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3.1. Pendahuluan

Kinetics (Greek jimesijor, forcing to move) is a branch of natural science that deals with the
rates and mechanisms of any processesphysical, chemical, or biological. Kinetic studies in
microbiology cover all dynamic manifestations of microbial life: growth itself, survival and
death, product formation, adaptations, mutations, cell cycles, environmental effects, and
biological interactions. Kinetics provides a theoretical framework for optimal design in
biotechnologies based on fermentation and enzyme catalysis, as well as on employment of
outdoor activity of natural microbial populations (wastewater treatment, soil bioremediation,

Contrary to simple rates measurements, kinetic studies require the perception of the
underlying basic mechanism sof studied processes. We will define mechanistic studies
asthose that interpret some complex process as an interplay of several simpler reactions, for
example, cell growth can be explained through activity of enzymes and microbial community
dynamics can be interpreted through behavior of individual cells and populations. Ideally,
mechanistic studies infer the coupling of experimental measurements with analysis of
simulating mathematical models. The models formalize postulated mechanisms, so that the
comparison of observations and the models predictions allows one to discard an incorrect

The quantitative studies in microbiology often involve the assessment of growth

stoichiometry. Stoichiometry [Greek rsoijgeiom, element] is the quantitative relationship
between reactants and products in a chemical reaction. In microbiology, stoichiometry stands
for a quantitative relationship between substrates and products of microbial processes,
including biomass formation (the consequence of complying with mass and energy
conservation laws). In practical terms, kinetic and stoichiometry are tightly linked to each
other, but stoichiometry mainly addresses problems of a static nature (how much? in what
proportion?), whereas kinetics considers the dynamics questions (at what rate? by which

3.2. Kinetika Pertumbuhan Mikroba

Merupakan suatu rangkaian reaksi kimia yang mengendalikan sintesis penyusunan biomassa
yang diperoleh pada akhir biakan secara menyeluruh dengan mengikuti prinsip kekekalan

Kesetimbangan reaksi pertumbuhan mikroba (Macrostoichiometry of Microbial


By analogy to simple chemical reactions, we can represent growth as a conversion of a

number of substrates (medium components) into cell mass and products. Growth of aerobic
heterotrophic microorganisms can be approximated by the following stoichiometric equation
(substrates = biomass + products) (1,2):
Here, microbial biomass is empirically expressed by by the gross formula CHpOnNqPoKv . .
. , for example, if some average microbial cell contains per dry cell weight (%) C 46, H 7.5,
O 31, N 11; and P, 1.3, then the biomass formula is CH1.9O0.5N0.2P0.01. The
stoichiometric quotients a1a5 . . . , b and Y (biomass yield) specify quantities of substrate
and products of microbial growth. If we know biomass yield and gross formulas of all
substrates and products, then quotients a1a6 . . . are easily calculated from conservation
conditions. There are at least two such conditions. First, the mass of each element (C, H, O,
N, P, K, . . . ) on the leftside of equation 1 should be equal to that on the right side (mass
balance). Second, if ionized substances are involved, we should take into account the balance
of charges to satisfy the condition of electroneutrality. Table 1 demonstrates some examples
of stoichiometric growth reactions relevant to biotechnology. Described formalism is useful
as a first step in biotechnological studies aimed at planning and optimizing microbial growth.
It estimates how much nutrient should be supplied to the fermenter to obtain the required
amount of biomass or target product. However, it should be absolutely clear that
stoichiometric equations like equation 1 are no more than an approximation to reality. The
most severe deviation stems from the fact that unlike chemical reagents, microbial cells are
characterized by changeable composition, and stoichiometric coefficients are not true
constants. One task of contemporary microbial stoichiometry is to find out the functional
relationships between stoichiometric parameters and internal (physiological) and external
(environmental) factors.

3.3 Pertumbuhan Mikroba (Microbial Growth) dalam kultur batch

Microbial cells use nutrients for growth, energy production and product formation as
indicated in the following expression;

Nutrients + microbial cells > cell growth + energy + reaction products

Nutrient : karbon , nitrogen, oksigen, fosfor, belerang, mineral

Produk reaksi : metabolit (primer dan sekunder), CO2, H2O dan enzim

Pertumbuhan mikroba merupakan pertambahan jumlah sel mikroba, dan berlangsung selama
nutrisi masih cukup tersedia. Pertumbuhan mikroba dapat diukur, dengan melihat kenaikan
biomassa atau jumlah sel. Selama pertumbuhan, mikroba menghasilkan metabolit
primer/sekunder berupa produk.

