MICROBES CULTIVATION IN

INDUSTRIAL SCALE AND ITS FACTORS

Group one
R. YUVITA RAKHMAN, VIA NUR FADILAH, TRI WIJAYANTI IRMA S.

INTRODUCTIONS
Production of antibiotics, alcohols, vinegar, amino acids, vitamins,
therapeutic antibodies, acetone and other solvents, and recombinant
proteins is accomplished by the large-scale cultivation of microbial
cells such as bacteria, algae, yeast, and fungus on industrial scale.
In all these industrial applications the metabolic activities or the
biochemical pathways are used for the production of specific
chemicals with the consumption of the substrates or a carbon
source such as sucrose. Here, the microbial culture acts as a factory,
where the substrate is the raw material

 The composition of the medium used for cultivation of

microorganisms is directly reflected in their physiological
phenotype and their fermentation performance, which in turn
affects the results of strain analyses and strain performance in
industrial applications.
For this reason, the successful development of strains for large
scale industrial production of heterologous proteins and lowvalue fuels, chemicals and materials merits the composition of
cultivation media in various steps of strain development to be
reconsidered.

MICROBIAL CULTURE
TECHNIQUE
A chemical reaction requires an appropriate temperature,
pressure, pH, and solvent system for maximum product
output. The microbial system also has to be provided with the
optimum environmental and nutritional conditions such as
temperature, pH, and the correct substrate for converting it into
the product.
The efficiency of microbial conversion of substrate into product is
comparatively less because a major part of the metabolic energy is
utilized for the generation of biomass by cell growth and
multiplication.
The chemical environment of a microbial cell is its
nutritional conditions in which it is growing. It also includes

NUTRIENTS FOR MICROBIAL

CULTURE
Like any other living system, microorganisms also require a source of
energy, carbon, nitrogen, oxygen, iron and other minerals, micronutrients,
and water for growth, and multiplication. All these nutrients that are
essential for microbial organisms are supplied in the form of nutrient
media.
For laboratory-scale cultivation we may use certain costly media
components, but for industrial purposes they should be economical and
readily available.
But in large-scale microbial cultivation for industrial purposes, the pH and
the dissolved salts present should be considered when formulating the
media requirements and its concentration

METABOLIC ENGINEERING, EVOLUTIONARY ENGINEERING

AND SYSTEMS BIOLOGY IN STRAIN DEVELOPMENT

Several studies have

pointed out that the
cultivation conditions and
media composition used for
the analysis of engineered
strains strongly influence
the data generated
Finally, genetic engineering
approaches to overcome
industrial media constraints
are also exemplified.

 Principle of
Evolutionary
engineering
Traditionally,
novel production
strains have
been developed
by mutagenesis,
breeding, and
the lately revived
concept of
evolutionary
engineering

MEDIA AND STRAIN

STABILITY
The typical industrial production strain is genetically undefined and
adapted to perform in rather poor, toxic, viscous, and nutrientlimited media.
Once desired traits have been established in recombinant
laboratory strains, the strains are either directly transferred to the
industrial production environment or as occurs much more
frequently a potential production strain has to undergo a new round
of metabolic engineering procedures.
The genetic stability of strains is an absolute requirement for
utilization in industrial processes

PENGERTIAN KULTIVASI
Upaya pemeliharaan bagi pertumbuhan mikroba. Untuk
berhasilnya kultivasi mikroba diperlukan teknik aseptik,
medium serta lingkungan fisik yang sesuai

Kultur
sel

Kultur secara
mikro

Kultur secara
makro
Mikroba = kelompok jasad hidup berukuran kecil
dengan kisaran ukuran sel sekitar 0,1 10 m

FAKTOR-FAKTOR YANG BERPENGARUH

TERHADAP KULTUR SEL
Medium kultur yang meliputi:
a. temperature,
b. pH
c. Komposisi medium
d. dissolved oxygen tension (DOT)

STUDY CASE
For example, it was found that a strain of the lactic acid bacterium
Streptococcus thermophilus engineered for enhanced exopolysaccharide
production a trait highly desirable in yoghurt production failed to express the
phenotype in milk without the addition of an extra nitrogen source. Similarly, a
genetically modified strain of the yeast Saccharomyces cerevisiae, which had
been communicated as the ultimate solution to the fermentation of
lignocellulose derived xylose [6,7], was found to require yeast extract,
additional hexose sugar and oxygenation to efficiently ferment the xylose
fraction in spent sulphite liquor [8]. Furthermore, heterologous protein
production in yeast is strongly influenced by the nitrogen-composition of the
production medium. Thus the final industrial environment must be considered
throughout the strain development process to avoid unfounded expectation and
more importantly to prevent costly investment into premature production
facilities.

