1

Chapter 1.

 Introduction
(Carl von Linne) (Ernst H. Haeckel)
-1750 -

(Carl Wose)
(Robert H. Whittaker) -3
- 5

(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.(eucar yotes, eucar ya):
, , , (protozoa)


1. .
.
.

2.
.
(form genus).

DNA nucleotide
3.
.

, , cytochrome,
4.
, lysine
.

5. organelle , ,
parameter .

6.
.


: , , , ,
1.
: ( colony)

2. , ,

, indole, H2S, , oxidase

, , gelatin , (methyl
3.
red), acetylmethyl carbinol(Voges-Prokauer ),
,

4. , ,

(, , )
5.
parameter .
nucleotide .
nucleotide ,
6.
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

4N-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():
- , ,

,,
,, CO2
Photolithotrophic autotroph
, ,
Photoorganotrophic heterotroph
, , CO2
Chemolithotrophic autotroph
, ,
Chemoorganotrophic heterotroph

Mixotroph ()
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 CH2 CH2
O OH OH
Ethylene oxide Ethylene glycol



UV,
Filtration
 (Pure culture)
Pure Culture Techniques
Quadrant Streak

Radiant Streak

T Streak

Continuous Streak




Colony


a. Micrococcus, b. Clostridium
,
c. Mycoplasma, d. E. coli
 1

Chapter 2.

: /
:

(carrier protein)


,

(channel protein) : (ion channel)


,
, ATP ,

M

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
.
.
 2

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)

Compound
Group Gasoline Diesel Light Crude Heavy Crude Bunker C
Class

Alkanes 45-55 35-45 - - -

Cyclo alkanes 5 30-50 - - -

Saturates
Waxes - 0-1 0-20 0-10 5-15

T
otal 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

Aromatics PAHs - 0-5 10-35 15-40 30-50

T
otal 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

2. Biodegradation :

, Biodegradation (Carbon Flux)

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

Mineralization
Biomass
CO2 + H2O

HC + O2
Carbon Flux by
+ N/P + Biodegradation

Biotransformation Residues
Feedback
or
Inhibition
(Acids, Ketons, ( :
Alcohols..) Cyclics, PAHs..)

,
,
(Feedback Inhibition).
, ,
( ).
.
 2000
. ,

Hydrocarbon Key enzyme

n- CH3 (CH2)n CH3 Pseudomonas

Nocardia
Aliphatics CH3-CH -(CH2)n -CH- CH3 Acinetobacter
iso- Mono-Oxygenase Corynebacterium
(Alkanes) CH3 CH3
Rhodococcus
cyclo- Candida, Yarrowia
Pseudomonas
mono-
Nocardia
Aeromonas
Aromatics Di- Di-Oxygenase Alcaligenes
Micrococcus
PAHs Mycobacterium
Sphingomonas

1 oxygenase enzyme
3. Hydrocarbon Uptake Mode :

Direct contact

H2O

H2O

(Pseudosolubilized oil)
H2O biosurfactant

Uptake
Emulsifying Activity (OD at 610nm)

1.6 Strains
Mode
WLH-3
1.4
WLH-2,3 Pseudo-
DJ-3 Group 1 EA , HP
DJ-3 solubilized
1.2 WLH-1
WLH-2
1 CL180 Direct
DJ-1 Group 2 WL-1, 2 EA , HP
Contact
0.8
IC-10
0.6 KH3-2
YS-7 Group 3 KH3-2, DJ-2 EA , HP Solubilized

0.4 WL-2
WL-1
WLH-1, DJ-1
0.2 Group 4 CL180, IC-10 MIXED MIXED
DJ-2 YS-7
0
0 20 40 60 80 100 EA : Emulsifying Activity
HP : Hydrophobicity
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., acetyl-CoA,
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
dehydrogenase.
MECHANISM
Round

CH3CH2CH2CH2CH2CH2CH2C~S-CoA
O Cofactor or Substrate
H
Dehydrogenase -C=C-C~S-CoA FAD
H
O TRANS

Hydratase HO
-C- CH2-C~S-CoA H2 O
H
O L-

Dehydrogenase -C- CH2-C~S-CoA NAD+

O O
Acyl Transferase R -C...CH3-C~S-CoA
HS-CoA
O O
S-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 COO

Phenylacetate
Phenylpyruvate Benzoate
Benzoate
Transport into Mitochondria depends on Carnitine

+
N(CH3)3
FA~CoA
Acyl transferase I
CH2
HS-CoA
Carnitine
FA~Carnitine H-C-OH

CH2

Translocase COO-
Carnitine

FA~Carnitine Carnitine
HS-CoA
Acyl transferase II
FA~CoA
 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 = 108 ATP
8 FADH2 = 16 ATP

8 NADH = 24 ATP
= 148 ATP
- 1 ATP
147 ATP
Palmitoyl-CoA + 7CoA + 7FAD + 7NAD+ + 7H2O

8 Acetyl-CoA 80 ATP
7 FADH2 10.5 ATP
7 NADH + 7H+ 17.5 ATP

108 ATP

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

R-3 R-2 R-1

CH3CH2 CH2CH2 CH2CH2 CH2CO~SCoA

FAD FAD FAD
NAD+ NAD+ NAD+
HS-CoA HS-CoA HS-CoA

4 Acetyl-CoA 40 ATP
3 FADH2 4.5 ATP
3 NADH + 3H+ 7.5 ATP

52 ATP
 3 2 1

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) Glucose (C6H12O6)

Hexanoic acid Glucose

Hexanoyl-CoA -1 ATP 2 pyruvates 2 ATP

2 NADH + H+ 5 ATP

Hexanoyl-CoA 2 pyruvates

3 Acetyl-CoA 30 ATP 2 Acetyl-CoA 20 ATP

2 FADH2 3 ATP 2 NADH + H+ 5 ATP
2 NADH + H+ 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 - C:N:P = 100:10:1
- (ppm) : 5,000 ppm - (ppm) : 1,500 ppm
2)
- (MPN) Ma (MPN/g) 1.E+06
- Q (m3) 4410
- MPN Mb=MaXQX1.8X1000000 7.938E+15
- () L (MPN/kg) 6.E+11
- W = Mb/L (kg) 13,230
3)
- Q (m3) 4410
- C1(ton) 31.51
- C2(ton) 11.91
- S(ton) 19.60

- (C16H34) Mbe (mol)= (12x16+1x34) 226.00
- Abe(kg-mol) = S / Mbe 86.72
- Ac = Abe x 16() 1387.53
4) N
- C:N:P 100:10:1()
- R 0.10
- N(kg-mol) = Ac x R 138.75
- (NH4)2SO4 Na(kg-mol) = N/2 69.38
- (NH4)2SO4 Mam = ((14+1x4)x2+32+16x4) 131.00
- (NH4)2SO4 Sa (kg) = Na x Mam 9088.35
5) P
- R2 0.01
- P (kg-mol) = Ac x R2 13.88
- KH2PO4 Pp (kg-mol) = P/1 13.88
- KH2PO4 Mp = (39+2+31+16x4) 136.00
- KH2PO4 Sp (kg) = Pp x Mp 1887.05
1. 1 13 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 ?