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
Growth of Microbes • Increase in number of cells, not cell size • One cell becomes colony of millions of cells
Growth of Microbes • Control of growth is important for – infection control – growth of industrial and biotech organisms – Biodegradation process
Factors Regulating Growth • Nutrients • Environmental conditions: temperature, pH, osmotic pressure • Generation time
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
Nutritional Categories • Carbon sources – CO2 = – organic = • Energy sources – sunlight = – organic =
autotroph heterotroph phototroph chemotroph
Chemoautotrophs Energy source (electron donor):inorganic carbon Carbon source: inorganic carbon Electron donor Carbon source Inorganic C
Electron acceptor
NH4+, Fe(II), Mn(II), H2S Carbon dioxide Organic carbon
H 2O
O2 aerobic respiration NO3, Fe(III), Mn(IV), SO4, CO2 anaerobic respiration N O, Fe(II), Mn(II), H S, CH 2 2 4
Chemoheterotrophs Energy source (electron donor):organic carbon Carbon source: organic carbon Electron donor Carbon source Organic C
Electron acceptor
O2 aerobic respiration NO3, Fe(III), Mn(IV), SO4, CO2
Carbon dioxide
H 2O anaerobic respiration
Photoautotrophs Energy source: light Electron donor: water Carbon source: inorganic carbon Electron donor Light (λ) Energy source Water Carbon source, electron acceptor Carbon dioxide Inorganic C (CO2)
Electron donor H 2O
Organic carbon
O2
Photoheterotrophs Energy source: light Electron donor: water Carbon source: organic carbon Energy source Light (λ) Electron donor Water Carbon source Organic C Organic C
Terminal electron acceptor Fe-S clusters in Photo System 1
CO2
Environmental Factors Influencing Growth • • • • •
Temperature O2 pH Osmotic Pressure Others: radiation, atmospheric pressure
Temperature Optima • • • •
Psychrophiles: cold-loving Mesophiles: moderate temperature-loving Thermophiles: heat-loving Each has a minimum, optimum, and maximum growth temperature
Fig. 7.8
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
Oxygen Requirements • Obligate aerobes – require O2 • Facultative anaerobes – can use O2 but also grow without it • Obligate anaerobes – die in the presence of O2
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
Measuring Bacterial Growth
Bacterial Division • Bacteria divide by binary fission • Alternative means – Budding – Conidiospores (filamentous bacteria) – Fragmentation
Fig. 7.13
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!
Fig. 7.14a
Plotting growth on graphs
Standard Growth Curve
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
Phases of Growth • Stationary phase – – nutrients becoming limiting or waste products becoming toxic – death rate = division rate
• Death phase – death exceeds division
Measuring Growth • Direct methods – count individual cells • Indirect Methods – measure effects of bacterial growth
Fig. i7.6
Fig. 7.17
Turbidity
Metabolic Activity
Dry Weight
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.
Energy yield from electron acceptor Terminal electron acceptor
ΔG (kcal/mol)
e• 6O2 • 24 NO3
6H2O aerobic respiration 12N2 anaerobic respiration
-686 -36
• SO4
H2S anaerobic respiration
-40
• CO2
CH2O photosynthesis
+115
Reduction Potential +0.85 O2/H2O
volts
+0.75 NO3/ N2 1.22
0.00
-0.22 -0.47
SO4/H2S 0.25 CH2O/CO2
1.28
Energy yield relationship between electron acceptor and electron donor Electron Reduction Electron Acceptor Potential (V) Donor O2
CH2O
Reduction Potential (V)
CO2
-0.47
Difference (V)
H 2O
+0.81
-1.28
NO3
N2
+0.75
-1.22
SO4
H 2S
-0.22
-0.25
The sign and magnitude of the difference represents how much free energy is available to the cell.
Growth Kinetics
Pertumbuhan Bakteri lag
log
stasioner
Endogenous
Concentration
bakteri
Makanan/substrat
Waktu
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
Pertumbuhan Bakteri
µmaks
µmaks/2
Ks
S, mg/L
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
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
Persamaan Monod 1/µ
1/Ks 1/µmaks 1/S
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)
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
Cell Yield Carbon source
Yield coefficient
glucose
0.4
Pentachlorophenol (PCP)
0.05
octadecane
1.49
Activated Sludge • Kondisi steady-state, neraca massa: – Biomassa: biomass in + biomass growth = biomass out – Food: food in – food consumed = food out Q0, S0, X0
V, S, X Reaktor
Q r, X u
Q0 + Qr X, S
Secondary Clarifier
Effluent Q 0 – Q w, Xe, S
Qu, Xu
Sludge treatment Q w, X u
• Biomassa: Q0X0 + (((µmaksSX)/(Ks + S)) – kdX) V = (Q0 – Qw)Xe + QwXu • Food: Q0S0 – ((µmaksSX)/y(Ks + S))V = (Q0 – Qw)S + QwS
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
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
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
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
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 Glutamate dehydrogenase Carbonic anhydrase
Substrate NH4+ Glutamate CO2
Km (mM) 57 0.12 12
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
Use linear plot and intercepts to determine Km and Vmax
1 ______
Vo
=
KM
1
_______
+ ______ Vmax[S] Vmax
Double-Reciprocal or Lineweaver-Burk Plot
From Lehninger Principles of Biochemistry
Enzyme Inhibitors Reversible versus Irreversible • Reversible inhibitors interact with an enzyme via noncovalent associations • Irreversible inhibitors interact with an enzyme via covalent associations
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
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
Competitive Inhibition Kmchanges while Vmax does not
Uncompetitive Inhibition Km and Vmax both change
Noncompetitive (Mixed) Inhibition Km and Vmax both change From Lehninger Principles of Biochemistry
