1

Chapter 1.

Introduction
(Carl von Linne)
-1750

(Robert H. Whittaker)
- 5

(Ernst H. Haeckel)
-

(Carl Wose)
-3


(Procaryote) (Eucaryote)

2.1

(Robert H. Whittaker)

Virus
:
.

3
(Carl Wose):

(Species)
(Genus)
(Family)
(Order)
(Class)
(,
Division)
(Kingdom)
(Domain)


Carolus Linnaeus (1707-1778)
: (Binomial nomenclature)

Saccharomyces cerevisiae( )
genus ( )

species( )

Basis for name

Saccharomyces cerevisiae (sugar fungus)
Clostridium acetobutylicum (acetone butanol)
Acetobacter aceti (veniger)
Streptococcus thermophilus ( )
Color
Aspergilus niger ( )
Size
Bacillus megaterium ( )

Trichodima reesei (Reese )
Geographic location Lactobacillus sanfrancisco

 (International Code of
Nomenclature of bacteria) .
(International Code of Botanical
nomenclature) .
(scientific name) 2
( ) , .
.
.
(underline) .

Woese (1977 )
16S ribosomal RNA 18S ribosomal RNA
2 .

Three domain
1. (archaebacteria, archaea):
methanogenes, extreme thermophiles, extreme halophiles,
2. (eubacteria, bacteria):
, , cyanobacteria
3. (eucaryotes, eucarya):
, , , (protozoa)


1.

.
.
.

2.

.
(form genus) .

3.
4.

DNA nucleotide
.
, , cytochrome ,
, lysine
.

5.

organelle , ,
parameter .

6.


1.

: , , , ,
: ( colony)

2.

, ,

3.

, indole , H2S , , oxidase

, , gelatin ,
(methyl red ), acetylmethyl carbinol (VogesProkauer ), ,

4.

, ,

5.

( , , )
parameter .

6.

nucleotide .
nucleotide ,
DNA , DNA
, .

 :
-
(Transmission Electron Microscope, TEM)
- 2000
(Scanning Electron Microscope, SEM)
- 10

- (water)
- (proteins)
- (lipids)
- (carbohydrates)
- (nucleic acids)
-


(Carbohydrates)


(Lipids)


(Proteins)

1.

(C), (H), (O), (N) (S) .

2.

, ,
, , .

3.

1g 4kcal .


(Amino acid)

 (Proteins)
(1) 1 :
(2) 2 :

ex) , , .

c=o
4 N-H
_-

_-

 (Proteins)
(3) 3 :

2 .
R ( , ,
, ) .
ex) , , .

(4) 4 :
3
.

R 3

.
ex)

(Nucleic acids)
DNA
RNA


1)
- (macronutrient): (95%
)
-> C, H, O, N, S, P, K, Ca, Mg, Fe
-> C H O N S P: , , ,
-> K:
-> Ca: ,
-> Mg: , ,
-> Fe: cytochrome ,
- (trace element, micronutrient): ,
,
-> Mn, Zn, Co, Mo, Ni, Cu
-
- ->

2) C,H,O
- , ,
- -> ->

Autotroph( ):
-> CO2
-> ( ), ( )

Heterotroph( )
-> (glucose)
->

: -> ,
-> , , , ( , ), (
)

3)
-
Phototroph( ): ( )
Chemotroph( ):
-
Lithotroph( ):
Organotroph( ):
- , ,

, ,

Photolithotrophic autotroph

, , CO2

Photoorganotrophic
heterotroph

, ,

Chemolithotrophic autotroph

, , CO2

Chemoorganotrophic
heterotroph

, ,

4) N, P, S
- :
-> , , , , ,

-> , (NH4), (NO3)

- :
-> , , ,
-> (PO4)
- :
-> ( , ), , thiamine
-> (SO4)
5) Growth factor( )
-
-
- 1) :
- 2) :
- 3) :
- ,


1. (pure culture)
-

->
-> colony
(splead plate) -> agar plate
(streak plate) -> agar plate
(pour plate) -> agar

Clean Bench (Laminar Flow)

2. (Difco manual )
-

1) (defined medium), (synthetic

medium)
-
- C,N,S,P,
-
2) (complex medium)
-
-
- peptone, yeast extract, malt extract
3) (selective medium)
- ( , , )
-
4) (differential medium)
-



Oven 1700C 90

1200C, 2 20

CH2 CH2 + H-OH

O
Ethylene oxide

UV,

CH2 CH2
OH
OH
Ethylene glycol

Filtration

 (Pure culture)

Pure Culture Techniques

Quadrant Streak

Radiant Streak

T Streak

Continuous Streak

Colony

a. Micrococcus, b.
Clostridium ,
c. Mycoplasma, d. E. coli

Chapter 2.

