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Microbial Growth Kinetics http://users.rowan.edu/~jahan/sophclinic/.../8%20JC
%20MicrobialGrowth.ppt
http://www.montana.edu/wwwmb/coursehome/mb433/Lectures/Lecture3.ppt
AJE Lecture Note
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Growth of Microbes • Increase in
number of cells, not cell size
• One cell becomes colony of millions of cells
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Growth of Microbes • Control of growth is important for
– infection control – growth of industrial and biotech organisms – Biodegradation process
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Factors Regulating Growth
• Nutrients • Environmental
conditions: temperature, pH, osmotic pressure
• Generation time
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Chemical Requirements • #1 = water! • Elements
– C (50% of cell’s dry weight) HONPS – Trace elements
• Organic – Source of energy (glucose) – Vitamins (coenzymes) – Some amino acids, purines and
pyrimidines
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Nutritional Categories
• Carbon sources – CO2 = autotroph – organic = heterotroph
• Energy sources – sunlight = phototroph – organic = chemotroph
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Chemoautotrophs
Inorganic C Organic carbon
Electron donor Carbon source
Electron acceptor O2 aerobic respiration H2O NO3, Fe(III), Mn(IV), SO4, CO2 anaerobic respiration
NH4+, Fe(II), Mn(II), H2S
Carbon dioxide
N2O, Fe(II), Mn(II), H2S, CH4
Energy source (electron donor):inorganic carbon Carbon source: inorganic carbon
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Chemoheterotrophs
Organic C Carbon dioxide
Electron donor Carbon source
Electron acceptor O2 aerobic respiration
H2O
NO3, Fe(III), Mn(IV), SO4, CO2 anaerobic respiration
Energy source (electron donor):organic carbon Carbon source: organic carbon
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Photoautotrophs
Inorganic C (CO2) Organic carbon Carbon source, electron acceptor
H2O O2
Light (λ)
Carbon dioxide
Electron donor
Electron donor
Energy source Water
Energy source: light Electron donor: water Carbon source: inorganic carbon
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Photoheterotrophs
Organic C CO2 Carbon source
Fe-S clusters in Photo System 1
Light (λ)
Organic C
Energy source
Terminal electron acceptor
Electron donor Water
Energy source: light Electron donor: water Carbon source: organic carbon
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Environmental Factors Influencing Growth
• Temperature • O2 • pH • Osmotic Pressure • Others: radiation, atmospheric pressure
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Temperature Optima
• Psychrophiles: cold-loving • Mesophiles: moderate temperature-loving • Thermophiles: heat-loving • Each has a minimum, optimum, and
maximum growth temperature
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Fig. 7.8
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Temperature Optima
• Optimum growth temperature is usually near the top of the growth range
• Death above the maximum temp. comes from enzyme inactivation
• Mesophiles most common group of organisms
• 40ºF (5°C) slows or stops growth of most microbes
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Oxygen Requirements
• Obligate aerobes – require O2
• Facultative anaerobes – can use O2 but also grow without it
• Obligate anaerobes – die in the presence of O2
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pH • Most bacteria grow between pH 6.5 and
7.5 • Acid (below pH 4) good preservative for
pickles, sauerkraut, cheeses • Acidophiles can live at low pH
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Measuring Bacterial Growth
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Bacterial Division
• Bacteria divide by binary fission • Alternative means
– Budding – Conidiospores (filamentous bacteria) – Fragmentation
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Fig. 7.13
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Generation Time
• Time required for cell to divide/for population to double
• Average for bacteria is 1-3 hours • E. coli generation time = 20 min
– 20 generations (7 hours), 1 cell becomes 1 million cells!
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Fig. 7.14a
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Plotting growth on graphs
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Standard Growth Curve
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Phases of Growth
• Lag phase – making new enzymes in response to new medium
• Log phase – exponential growth – Desired for production of products – Most sensitive to drugs and radiation during
this period
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Phases of Growth
• Stationary phase – – nutrients becoming limiting or waste products
becoming toxic – death rate = division rate
• Death phase – death exceeds division
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Measuring Growth
• Direct methods – count individual cells • Indirect Methods – measure effects of
bacterial growth
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Fig. i7.6
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Fig. 7.17
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Turbidity
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Metabolic Activity
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Dry Weight
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Energy Yield In a chemical reaction, only part of the energy is used to do work.
Energy available for work is called “free energy” or ΔG. The rest of the energy is lost to entrophy. ΔG = -RT log Keq where Keq = [C] [D] / [A] [B] from rxn:
A + B C + D
If logKeq is a negative value, this means the reaction can only proceed if energy is added (endothermic rxn).
