fatty acid content of all lipids is determined by the particular type of biosynthetic pathway of a
given species
• the variable properties that make an organism’s CFA composition distinctive include
1) quantitative differences in CFA content
2) presence of unique CFAs
DNA-DNA Similarity

Comparison of overall DNA-DNA

hybridization of the genome
between two (or more) organisms

“The Gold Standard”

•strains with DNA hybridization values above 60-70% are considered same species
•strain with hybridization values above 20% probably reside in the same genus
•useful tool to distinguish closely related bacteria
•does not reveal phylogenetic history
What does 16S rRNA gene similarity vs DNA-DNA similarity tell us?

Rossello-Mora & Amann 2001

Gillis et al.; Bergey’s Manual
 What is a Bacterial / Archaeal Species?

A collection of similar strains

that differ sufficiently from other groups of strains and shares common
ancestry
Genus = collection of different species, each sharing some major property that define
the genus

How do you define sufficiently?

General convention (for species):

> 97-98% 16S rRNA gene similarity
> 60-70% whole genome DNA homology
 Eukaryotic Microbes

• Fungi, algae, protozoa: lack tissue differentiation which distinguishes them from plants and animals
• extremely diverse with tens of thousands of species
 Fungi

• heterotrophic eukaryotes: saprophytes or parasites

• all transitions from motile unicellular to multicellular organisms
• approximately 70,000 known species
1,500,000 estimated total species (Bull et al., 1992)
• chitin (polymer of glucose derivative) cell wall
sexual life cycles
mitosis can take place in haploid, diploid or in both phases:
Haplont, Diplont, Haplodiplont, Haplodikaryont, Dikaryont
parasexual cycle: recombination takes place in the course of mitotic divisions
• Taxonomy and classification: Myxomycetes
(slime molds)
Zygomycetes (Phycomycetes, lower fungi)
Rhizopus nigricans (production of citric acid)
Ascomycetes
Saccharomyces cerevisiae, yeast Basidiomycetes
wood-rotting fungi, edible fungi (mushrooms) Fungi
imperfecti, (Deuteromycetes)
lost capacity for sexual propagation or is yet to be observed
Penicillium, Aspergillus
Fungal hyphae and mycelia
Asexual Fungal Spores:

Prescott et al.
Microbiology
 Why is Microbial Diversity Important?
Important values to the Earth’s ecosystem and to society:

1. Microbes are critical to the sustainability of life on Earth, including recycling of

elements, maintenance of climate, degradation of wastes
2. Discoveries of microbial diversity expand the frontiers of knowledge about the
limits and strategies of life
3. Microbes represent the largest untapped reservoir of biodiversity for potential
discovery of new biotechnology products, such as biopharmaceuticals, new
enzymes, or organisms that carry out novel processes
4. Microbes often have key roles in conservation of “higher” organisms and in
restoration of degraded ecosystems
Putting Microbes to Use:
 MICROBIAL DIVERSITY AND BIOPROSPECTING
Biodiversity as a source of innovation in biotechnology
Biotechnology = the art of exploiting biology
• International Convention on Biological Diversity defines genetic resources as
“genetic material of actual of potential value”, inclusive of whole organisms, cell
component and extracts, and nucleic acids.
• Microbial diversity as a major resource for biotechnological products and
processes
[Link] and beverage biotechnology
[Link] (amino acids, antibiotics, biopharmaceuticals,…)
[Link]
[Link] synthesis
[Link] cell protein
[Link] biotechnology
[Link] fuels
• Search for and discovery of novel microorganisms and properties = microbial
bioprospecting
 Taxonomic diversity of some useful/important bacteria
• bacteria characterized by tremendous metabolic
versatility:
degradation of various compounds
different end products of fermentation
Main structures of prokaryotic and eukaryotic cells
 Proteobacteria (purple bacteria)
Gamma division
• Enteric bacteria: Escherichia coli, Salmonella, Shigella, Proteus, Yersinia
• generate ATP ether by oxidative degradation of organic compounds or by fermentation of
simple sugars under anaerobic conditions
• Fluorescent pseudomonads
• obligately respiratory (aerobic or denitrifying)
• some species famous for using a wide variety of organic compounds as carbon and energy sources
• several plant pathogens
Beta division
• Thiobacillus & Acidithiobacillus
• chemoautotrophic bacteria that live by oxidizing reduces sulfur compounds
• industrial extraction of ores
Alpha division
• Rhizobium
• nitrogen-fixing chemoheterotrophs that fix nitrogen in symbiosis with legumes
• Zymomonas
• fermentation of sugars to ethanol using the Entner-Doudoroff pathway
• Gluconobacter, Acetobacter
• oxidize ethanol to acetic acid, used in manufacture of vinegar
 Gram-positive bacteria
Clostridial branches
• “genus” Clostridium being split into several genera
• strictly anaerobic, obtain energy only by substrate-level phosphorylation (lack
cytochrome system and mechanism for electron transport)
• form endospores
• ferment sugars with butyric acid as major end product
• acetone-butanol fermentation, C. acetobutylicum
• fermentation of amino acids (Stickland reaction)
• pathogenic clostridia; C. botulinum, C. tetani, C. perfringens
Lactic acid bacteria
• Lactobacillus, Leuconostoc, Pediococcus
• obtain energy by fermentation of simple sugars to lactic acid (homofermentative) or to lactic acid,
ethanol and CO2 (heterofermentative)
• aerotolerant anaerobes, grow best at low oxygen tension in habitats rich in sugars,
peptides, purines/pyrimidines and vitamins
• tolerate low pH
• Lactobacillus acidophilus
fermentation of vegetables and milk
generate ATP by converting glucose into two molecules of lactic acid
Bacillus
• rod-shaped endospore-forming aerobic bacteria, chemoheterotrophic
• B. anthracis, B. cereus, but majority are harmless saprophytes
• insect pathogens B. thuringiensis ➞ microbial insecticides
• antibiotics synthesized by B. subtilis (bacitracin) and B. polymyxa (polymyxin)
Actinomycetes and related genera
• Propionibacterium found in Emmental cheese
• ferment lactate to propionate, acetate and CO2
• Arthrobacter and Coryneform group
• important in the degradation of unusual compounds in soil (Rhodococcus, Nocardia,
Mycobacterium)
• Mycobacterium tuberculosis
• production of glutamic acid by Corynebacterium glutamicum
• Streptomycetes
• antibacterial substance (streptomycin) isolated from cultures of Streptomyces griseus (Schatz and
Waksman, 1944) and many others
• antibiotics: tetracycline, erythromycin, neomycin, gentamycin,...
Thermus-Deinococcus
• Thermus
• thermophilic eubacteria, optimal growth temperature 70-72̊C, Taq polymerase
• Deinococcus
• unusual organism with extremely high resistance to ionizing radiation
Waites et al. Industrial Microbiology
Microbial cell factories