: /
:
(carrier protein)


,
(channel protein) : (ion channel)


,
, ATP ,


1.
- ->
- ->
-
1) Diffusion( )
- Passive diffusion( ):
->
->
->
-> , ,
,

- Facilitated diffusion( )
->
-> (permease)
-> (glycerol), (sugar)
,


(1) (passive transport):
(Facilitated diffusion): ; ,
(carrier): , Glucose transporter
(channel): , Na+, K+, Cl-, Ca2+-gated channel

(2) (active transport): ,

: Na+ - K+ ATPase (pump)

2) Active transport( )
-

-> ABC transporter(ATP-binding casette)

Symport( ):
Antiport( ):


(1) (coupled transporter)
,
(2) ATP (ATP-depnedent pump)
ATP ,
Na+-K+ ATPase Ca2+ ATPase H+ ATPase

3) Group translocation( )
-
- PTS (phosphotransferase system; )
- PEP(phosphoenolpyruvate)


- ( )
-Siderophore :

, Siderophore
Fe3+
Fe(OH)3
.
.

Chapter 1.

BIODEGRADATION of
HYDROCARBON

1. (Crude Oil) ?

,
(hydrocarbons, >75%) (Sulfuric compounds, 4%),
(oxygenic compounds, 2%), (nitrogenic compounds, 1%),
(V, Fe, Ni, Cu, K, Na, Ca, As, Si) .
.
unit : percent, except for metals(ppm)
Group

Compound

Gasoline

Diesel

Light Crude

Heavy Crude

Bunker C

Alkanes

45-55

35-45

Cyclo alkanes

30-50

Waxes

0-1

0-20

0-10

5-15

Total

50-60

65-95

55-90

25-80

20-30

BTEX

15-25

0.5-2

0.1-2.5

0.01-2

0-1

PAHs

0-5

10-35

15-40

30-50

Total

25-40

5-25

10-35

15-40

30-50

Resins

0-2

0-10

2-25

10-20

Asphaltenes

0-10

0-20

5-20

Metals

30-250

100-500

100-2000

Sulfur

0.02

0.1-0.5

0-2

0-5

2-4

Saturates

Aromatics

Class

2. Biodegradation :
, Biodegradation (Carbon Flux)

, (Mineralization; CO2 & H2O), (Biotransformation)

.
Mineralization
Mineralization

Biomass
Biomass

CO2++HHO
2O
CO
2
2
HC
2
HC++OO
2

Carbon Flux by
Biodegradation

++N/P
N/P++

Biotransformation
Biotransformation

oror
(Acids,Ketons,
Ketons,
(Acids,
Alcohols..)
Alcohols..)

Feedback
Inhibition

Residues
Residues
( (
: :
Cyclics,
Cyclics,PAHs..)
PAHs..)

,
,
(Feedback Inhibition).
, ,
( ).
.

 2000
. ,

Hydrocarbon
nAliphatics
(Alkanes)

iso-

Key enzyme

CH3

(CH2)n

CH3

CH3 -CH -(CH2)n -CH- CH3

CH3

CH3

Mono-Oxygenase

cyclomonoAromatics

DiPAHs

Di-Oxygenase

Pseudomonas
Nocardia
Acinetobacter
Corynebacterium
Rhodococcus
Candida, Yarrowia
Pseudomonas
Nocardia
Aeromonas
Alcaligenes
Micrococcus
Mycobacterium
Sphingomonas

1 oxygenase enzyme

3. Hydrocarbon Uptake Mode :

Direct contact

H2O

H2O
biosurfactant

(Pseudosolubilized oil)

Emulsifying Activity (OD at 610nm)

H2O

1.6

Strains

Uptake
Mode

WLH-3

1.4

DJ-3

1.2

WLH-1

WLH-2

1
DJ-1

0.8

CL180

Group 1

WLH-2,3
DJ-3

EA , HP

Pseudosolubilize
d

Group 2

WL-1, 2

EA , HP

Direct
Contact

Group 3

KH3-2, DJ-2

EA , HP

Solubilize
d

Group 4

WLH-1, DJ-1
CL180, IC-10
YS-7

MIXED

MIXED

IC-10

0.6

KH3-2

YS-7

0.4

WL-2
WL-1

0.2
DJ-2

0
0

20

40

60

80

Hydrophobicity (%)

100

EA : Emulsifying Activity
HP : Hydrophobicity

Main principle of aerobic degradation of

hydrocarbons:
growth associated processes.
(1)Metabolic processes

for optimizing the

contact between the microbial cells and
the organic pollutants. The chemicals must
be accessible to the organisms having
biodegrading activities. For example,
hydrocarbons are water-insoluble and their
degradation requires the production of
biosurfactants.

