Advanced Redox Technology (ART) Lab

고도산화환원 환경공학 연구실

http://artlab.re.kr

Chapter 1. Basics of Microbiology

All the figures and tables in this material are from the reference below unless specified otherwise.
Reference: Bruce E. Rittmann and Perry L. McCarty, "Environmental Biotechnology: Principles
and Applications", McGraw-Hill, 2001.

Changha Lee

School of Chemical and Biological Engineering

Seoul National University
 Intro: Environmental Biotechnology

√ Environmental Biotechnology
i) applies the principles of microbiology to solve environmental problems.
(e.g., water & wastewater treatment, energy production from organic waste)

ii) links the principles of microbiology with engineering fundamentals involving

reaction kinetics & engineering as well as conservation of energy & mass.

iii) is always concerned with mixed culture, open, nonsterile systems.

(the major difference between environmental biotechnology and other disciplines that
feature biotechnology)

iv) can be successful depending on how individual microorganisms with desired

characteristics can survive in competition with other organisms.

- How desired functions can be maintained in complex ecosystems.

- How the survival and proliferation of undesired microorganisms can be prevented.

(e.g., Filamentous bacteria in Activated Sludge Process)
 1.1 The Cell
√ Cell: the fundamental building block of life,
an entity separate from other cells and its environment

√ Essential components of cells:

• Cell wall: a structure that confers rigidity to the cell and protects the membrane
• Cell membrane: a barrier between the cell and the environment
• Cytoplasm (세포질): water and macromolecules that the cell needs to function
• Chromosome (염색체):
-A genetic element carrying genes essential to cellular function.

-Prokaryotes typically have a single chromosome consisting of a circular DNA molecule.

-Eukaryotes typically have several chromosomes, each containing a linear DNA molecule.

• Ribosomes (리보솜) :convert the genetic code into proteins (mainly enzymes) that
carry out the cell’s reactions
• Enzymes :catalysts that carry out the desired biochemical reactions.
 1.1 The Cell
• DNA: Deoxyribonucleic acid
DNA stores and replicates the genetic information.

Double stranded helix structure

Nucleotide: phosphate + sugar + nitrogenous base

Source: Wikipedia
 1.1 The Cell
• Transcription
: the process in which a particular segment of DNA is copied (transcribed) into RNA
(ribonucleic acid) by the enzyme RNA polymerase (RNAP)

• Translation
: the process in which
ribosomes synthesize
proteins

Source: Wikipedia
 1.1 The Cell
: Cells that have
Prokaryotes their chromosomes
(원핵생물) inside a nucleus

: Cells that do not (Eukarya, Eukaryotes, 진핵생물)

contain their
chromosome inside
a nucleus

• Environmental biotechnology is mainly

interested in microorganisms in Bacteria and
The root Archaea domains, and some algae and
protozoa (& plants?) in the Eukarya domain.

▲ Phylogenetic tree of life as determined from comparative rRNA sequencing

 1.1 The Cell

▲ Prokaryotic cells

- Simple and small (0.15 m), ancient cells

- Lack of organelles (such as nucleus, mitochodrion)

They both have DNA,

ribosomes, cytoplasm, cell
membranes (cytoplasmic or
plasma membranes).
▲ Eukaryotic cells
- Complex and large (10100 m), evolved cells
- Many membrane-bound organelles
 1.2 Taxonomy and Phylogeny
√ Taxonomy
• Taxis (arrangement) + Nomos (law)

• Taxonomy relies on observable properties (phenotype)

i) morphology,
ii) transformation (ability to use or convert a given chemical into another one)
iii) Staining (the manner in which it interacts with dyes )

e.g., taxonomic ranks of Pseudomonas fluorescens

Formal rank Example
Domain Bacteria
Division (Phylum) Proteobacteria
Class Gammaproteobacteria
Order Pseudomonadales
Family Pseudomonadaceae
Genus Pseudomonas
Species Pseudomonas fluorescens
 1.2 Taxonomy and Phylogeny

√ Phylogeny

• Phylogeny is the method of classification based on genetic characteristics (encoded in

DNA and RNA).

• The sequences of base pairs in an organism’s 16S ribosomal RNA (rRNA) are important
(used to distinguish two domains, the Bacteria and the Archaea).

* The number 16S refers to Svedberg units, which are units of sedimentation coefficients
of ribosome subunits or intact ribosomes when subjected to centrifugal force in an
ultracentrifuge.