Pertumbuhan sel mikroba biasanya mengikuti suatu pola pertumbuhan tertentu berupa kurva
pertumbuhan sigmoid (model Monod). Microbial Growth Kinetics describe how the microbe
grows in the fermenter. This information is important to determine optimal batch times.The
growth of microbes in a fermenter can be broken down into four stages: Lag Phase,
Exponential Phase, Stationary Phase and Death Phase (Figure 1).

Figure 1: Growth curve of a batch culture. (a) Acceleration phase, (b) Retardation phase and
(c) Declining phase.

Gambar 2.8. Batch Fermentor

FASE LAG (Fase Adaptasi)

Fase lag merupakan suatu periode penyesuaian terhadap medium-------tidak terjadi

perbanyakan jumlah sel.

FASE LOG (Fase Eksponensial)

Pada fase eksponensial atau logaritmik, sel membelah dengan kecepatan konstan dan terjadi
pertambahan jumlah sel menjadi 2 kali lipat (generation time). Laju pertumbuhan pada fase
eksponensial mengikuti persamaan diferensial orde pertama (Shuler and Kargi, 1992).


X = X0 pada t = 0

X adalah konsentrasi biomassa (g/l), t adalah waktu (jam) dan adalah laju pertumbuhan
spesifik (jam-1). Integrasi persaman di atas menghasilkan

ln X = t + ln X0

Pada fermentasi batch, laju pertumbuhan spesifik adalah konstan dan dipengaruhi oleh
perubahan konsentrasi nutrien. Pada konsentrasi nutrien awal yang rendah akan
menghasilkan laju pertumbuhan yang lebih kecil dari laju pertumbuhan spesifiknya.
Hubungan laju pertumbuhan dengan konsentrasi substrat (S) ditunjukkan oleh Gambar 2.7.
Pada daerah A terdapat pembatasan oleh substrat. Pada kondisi ini peningkatan konsentrasi
substrat akan meningkatkan laju pertumbuhan mikroba. Pada daerah B tak terdapat
pembatasan, dijumpai pada fasa eksponensial. Pada daerah C terjadi penghambatan oleh
substrat. Pada saat S mendekati 0, berbanding lurus dengan S, sedangkan jika S berlebihan
tidak bergantung pada S, = m ( laju pertumbuhan spesifik maksimum) (Mangunwidjaja,

Gambar 2.7 Pengaruh konsentrasi substrat terhadap laju pertumbuhan spesifik
(Mangunwidjaja, 1994)


Starts at the end of the Deceleration Phase, when the net growth rate is zero (no cell division,
or growth rate is equal to death rate). Cells are still metabolically active, and can produce
secondary metabolites. Selama fase ini, jumlah sel yang hidup tetap konstan tetapi akhirnya
menuju periode penurunan populasi. Many antibiotics and some hormones are produced as
secondary metabolites. Secondary metabolites are produced as a result of metabolite
deregulation. Dihasilkan metabolit sekunder untuk pertahanan diri bakteri. Primary
metabolites are growth-related products, while secondary metabolites are non-growth-
related (lihat Gambar 1). Pada fase ini laju pertumbuhan adalah nol (tidak ada pembelahan
sel) atau laju pertumbuhan sama dengan laju kematian. Konsentrasi massa se tetap, namun
jumlah sel yang hidup akan berkurang, terjadi lisis sel dan sebagian sel dapat tumbuh pada
produk hasil lisis sel tersebut. Walaupun laju pertumbuhan adalah nol selama fase stasioner
tetapi metabolisme sel masih aktif dan menghasilkan metabolit sekunder, sebagai hasil dari
perubahan pengendalian selular karena terbatasnya konsentrasi nutrien esensial. Produksi
metabolit sekunder (antibiotik, hormon) justru meningkat pada fase stasioner. Selama fase
stasioner, sel mengkatabolisme nutrisi yang tersimpan dalam sel (endogenous metabolism)
sehingga diperoleh energi (maintenance energy) untuk pemeliharaan membrane sel,
transportasi nutrien, gerak dan perbaikan struktur sel yang rusak. Pertumbuhan mikroba akan
terhenti selain disebabkan oleh terbatasnya konsentrasi nutrien esensial dan terakumulasinya
produk yang bersifat toksik juga disebabkan oleh terbentuknya produk yang menghambat
pertumbuhan. Penghambatan ini tergantung pada jenis dan konsentrasi produk
penghambatnya. Produksi etanol oleh ragi merupakan contoh produk penghambat
pertumbuhan. Hal tersebut dapat dicegah dengan cara mengencerkan medium yang tercemar
toksik, menambahkan komponen kimia yang membentuk kompleks dengan produk
penghambat dan tidak termetabolisme, dan memindahkan secara berkesinambungan produk
penghambat dari dalam reaktor (Shuler and Kargi, 1992).During the stationary phase, the cell
catabolizes cellular reserves for new building blocks and for energy-producing monomers.
This is called endogenous metabolism. The cell must expend maintenance energyin order to
stay alive. The equation that describes the conversion of cellular mass into energy, or the loss
of cell mass due to lysisduring the stationary phase is:


Pada saat medium kehabisan nutrien maka populasi bakteri akan menurun jumlahnya dan
jumlah sel yang mati lebih banyak dari pada sel yang hidup. The death or decline phase is
characterized by the expression:

3.4. Pertumbuhan Mikroba (Microbial Growth) dalam kultur kontinyu

a continous process is given in figure 2.9.

Gambar 2.8. Continous Fermentor (CSTR)

Model kinetika berperan penting untuk memantau dan memprediksi proses fermentasi. Model
kinetika terdiri dari kinetika pertumbuhan, penggunaan substrat dan pembentukan produk.
Model kinetika pertumbuhan dibagi menjadi dua tipe yaitu unstructured dan structured.
Model unstructured lebih sederhana karena biomassa sel dipandang sebagai bentuk yang
seragam (single component) tanpa dinamika internal sel sehingga laju reaksi hanya
tergantung pada kondisi fasa liquid dalam reaktor. Sedangkan Model structured memasukkan
faktor internal sel seperti perubahan komposisi biomassa, sehingga model ini lebih kompleks
(Bailey and Ollis, 1987).

Model unstructured yang sering digunakan untuk menggambarkan kinetika pertumbuhan

adalah persamaan Monod. Model ini mengekspresikan bahwa laju pertumbuhan spesifik
mikroba akan meningkat jika konsentrasi substrat meningkat. Namun laju pertumbuhan
spesifik akan turun pada konsentrasi substrat yang terlalu tinggi. Persamaan Monod
menggambarkan laju pertumbuhan spesifik merupakan fungsi dari konsentrasi substrat
pembatas (S):


Model Monod ditunjukkan oleh Gambar dibawah ini (Kurva pertumbuhan ini dihasilkan dari
persamaan di atas).

Ks adalah tetapan kejenuhan, yaitu konsentrasi substrat pada = m . Nilai Ks bergantung
pada jenis mikroba dan jenis substrat yang digunakan. Untuk substrat gula, nilai Ks berkisar
1-100 mg/l. Sedangkan untuk substrat nitrogen nilai Ks lebih rendah dari substrat gula.
Secara umum, bila S>3Ks, maka = m (Mangunwidjaja, 1994). Modifikasi persamaan (2.3)
menghasilkan bentuk berikut :

Persamaan kinetika pertumbuhan mikroba yang menggabungkan persamaan (2.1) dan (2.3).
Persamaan tersebut memiliki bentuk sebagai berikut:

Parameter kinetika lain dapat dirumuskan untuk menjelaskan lebih lanjut tentang kinetika
pertumbuhan mikroba yaitu koefisien perolehan/yield. Koefisien yield pertumbuhan
dirumuskan sebagai jumlah sel kering yang dihasilkan per jumlah substrat yang dikonsumsi

YX/S = X/S (2.9)

3.5. Kinetika Pembentukan Produk

Pembentukan produk mikrobial dapat digolongkan dalam 3 pola yaitu :

1. Pola pembentukan produk yang berasosiasi dengan pertumbuhan. Laju pembentukan
spesifik produk berbanding lurus dengan laju spesifik pertumbuhan. Enzim merupakan
contoh produk yang dihasilkan dari pola pertumbuhan di atas (Growth-associated

2. Pola pembentukan produk yang tidak berasosiasi dengan pertumbuhan. Pembentukan

produk terjadi pada fasa stasioner pada saat laju pertumbuhan adalah nol. Laju spesifik
pembentukan produk adalah konstan. Antibiotik merupakan metabolit sekunder yang
terbentuk melalui pola pembentukan produk yang tidak berasosiasi dengan pertumbuhan
(Non growth-associated product)

3. Pola campuran, produk terbentuk selama pertumbuhan yang lambat dan fase stasioner.
Laju spesifik pembentukan produk mengikuti persamaan Luedeking-Piret. Asam laktat,
xanthan gum merupakan contoh metabolit sekunder yang diproduksi melalui pola ini
(Mixed-growth-associated Product).Tiga jenis pola pembentukan produk tersebut
ditunjukkan pada gambar dibawah ini.