When logKeq is a negative value, ΔG is positive. If logKeq is a positive value, this means the reaction is favored and, in fact, gives off energy (exothermic rxn).
When logKeq is a positive value, ΔG is negative.
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Energy yield from electron acceptor
• 6O2 6H2O aerobic respiration -686 • 24 NO3 12N2 anaerobic respiration -36
• SO4 H2S anaerobic respiration -40 • CO2 CH2O photosynthesis +115
ΔG (kcal/mol)Terminal electron acceptor
e-
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Reduction Potential vo
lts
+0.85
+0.75
0.00
-0.22 -0.47 CH2O/CO2
SO4/H2S
NO3/ N2
O2/H2O
1.28 1.22
0.25
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Energy yield relationship between electron acceptor and electron donor Electron Reduction Electron Reduction Difference Acceptor Potential (V) Donor Potential (V) (V) O2 H2O +0.81 CH2O CO2 -0.47 -1.28 NO3 N2 +0.75 -1.22 SO4 H2S -0.22 -0.25
The sign and magnitude of the difference represents how much free energy is available to the cell.
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Growth Kinetics
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Pertumbuhan Bakteri
lag log stasioner Endogenous
Concentration
Waktu
bakteri
Makanan/substrat
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Pertumbuhan bakteri
• Pers matematis pertumbuhan bakteri : Persamaan Monod (1920)
µ = µmaksS/(Ks + S) dimana:
µ = laju pertumbuhan spesifik, 1/waktu S = konsentrasi substrat, mg/L Ks = half saturation konstant
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Pertumbuhan Bakteri
µmaks
µmaks/2
S, mg/L Ks
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Pertumbuhan Bakteri
• Rumus umum (heterotroph bacteria): – µmaks <, Ks > : less biodegradable – µmaks >, Ks < : biodegradable – Contoh:
• Glukosa: µmaks = 0,37 – 0,77 dan Ks = 11 – 108 mg/L • Skim milk: µmaks = 0,10 – 0,12 dan Ks = 100 – 110 mg/L
• Autotroph bacteria dalam nitrifikasi: – µmaks << : lambat – Ks <<: independent
• Penentuan laju pertumbuhan dilakukan pada fase log: dX/dt = kX, Xt = X0ekt sehingga k = ln Xt – ln X0/(t – t0) Dimana:
X = biomassa t = waktu k = laju pertumbuhan
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Persamaan Monod
• Mencari harga k: y = a + bx, dimana:
y = ln (A600) x = waktu a = intial ln (A600) pd t = 0 b = gradien, k
• Mencari harga Ks dan µmaks: persamaan Monod diubah menjadi
persamaan double reciprocal: 1/µ = (Ks/µmaks) (1/S) + 1/µmaks, sehingga:
– Pada saat 1/S = 0, maka 1/µ = 1/µmaks
– Pada saat 1/µ = 0, maka (Ks/ µmaksS) = - 1/µ -1/S = 1/Ks
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Persamaan Monod 1/µ
1/S
1/µmaks
1/Ks
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Persamaan Monod
• Jumlah biomass dalam reaktor menerus: dX/dt = kX – kdX, kd = koefisien decay dX/dt = ((µmaksSX)/(Ks + S)) – kdX
• Makanan: Food consumption ≅ biomass production dX/dt = rx = - y dS/dt, dimana y = decimal fraction yg menunjukkan perbandingan berat
biomass per kg substrate: - Aerobik: 0,4 – 0,8 - Anerobik: 0,08 – 0,2 dS/dt = -rx/y = (µmaksSX)/y(Ks + S)
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Cell Yield (Y)
• Not all of the carbon added as the carbon source is converted to cell biomass
• A fraction is respired as CO2 during the transformation of the carbon to energy (ATP)
• Cell yield coefficient is defined as the amount of biomass produced per unit substrate consumed
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Cell Yield
Carbon source Yield coefficient
glucose 0.4
Pentachlorophenol (PCP) 0.05
octadecane 1.49
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Activated Sludge
• Kondisi steady-state, neraca massa: – Biomassa: biomass in + biomass growth = biomass out – Food: food in – food consumed = food out
• Biomassa: Q0X0 + (((µmaksSX)/(Ks + S)) – kdX) V = (Q0 – Qw)Xe + QwXu
• Food: Q0S0 – ((µmaksSX)/y(Ks + S))V = (Q0 – Qw)S + QwS
Reaktor Secondary Clarifier
Effluent Q0, S0, X0
V, S, X Q0 + Qr X, S
Qu, Xu Qr, Xu
Qw, Xu Sludge treatment
Q0 – Qw, Xe, S
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Activated Sludge
• X0 dan Xe sgt kecil dibandingkan di reaktor, X0 dan Xe ≅ 0, sehingga persamaan neraca massa biomass menjadi:
V (((µmaksSX)/(Ks + S)) – kdX) = QwXu (µmaksS)/(Ks + S) = ((QwXu)/VX) + kd
• Penyederhanaan neraca massa food: (µmaksS)/(Ks + S) = ((Q0/V)(y/X)(S0 – S)) – kd note: V/Q0 = hydraulic retention time = θ (VX)/(QwXu) = θc = sludge age (umur lumpur)
• Susbtitusi persamaan neraca massa biomass dan food: 1/θc = (y(S0-S)/θX) – kd X = (θcy(S0-S))/(θ (1+kdθc)) θ < X besar: tdk mungkin, sehingga apabila θ terlalu singkat: wash out dan tdk ada pertumbuhan (X turun) So mendekati S: tdk ada pengolahan
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Ensimologi - 1
• Ensimologi: ilmu yang mempelajari karakteristik dan perilaku ensim • Ensim: katalis organik (biokatalis) yang dibentuk dari protein dan
dihasilkan oleh sel makhluk hidup yang sensitif terhadap perubahan temperatur
• Keberadaannya: – Extra selular: bekerja diluar sel dengan tujuan untuk mereduksi senyawa2
kompleks sehingga mudah di dialisis oleh dinding sel – Intra selular: untuk melangsungkan reaksi biokimia di dalam sel
• Berdasarkan cara diproduksinya: – Konstitutif: diproduksi secara kontinu – Indusif: diproduksi karena respon thd stimulus yang diaplikasikan dari
luar • Nomenclature: diakhiri dengan “ase”, contoh dehalogenase atau
sucrase yang merubah sucrosa menjadi glukosa dan fructosa
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Ensimologi - 2
• Aktivitas ensim tgt dari: – Kofaktor: struktur tambahan yang diperlukan oleh ensim
• Logam • Molekul organik
– Temperatur: terlalu rendah inactive, terlalu tinggi denaturasi – pH – Mikro + makro nutrien – Inhibitor dan inducer
• Bidang TL: – Immobilisasi ensim – Reaksi akan jauh lebih cepat – Target: mineralisasi bukan biotransformasi
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Ensimologi - 3
• Kinetika ensim: Persamaan Michaelis-Menten Vx = Vmax Sx/(Sx + Km) Vmax = maksismum specific activity Km = Michaelis-Menten konstan Sx = Substrat
• Enzim Specificity: kemampuan ensim untuk mendegradasi senyawa yang serupa dengan substrat utamanya
• Enzim purifikasi: Proses pemurnian ensim dalam kaitannya dengan karakterisasi ensim
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Understanding Km • Km is a constant • Km is a constant derived from rate constants • Km is, under true Michaelis-Menten conditions,
an estimate of the dissociation constant of E from S
• Small Km means tight binding; high Km means weak binding
Enzyme Substrate Km (mM) Glutamate dehydrogenase NH4
+ 57 Glutamate 0.12
Carbonic anhydrase CO2 12
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Understanding Vmax
The theoretical maximal velocity • Vmax is a constant • Vmax is the theoretical maximal rate of the
reaction - but it is NEVER achieved in reality • To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate • Vmax is asymptotically approached as
substrate is increased
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Double-Reciprocal or Lineweaver-Burk Plot
1 KM 1 ______ = _______ + ______
Vo Vmax[S] Vmax
From Lehninger Principles of Biochemistry
Use linear plot and intercepts to determine Km and Vmax
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Enzyme Inhibitors
Reversible versus Irreversible • Reversible inhibitors interact with an
enzyme via noncovalent associations • Irreversible inhibitors interact with an
enzyme via covalent associations
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Classes of Inhibition Two real, one hypothetical
• Competitive inhibition - inhibitor (I) binds only to E, not to ES
• Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition
• Noncompetitive (mixed) inhibition - inhibitor (I) binds to E and to ES
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Inhibitor (I) binds only to E, not to ES
Inhibitor (I) binds only to ES, not to E.
This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition
Inhibitor (I) binds to E and to ES.Enzyme Inhibition
From Lehninger Principles of Biochemistry
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Competitive Uncompetitive Noncompetitive Inhibition Inhibition (Mixed) Inhibition
Kmchanges while Vmax does not
Km and Vmax both change
Km and Vmax both change
From Lehninger Principles of Biochemistry