• Phylogeny relates organisms based on their evolution history, while taxonomyrelates

organisms based on observable characteristics of the cells.
 1.2 Taxonomy and Phylogeny

√ Prokaryotes
• Cells that do not contain their chromosome inside a nucleus
• Single cellular organisms
• Bacteria (the cell wall contains peptidoglycan (murein))
Archaea (it does not)

√ Eukaryotes
• Cells that have their chromosomes inside a nucleus
• Single cellular (algae & protozoa) or multi-cellular organisms
1.2 Taxonomy and Phylogeny
peptidoglycan
 1.3 Prokaryotes - Bacteria
√ Morphology

: Shape, size, structure, spatial relationship to one another

Shape: Coccus (spherical), Bacillus (rods), Spirillium (helical shape)

1.3 Prokaryotes - Bacteria
 1.3 Prokaryotes - Bacteria
√ EPS (Extracellular Polymeric Substances)
• Organic polymers of microbial origin which in biofilm systems are frequently
responsible for binding cells and other particulate materials together (cohesion)
and to the substratum (adhesion)
*They are of much significance in environmental biotechnology
• They are excreted from the cell and do not diffuse away from the cell because of
viscous nature
• They increase the viscosity of the surrounding fluid and can also help to hold
bacteria together in aggregates or bacterial “flocs”.
• They form capsule or slime layer (= biofilm).

• Different classes of organic molecules such as polysaccharide, proteins, nucleic

acids, (phospho)lipids, and other polymeric compounds, which have been bound to
occur in the intercellular spaces of microbial aggregates.
• Important to stress that EPS is composed by a number of different organic
macromolecules of different origin ( humic substances attached). it is basically
impossible to perform any extraction that extracts only the exopolymers produced
by the microbes present in the aggregate.
 1.3 Prokaryotes - Bacteria
√ EPS (Extracellular Polymeric Substances)

-Physiological
states of activated
sludge

-Architecture of bio-
cake

-Membrane
permeability

▲ Definition of cell biomass and extracellular polymeric substances(EPS)

*The EPS may arise from microbial production, lysis and hydrolysis, or from
attachment ,e.g., substances from water phase to bioaggregates
 1.3 Prokaryotes - Bacteria
√ Chemical composition
1.3 Prokaryotes - Bacteria
 1.3 Prokaryotes - Bacteria

√ Reproduction and Growth

• Bacteria normally reproduce through binary fission, in which a cell divides into two.
This asexual reproduction occurs spontaneously after a growing cell reaches a certain
size.

• Generation time: the interval of time required for the formation of two cells from one
- It may be as short as 30 min, as with E.coli.
- The weight would increase from 10-13g to that of a human child (18kg) in a single day

*Environmental and nutritional limitations of the culture flask generally limit the
growth long before such a mass increase occurs,
but the potential for such rapid growth is inherent in the bacterial cell.
 1.3 Prokaryotes - Bacteria

√ Classes of bacteria based on energy and carbon-source

• Phototrophs: use energy from light

• Chemotrophs: obtain energy from chemical reactions
- Chemoorganotrophs: use organic chemicals for energy
- Chemolithotrophs: use inorganic chemicals for energy

• Autotrophs: use inorganic carbon (e.g.,CO2) for cell synthesis

• Heterotrophs: use organic compounds for cell synthesis.
 1.3 Prokaryotes - Bacteria
√ Environmental condition for growth
• Temperature
Psychrophile (-5~20℃), Mesophile (8~45℃),
Thermophile (40~70℃), Hyperthermophile (65~110℃)

• pH
- pH range for growth: 6~8
for most bacteria
• Oxygen
- Aerobic, Anaerobic
• Salt
- Halophiles (3.5% NaCl),
Extreme halophile
(10~30% NaCl)
 1.3 Prokaryotes - Bacteria

√ Proteobacteria = Purple bacteria

• The most studied and important group of bacteria for environmental biotechnology
• Traditional Gram-negative bacteria
• Largest number of species
• Alpha, beta, gamma, delta, epsilon subgroup (by 16S rRNA)
• Diverse physiology: phototrophic, chemolithotropic, chemoorganotrophic
• They have ability to transform a great variety of inorganic and organic pollutants into
harmless minerals．
• They are the causative agents of disease (Pathogens)
1.3 Prokaryotes - Bacteria
 1.3 Prokaryotes - Archaea

• Bacteria : The cell wall contains peptidoglycan

Archaea : The cell wall does not contain peptidoglycan

• Microorganisms that convert acetate and hydrogen to methane (methanogens).