(2)The initial intracellular attack of organic

pollutants is an oxidative process, the
activation and incorporation of oxygen is
the enzymatic key reaction catalyzed by
oxygenases and peroxidases.
(3)Peripheral degradation pathways convert
organic pollutants step by step into
intermediates of the central intermediary
metabolism, e.g., the tricarboxylic acid
cycle.
(4)Biosynthesis of cell biomass from the
central precursor metabolites, e.g., acetylCoA, succinate, pyruvate, Sugars required
for various biosyntheses and growth must
be synthesized by gluconeogenesis.

Initial attack on xenobiotics by oxygenases

Growth-Associated Degradation of Aliphatics

Fig. Peripheral pathways of

alkane degradation. The main
pathway
is
the
terminal
oxidation
to
fatty
acids
catalyzed
by
n-alkane
monoxygenase,
alcohol
dehydrogenase and aldehyde

MECHANISM
Round

CH3CH2CH2CH2CH2CH2CH2C~S-CoA
O
Dehydrogenas
e

FAD
TRANS

HO

Hydratase

-C- CH2-C~S-CoA
H
O

Dehydrogenase
Acyl
Transferase

H
-C=C-C~S-CoA
H
O

Cofactor or
Substrate

-C- CH2-C~S-CoA
O
O

R -C...CH3-C~S-CoA
O
O
S-CoA

H2 O
LNAD+

HS-CoA

Growth-Associated
Degradation of
iso-alkanes
(,-Oxidation)

Degradation
of
a
broad
spectrum of aromatic natural
and xenobiotic compounds into
two central intermediates:
Catechol and protocatechuate.

Knoops Experiment
Diet
(even chain)

(odd chain)

CH2CH2CH2COO

CH2CH2COO

Urine

CH2COO
Phenylpyruvate
Phenylacetate

COO
Benzoate
Benzoate

Transport into Mitochondria depends on Carnitin

+
FA~CoA

Acyl transferase I

CH2

HS-CoA
FA~Carnitine

N(CH3)3

Carnitine

H-C-OH
CH2

Translocase

COOCarnitine

FA~Carnitine

Carnitine

HS-CoA
FA~CoA

Acyl transferase II

THE ENERGY STORY

PART I
Glucose
C6H12O6 + 6O2 6CO2 + 6H2O

Ho = -2,813 kJ/mol
= - 672 Cal/mol
= 3.74 Cal/gram

Stearic Acid
C18H36O2 + 26O2 18CO2 + 18 H2O

Ho = -11,441 kJ/mol
= - 2,737 Cal/mol
= 9.64 Cal/gram

On a per mole basis a typical fatty acid is 4

times more energy rich that a typical
hexose

Energy Story Part II

1.0 g glucose
= 3.7 kcal (15.5 kJ)
1.0 g stearic acid = 9.7 kcal (40.5 kJ)

ENERGY CONSERVATION
Stearic Acid (C18 satd)
9 Acetyl CoA
8 FADH2
8 NADH

= 108 ATP
= 16 ATP
= 24 ATP
= 148 ATP
- 1 ATP
147 ATP

Palmitoyl-CoA + 7CoA + 7FAD + 7NAD + + 7H2O

8 Acetyl-CoA
7 FADH2
7 NADH + 7H+

80 ATP
10.5 ATP
17.5 ATP
108 ATP

Octoyl-CoA + 3HSCoA + 3FAD + 3NAD + + 7H2O

R-3
R-2
R-1

CH3CH2 CH2CH2 CH2CH2 CH2CO~SCoA

FAD
NAD+
HS-CoA

FAD
NAD+
HS-CoA

4 Acetyl-CoA
3 FADH2
3 NADH + 3H+

FAD
NAD+
HS-CoA
40 ATP
4.5 ATP
7.5 ATP
52 ATP

CH3CH2 CH2CH2 CH2CH2 CH2CO~SCoA

FAD
NAD
HSCoA

CH3CH2 CH2CH2 CH2CO~SCoA

FAD
NAD
HSCoA

CH3CH2 CH2CO~SCoA
FAD
NAD
HSCoA

CH3CO~SCoA

CH3CO~SCoA

Hexanoic acid (C6H12O2)

Hexanoic acid
Hexanoyl-CoA

Glucose
-1 ATP

2 pyruvates

2 ATP

2 NADH + H+

5 ATP

2 pyruvates

Hexanoyl-CoA
3 Acetyl-CoA
2 FADH2
2 NADH + H+

Glucose (C6H12O6)