What are the important traits of an ideal microorganism for industrial

bioproduction?

Take lactic acid production by microorganisms from lignocellulose

biomass as an example for discussion
Microbial cell factories

Broad substrate range

Robust
Tolerate inhibitors from lignocellulose treatment
Efficient producer: high product concentration, high production rate
(product/L.h), high production yield (product/substrate)
Tolerate end product
Low maintenance (withstand high shearing rate)
Can recycle
Safe to work with
Easy to control contamination
Microbial cell factories – Improvement of microorganisms

Parental strain

Improvement Screening

Selection,
- Classical strain characterization
engineering
- Metabolic
engineering Selection of the
best strain
Microbial cell factories – Parental strain

• Obtain from a culture collection

• Isolation → bioprospecting
Microbial cell factories – Strain improvement target
Microbial cell factories – Metabolic engineer

Metabolic engineer: Precise, direct

changes to the genome
- Heterologous gene expression
- Homologous gene up-regulation
- Mutant gene expression
- Gene deletion
Microbial cell factories – Metabolic engineer

Metabolic engineered E. coli for 1,4-butanediol production

 Culture collections for microbes

Preservation
• maintain organism alive, uncontaminated and without variation or mutation
(as close as possible to original isolate)
 Culture Collections
Preservation
• maintain organism alive, uncontaminated and without variation or mutation (as close as
possible to original isolate)
• no single preservation procedure is appropriate for all organisms
1. Periodic transfer to fresh agar slants, kept after growth at 4°C
- mutants or contaminants may accumulate
2. Lyophilization
- microbial cells mixed with cryoprotectant (20% wt/vol skim milk powder or 12% sucrose), frozen
and water removed under vacuum
- can be shipped without refrigeration
3. Frozen at -75 to -156°C
- slowly frozen in 10% glycerol to prevent formation of ice crystals
- viable for years or decades
4. Spore-forming bacteria and fungi can be preserved by slowly air-drying spores at ambient
temperatures
ATCC American Type Culture Collection
DSMZ Deutsche Sammlung von Microorganismen und Zellkulturen
NRRL Agricultural Research Service Culture Collection
National Center fro Agricultural Utilization Research
JCM Japan Collection of Microorganisms
 American Type Culture Collection (ATCC)

[Link]

Culture Collections:
Bacteria
Bacteriophages
Cell Lines & Hybridomas
Filamentous Fungi & Yeast
Plant Seeds
Protozoa & Algae
Viruses & Antisera (Animal – Plant)

Bacteria Collection
The ATCC Bacteriology Collection is the most diversified assemblage of prokaryotes
in the world, containing nearly 18,000 strains in more than 750 genera. Every
important prokaryotic physiological group is represented. The Collection holds more
than 3,600 type cultures of validly described species, forming the basis for
systematic bacteriology, and nearly 500 bacteriophages
ATCC is a global nonprofit bioresource center.
Summary

• Industrial microbiology: put microorganisms to work, exploiting

metabolic diversity of microorganisms

• Basis of bioprospecting is the diversity of microorganisms

• Microorganisms as microbial cell factories: improving production strain

and preservation
 Questions for thought
What is a bacterial species?

What are some of the key methods used to delineate a species? Briefly describe and
compare the information that they provide and their “taxonomic resolution”.

What is the underlying principle in the 3-domain division of life? Is this division
reflected in cellular structure, how? How might this division be reflected in metabolic
functions and how these are applied?

Reading:
Zheng, Y., Saitou, A., Wang, C., Toyoda, A., Minakuchi, Y., Sekiguchi, Y., Ueda, K., Takano, H., Sakai, Y., Abe, K.,
Yokota, A., & Yabe, S. (2019). Genome Features and Secondary Metabolites Biosynthetic Potential of the Class
Ktedonobacteria. Frontiers in Microbiology, 10, 447612. [Link]