CH3COO + H2O  CH4+ HCO3

4H2 + CO2  CH4+ 2H2O

• Archaea are characterized by a large number of extremophiles, organisms living

under extreme environmental conditions (thermophiles, halophiles, etc.)
 1.4 Eukarya (Eukaryotes) - Fungi

√ Fungi

• All are organolithotrophic, and none are phototrophic.

• They are known to decompose a great variety of organic materials that tend to resist
bacterial decay. They have the key oxidative enzyme (peroxidase), which helps to
break lignins.
e.g., leaves, dead plants, dead trees, and other lignocellulosic organic debris.

• They are specially important for degradation of toxic compounds.

• But, slow decomposition rate  making less attractive for engineered systems
 1.4 Eukarya (Eukaryotes) - Algae
√ Algae
• Oxygenic phototrophs
the main source of oxygen in natural water bodies

• Problems
- Production of taste and odor compounds, toxins in water supplies
- Filter clogging at water treatment plants
- Decreased clarity of lakes (produces turbidity)

• Most species are phototrophs, autotrophs.

• As algae grows, pH tends to increase

e.g., HCO3 + H2O  CH2O + O2 + OH

• A pH greater than 8.5 to 9 is detrimental to algae growth.

(a few species can continue to extract inorganic carbon from water until pH reaches 1011.)

• Algae generally prefer a near neutral to alkaline pH.

• Algae (C:50%, N: 10%, P:2%)

Under nitrogen or phosphorous limiting conditions, the rate of Algae growth is reduced.
 1.4 Eukarya (Eukaryotes) - Protozoa
√ Protozoa
• Single-celled, heterotropic eukaryotes that can pursue and ingest their food.

• Generally feed on bacteria and other small particulate matter.

• Can be differentiated according to their method of locomotion.

• In biological treatment systems, protozoa act to polish effluent streams by helping

to cleanse them of fine particulate materials that would otherwise leave in the
effluent.
- These organisms ingest colloidal and soluble organic material and typically help to
clarify secondary effluent. In their absence colloid concentrations are higher.

Group Common name Locomotion

Sarcodina Ameba Pseudopodia
Masigophora Flagella One or more flagella
Ciliophora Ciliates Cilia
Sporozoa nonmotile
 1.4 Eukarya (Eukaryotes)
– Other Multicellular Organisms

• Mutulcellualr, strictly aerobic and ingest small particulate organic matters

(bacteria, algae, other living or dead organic particles of similar size).
 1.5 Viruses
• Viruses are generally not considered to be "living" entities, as they are unable, on
their own, to replace their parts or to carry out metabolism.

• Viruses range in size from about 15-300 nm.

• Bacteriophages
- Viruses that infect prokaryotic cells
- They are prevalent in biological wastewater treatment systems.
- When bacteria are infected with phages, the bacteria cell bursts open, releasing them for
infectious of other cells. So they have been suspected of causing process upsets by killing
needed bacteria.
1.6 Infectious Disease
 1.6 Infectious Disease

√ Protozoan-related diseases
• Giardia lamblia (Giardiasis –a foul smelling diarrhea)

- Giardia cysts are resistant to disinfection.

- Warning to wilderness campers to boil or filter water taken from seemingly pristine
environments.

- 1965-81, 53 waterborne outbreaks of Giardiasis in USA

(20,000 people were affected)

• Cryptosporidium parvum

- C. Parvum oocysts is highly resistant to chlorination.

- In 1993, about 370,000 people in Milwaukee, Wisconsin, developed a diarrheal

illness (100 died)
 1.7 Biochemistry

√ Biochemistry:
the study of chemical processes (reactions) within and relating to living organisms

• Microorganisms grow at the expense of potential energy stored in inorganic or organic

molecules or present in radiant energy (light)

• In order to obtain the energy, organisms transform the chemicals through oxidation and
reduction reactions.
- Enzymes catalyze all the key reactions

• Only through an understanding of the microorganisms' energy-yielding and energy-

consuming reactions (biochemistry), we can create the conditions that lead to
environmental protection and improvement.
 1.8 Enzymes
√ Enzymes: Organic catalysts produced by microorganisms
• Speed up the rate of the thousands of energy-yielding and cell-building reactions

• The largest and most specialized group of protein molecules within the cell.
• M.W: generally 10,000 ~ a million
• Structure: primary, secondary, and tertiary structure
• Enzyme denaturation or inactivation: by heat or chemicals
 1.8 Enzymes
• Nomenclature : commonly named by adding the suffix –ase
e.g., dehydrogenase, hydroxylase, proteinase, oxido-reductase, etc.