3.6. Bioreactor classication

The various types of bioreactor can be classied according to the mode of operation as batch,
semi-continuous or continuous reactors. As mentioned in the previous section, in a batch
reactor all the materials needed by the cell culture are introduced at the beginning of the
process and there is no introduction or removal of substances or fresh medium. In
semicontinuous or fed batch operation, additional substrate is fed into the bioreactor, thus
prolonging operation by providing an additional continuous supply of nutrients to the cells.
No material is removed from the reactor, apart from normal sampling, and therefore the total
quantity of material within the reactor will increase as a function of time. However, if the
feed is highly concentrated, then the reactor volume will not change much and can be
regarded as essentially constant. In continuous operation, fresh medium is added
continuously to the bioreactor, while at the same time depleted medium is continuously
removed. The rates of addition and removal are such that the volume of the reactor contents

is maintained constant. The depleted material contains any products that have been excreted
by the cells and, in the case of suspended-cell culture, also contains e_uent cells from the

Otherwise, on the basis of the mixing technology, we can distinguish three main bioreactor
types: stirred tank, airlift and orbitally shaken bioreactor. The stirred tank reactor is
composed of a vessel and a mixer, such as a stirred or a turbine, usually mounted with a shaft
at the top of the vessel to achieve mixing inside the tank. This is the most popular in industry
and represents a well established technology. In the airlift reactor the culture medium is kept
mixed and gassed by the introduction of air or another gas mixture at the base of the vessel
equipped either with a draught tube or another device by which the reactor volume is
separated into a gassed and an ungassed region with different density, thus generating a
vertically circulating flow. In general, these reactors are less expansive than the stirred tank
bioreactors, however the mixing properties are likely to be more critical. The orbitally shaken
bioreactor is a vessel, usually cylindrical, put in motion by a shaker. The mixing is archived
by the fluid motion resulting from friction force which is exerted by the liquid-contacting
vessel wall, and most of all, by the high inertial forces due by the shaking movement of the
tank. The shaken bioreactors have been widely used in the biotechnological field since they
are easily to handle and inexpensive. In spite of their large practical importance, very little is
known about the characteristic properties of shaken cultures. These are the reasons why in the
last years many studies about cell growth have been carried out using this alternative
technology [31] and models for shaken bioreactors have been recently proposed [4]. In this
work, in particular, we focus on cell growth in shaken bioreactors under batch conditions.



Fermentation process Medium requirements Medium formulation of optimal growth and
formation Simple and complex media Design of various commercial media for industrial
fermentations Microbial growth Applications of fermentation Microbial biomass
metabolites Microbial enzymes Transformation process Recombinant products Main
parameters in fermentation processes Mode of operation of fermentation processes Basic
configuration of fermenter and axillaries.
Methods of immobilization Kinetics of immobilized enzymes Thermal death kinetics of
microorganisms Batch and continuous heat sterilization of liquid media Filter sterilization
liquid media Air sterilization and design of sterilization equipment.
Stoichiometry of cell growth and product formation Elemental balances Degrees of
reduction of
Substrate and Biomass Available Electron Balances Yield coefficients of Biomass and
formation Maintenance coefficients energetic analysis of microbial growth and product
Oxygen consumption and heat evolution in aerobic cultures Thermodynamic efficiency of
Modes of operation Batch, Fed Batch and Continuous Cultivation Simple unstructured
models for microbial growth Monad model Growth of filamentous organisms Product
kinetics Leudeking-piret models, substrate and product inhibition on cell growth and

Overview of response surface methodology Factorial design Central composite design
level design Plackett-Burman design and Taguchi design of experiments.
L: 60 Total: 60
1. Stanbury P. F., Hall S. J. and Whitaker A., Principles of Fermentation Technology,
& Technology Books, 1999.
2. Blanch H. W. and Clark D. S., Biochemical Engineering, CRC Press, 1997.
1. Anderson M.J. and Whitcomb P.J., RSM Simplified: Optimizing Processes Using
Surface Methods for Design of Experiments, Productivity Press, 2005.
2. Jack P.C. Kleijnen, Design and Analysis of Simulation Experiments, Springer, 2008.
3. Roy R.K., Design of Experiments Using the Taguchi Approach: 16 Steps to Product and
Process Improvement, Wiley-IEEE, 2001.
4. Shuler M.L. and Kargi F., Bioprocess Engineering: Basic Concepts, Second Edition,
Prentice Hall, 2001.