30 ATP
3 ATP

2 Acetyl-CoA

20 ATP

2 NADH + H+

5 ATP

5 ATP
37 ATP

32 ATP

Mwt = 116

Mwt = 180

ATP per Gram = 0.32

ATP per Gram = 0.17

Table. Comparison of experimental molar growth yield with theoretical molar

growth yield predicted on the basis of available electrons for selected
organic compounds.

a. Av. e2 is the number of available electrons mol21 substrate.

b. Ym exp is the molar yield determined experimentally.
c. Yav.eexp is the equivalent electron yield calculated according to the Payne relation (Yav.eexp. Ym exp/av. e2). It is expressed as g of dry cell weight per available electron.
d. Ytheo m is the theoretical molar yield determined using theoretical values of Yav.e-.
e. The energy discrepancy index (de) is the ratio giving theoretical values to the experimental values (de . Ytheo m /Ym exp . Yav. e-/Yav. eexp). A de value .1 indicates that
energy is dissipated during the metabolism.
f. From Payne (1970).
g. For regular substrates, Yav.e- . 3.07 gdw e2 av.21 (Payne and Wiebe, 1978).
h. From Johnson (1967).
i. For hydrocarbons, Yav.e- . 2.08 gdw e2 av.21 (Payne, 1978).
j. From Tidswell et al. (1996).
k. This study.
l. From Hanson et al. (1999). MEGDE, monoethylene glycol dodecyl ether; DEGDE, diethylene glycol dodecyl ether; TEGDE, triethylene glycol dodecyl ether; OEGDE,
octaethylene glycol dodecyl ether. ND, not determined.

1)
- : 4,410 m3
- (ppm) : 5,000 ppm
2)
- (MPN)
-
- MPN
- ( )
-
3)
-
-
-
-

- (C16H34)
-
-
4) N
- C:N:P 100:10:1( )
-
-
- (NH4)2SO4
- (NH4)2SO4
- (NH4)2SO4
5) P
-
-
- KH2PO4
- KH2PO4
- KH2PO4

- C:N:P = 100:10:1
- (ppm) : 1,500 ppm
Ma (MPN/g)
Q (m3)
Mb=MaXQX1.8X1000000
L (MPN/kg)
W = Mb/L (kg)
Q (m3)
C1(ton)
C2(ton)
S(ton)

1.E+06
4410
7.938E+15
6.E+11
13,230
4410
31.51
11.91
19.60

Mbe (mol)= (12x16+1x34)

Abe(kg-mol) = S / Mbe
Ac = Abe x 16( )

226.00
86.72
1387.53

R
N(kg-mol) = Ac x R
Na(kg-mol) = N/2
Mam = ((14+1x4)x2+32+16x4)
Sa (kg) = Na x Mam

0.10
138.75
69.38
131.00
9088.35

R2
P (kg-mol) = Ac x R2
Pp (kg-mol) = P/1
Mp = (39+2+31+16x4)
Sp (kg) = Pp x Mp

0.01
13.88
13.88
136.00
1887.05

1. 1 1 3 1
?
2. C6H12O6(Glucose) + O2 CO2 + H2O
- , , , ?
- Respiration coefficient (RQ = CO2 eliminated / O2 consumed) ?
3. C6H34(Hexadecane) + O2 CO2 + H2O
- , , , ?
- Respiration coefficient (RQ = CO2 eliminated / O2 consumed) ?
4. C6H12O6(Glucose) + NH3 + O2 CH1.74N0.2O0.45(Biomass) + CO2 + H2O
- , , , , ?
- Glucose Biomass (g Biomass / g Glucose) ? Biomass (23.74
g/mol)
5. C6H34(Hexadecane) + NH3 + O2 CH1.74N0.2O0.45(Biomass) + CO2 + H2O
- , , , , ?
- Biomass (g Biomass / g Hexadecane) ? Biomass (23.74 g/mol)
6. C6H12O6(Glucose) + NH3 ( ) CH1.74N0.2O0.45(Biomass) + 0.43
C3H8O3(Glycerol)
+ 1.54 CO2 + 1.3 C2H5OH(Ethanol) + H2O
- , , ?
- Glucose Biomass (g Biomass / g Glucose)
Ethanol (g Ethanol / g Glucose) ? Biomass (23.74 g/mol)

7. .
- : 2,000
- : 5,000 ppm (TPH : Total Petroleum Hydrocarbon ( )
(Landfarming)

3 TPH 2,000 ppm .

, (N, P source) .

- : 1.4 ( )
- : 106 CFU/g soil
- : 108 CFU/ml ( 1 )
- : C16H34 ( ) (MW : 226)
- : (C)/ (N)/ (P) = 100/10/3
-
: (NH4)2SO4(MW : 131)
: KH2PO4 (MW : 136)
?
(NH4)2SO4 ?
KH2PO4 ?