• Major source of energy : Redox (reduction & oxidation) reactions

• Redox reactions involve

the transfer of electrons
- Primary electron donor
- Electron carrier (shuttle)
- Terminal electron acceptor
 1.8 Enzymes - Enzyme Reactivity
√ Enzyme reaction: lock-and-key fashion

• The rate (or kinetics) of an enzyme-catalyzed reaction is governed by the same principles
that govern other chemical reactions.

• The complicated biological mechanisms discussed are rarely known in sufficient detail to
allow formulation of an analytical kinetic expression.

• Certainly in wastewater treatment where the biomass is bacteriological zoo and the
substrates are a mixture of household and industrial wastes, any kinetic expression for
the biological reaction rates must be based upon a number of simplifying assumptions.

• Microorganisms are “bags full of enzymes” so that it is not surprising that the growth rate
of microorganisms (Monod equation) is related to the reactions of the catalysts (enzymes)
that mediate many reactions (Michaelis and Menten equation).
 1.8 Enzymes - Enzyme Reactivity

√ Michaelis and Menten model

• Assumption:
- The substrate S is reversibly combined with enzyme E to form a complex ES
- The reverse reaction between free product (P) and enzyme (E) is negligible during the
initial course of the reaction.
- The transformation of the ES complex into the free product (P) and Enzyme (E) is rate-
determining
 1.8 Enzymes - Enzyme Reactivity
• General theory of enzyme reaction and kinetics: Michaelis-Menten equation

k1
E  S ES [1.3]
k 1
k2
ES EP [1.4]

• The rate of formation of ES from equation 1.3:

dES
 k 1 (E 0  ES )S [1.5]
dT
E0 : total enzyme conc. k : rate constant
E 0  ES = free enzyme conc.

• The rate of breakdown of ES:

dES
  k 1 ES  k 2 ES [1.6]
dt
 1.8 Enzymes - Enzyme Reactivity
• At steady state : equation [1.5] is equal to [1.6]

k1 (E0  ES)S = k-1ES + k2 ES [1.7]

S(E 0  ES ) k 1  k 2
  KM [1.8]
ES k1
E0  S
ES  [1.9]
KM  S

√ KM: Michaelis-Menten coefficient

• affinity between the substrate and the enzyme
• a low value of KM: strong affinity
• a high value of KM: poor affinity
 1.8 Enzymes - Enzyme Reactivity

v  dP / dt  dS / dt  k 2 ES [1.10]
• v: the rate of formation of product P

k 2 E0S
v [1.11]
KM  S
• If the substrate concentration is very high (KM << S)

vm  k2 E0 [1.12] vm: maximum velocity

• Dividing equation 1.11 by 1.12

S
v  vm [1.13]  Michaelis-Menten equation
KM  S

• quantitative relationship between the substrate concentration and the reaction rate
 1.8 Enzymes - Enzyme Reactivity

• For an important case where v=1/2vm,

1 𝑆
= [1.14]
2 𝐾𝑚 + 𝑆
𝑣𝑚
𝑆 = 𝐾𝑚 , 𝑤ℎ𝑒𝑛 𝑣 = [1.15]
2

• Thus, the coefficient, Km equals the

substrate concentration S at which
the velocity of the reaction is one-half
of the maximum velocity.
(The unit of Km = The unit of S)
 1.8 Enzymes - Enzyme Reactivity

• When enzyme transforms more than one substrate, the value for Km and Vm are different
for each substrate.

• For a single substrate–enzyme reaction, Km is generally 10-5 ~ 10-2 M. Then, it only

requires 10-5 ~ 10-2 M of S to allow an enzyme to operate half of Vm.

𝒗𝒎
• If 𝑲𝒎 ≫ 𝑺 , 𝒗 = 𝑺 = 𝑲′ [𝑺]  First-order decay
𝑲𝒎

• If 𝑲𝒎 ≪ 𝑺 , 𝒗 = 𝒗𝒎  Zero-order decay
 1.8 Enzymes - Enzyme Reactivity

√ Effects of pH and temperature on enzyme reactivity

• pH
- Many enzymes have optimal activity at neutral pH
The enzyme activity decreases with either increasing or decreasing pH from this optimal
point.
- Some others have optima at higher or lower pH values.

• Temperature
- Rates of reaction roughly double for each 10℃ increase intemperature
- Greater than the optimum temperature: the enzyme begins to denature and the enzyme’s
activity then deteriorates and ceases.
 1.8 Enzymes - Enzyme Reactivity
• Chemical agents reduce the enzyme reactivity
(So toxic chemicals can adversely affect a biological treatment process.)
• Chemical agents does not destroy the enzyme, and the reactivity can be reversed if the
agent is removed (reversible inhibition)
• Reversible inhibition : competitive and noncompetitive inhibition

• Competitive inhibition : a chemical that is similar in structure to the normal enzyme

substrate competes with the substrate for the active site on the enzyme

E + S E S

E + I E I

S
v  vm [1.16] I : concentration of inhibitor
I
K M (1  ) S KI : competitive inhibition coefficient
KI
 1.8 Enzymes - Enzyme Reactivity

• Noncompetitive inhibition
-The chemical agent acts by complexing a metallic activator or binding at a place on the
enzyme other than the active site. The enzyme is then less active toward its substrate

E + S
E S

E + I E
I

S 1 I : concentration of inhibitor
v  vm  [ 1 .1 7 ]
K  S I KI : competitive inhibition coefficient
M 1
KI
 Homework

Derive the equations below (1.16 & 1.17) based on the enzyme
reactions including those of the inhibitor

S S 1
v  vm [1.16] v  vm  [ 1 .1 7 ]
I K  S I
K M (1  ) S M 1
KI KI

And KI = ?
 1.9 Energy Capture
√ Electron and Energy Carriers
• All living organisms, including the microorganisms, capture energy released from
oxidation-reduction reactions.
• Electrons are transferred from primary e-donor to the final e-acceptor via e- carriers
(e.g., NAD+/NADH, NADP+/NADPH).
• The transfer steps have a free-energy release that the cells capture in the form of
energy carriers (ATP-ADP).
 1.10 Metabolism
√ Metabolism : The total sum of all the chemical processes of the cell

• Catabolism (이화작용): energy-generating reactions

- All the processes involved in oxidation of substrates or use of sunlight in order to obtain
energy
- It provides the energy required for 1) anabolism, 2) motion, and 3) any other energy-
requiring processes

• Anabolism (동화작용): biosynthetic reactions

- All the processes for the synthesis of cellular components from carbon sources

• Metabolites : Intermediates that form in metabolism

• Energy coupling : transfer of energy between catabolism and anabolism
 1.10 Metabolism
Stage I
Degradation of large or complex molecules

Stage II
Converted into a smaller number
of simpler compounds

Catabolism ★ fatty acids and amino acids

 acetyl-CoA
/ Anabolism
★ hexose and pentose sugars and glycerol
 glyceraldehyde-3-phosphate
and pyruvate
 acetyl-CoA

Stage III
Electron (NADH)
Ultimately Oxidized to CO2 and H2O
Energy (ATP)
“the citric acid cycle” or “Krebs cycle”
Stage III generates the largest amounts of
electrons and energy for the cells
1.10 Metabolism

Source: Wikipedia
 1.10 Metabolism - Catabolism

√ Catabolism in chemotrophs depends on redox reactions

• Oxidation removes electrons, and reduction adds electrons

• Electron donors
- materials that are oxidized

- considered to be the energy substrate or “food”

- compounds containing carbon in a reduced state : organic chemicals

- compounds containing other elements in a reduced state : reduced inorganic compounds, such as
ammonia, hydrogen, or sulfide

• Electron acceptors
- materials that are reduced

- include primarily oxygen, nitrate, nitrite, Fe(III), sulfate, and carbon dioxide

- chlorate, perchlorate, chromate, selenite, and chlorinated organics (tetrachloroethylene,

chlorobenzoate)

 environmental concern, growing interest

 1.10 Metabolism - Catabolism

√ The quantity of energy : depends upon the chemical properties of the electron
donor and acceptor

△G0′ : the free energy released under standard condition (at pH 7)

= -106.12 kJ/e- eq.
 1.10 Metabolism - Catabolism
• Methanogenesis, an anaerobic process, provides much less energy per electron
equivalent than the aerobic oxidation does.
• Glucose contains more energy than acetate
 1.10 Metabolism - Catabolism
• The chemolithotrophic oxidation of hydrogen produces more energy than
chemoorganotrophic oxidation of acetate, but less than that of glucose
-27.40 - 78.72 = -106.12 kJ/e- eq for acetate and oxygen

- 41.35 - 78.72 = -120.07 kJ/e- eq for glucose and oxygen

 1.10 Metabolism - Catabolism
√ Relative free energy available from
oxidation/reduction couples

• “Fermentation“
- Use of organic compounds as electron donors and acceptors

e.g., facultative or anaerobic bacteria

- glucose (reactant) – ethanol (product)

- glucose (reactant) – acetate (product)

-120 kJ/e- eq

• Objective of catabolic reaction

- To capture as much of the energy released as possible