Mikroorganisme Yang Cocok Untuk Proses

Industry
1. Mampu tumbuh dengan cepat.
2. Menghasilkan produk yang banyak dalam
suatu periode singkat.
3. Unggul, Sifat unggul yang ada harus
dapat dipertahankan untuk menghasilkan
dan
mempertinggi
produk
yang
diharapkan.
4. Stabil, Pada kondisi yang diberikan,
mikrobia harus mempunyai sifat-sifat
yang tetap, tidak mengalami perubahan
karena mutasi atau lingkungan.
5. Bukan pathogen.

SIFAT PENTING LAIN YANG HARUS DIMILIKI

MIKROORGANISME INDUSTRI
Tidak berbahaya bagi manusia, dan secara ekonomik produknya penting
bagi hewan dan tumbuhan.
Harus non-patogen dan bebas toksin, atau jika menghasilkan toksin,
harus cepat di-inaktifkan.
Mudah dipindahkan dari medium biakan.
Mikroorganisme industri harus dapat direkayasa secara genetik.
Memilki karakteristik genetik yang stabil.

TEKNIK DASAR KULTUR MIKROBA

Dapat ditumbuhkan dalam medium padat (misal: tempe, tape, oncom, jamur
konsumsi) dan medium cair (misal: untuk menghasilkan antibiotik, etanol, asam
asam amino, nata de coco).
Berdasarkan komposisi medium untuk menumbuhkan mikroba:
-medium lengkap
-medium minimal

TYPES OF MICROBIAL CULTURES

The culturing of the microbial system can be achieved in different ways.
The type of culture method sometimes depends on the type of the
microbial system or on the type of the product that we expect. For
example, one can get two entirely different products from the same
organism by changing the nutritional and other parameters or even
culturing vessels.

TEKNIK KULTIVASI MIKROBA

1. KULTUR BATCH (sekali panen)
Mikroba ditumbuhkan dalam
medium sampai mencapai fase
pertumbuhan maksimal kemudian
dipanen produknya (sel/biomassa
atau produk metabolitnya).
This is a small-scale laboratory
experiment in which a microbial
culture is growing in a small
volume flask. In batch cultures,
the nutrients are not renewed and
the exponential growth of cells is
limited to a few generations.
Kurva pertumbuhan mikroba
dalam kultur tertutup (batch

APLIKASI KULTUR BATCH

Digunakan untuk memproduksi biomassa, metabolit
primer dan metabolit sekunder
Untuk produksi biomassa digunakan kondisi kultivasi
yang mendukung pertumbuhan biomassa, sehingga
mencapai maksimal
Untuk prodiksi metabolit primer kondisi kultivasi
harus dapat memperpanjang fase eksponensial yang
dibarengi dengan sintesis produk
Untuk produksi metabolit sekunder kondisi kultivasi
harus dapat memperpendek fase eksponensial dan
memperpanjang fase stasioner

TYPES OF MICROBIAL CULTURES

2. Fed-batch culture.
This method is specially suited for cultures in which a high concentration of
substrate is inhibitory to cell multiplication and biomass formation. In such
situations the substrate can be fed at low concentrations to achieve cell
growth. This method can easily produce a high cell density in the culture
medium, which may not be possible in a batch fermentor or shake flask
culture. This is especially important when the product formation is
intracellular to achieve maximum product output per biomass.

TYPES OF MICROBIAL CULTURES

3. Continuous culture. Bacterial cultures can be maintained in a state of
exponential growth over long periods of time using a system of continuous
culture, designed to relieve the conditions that stop exponential growth in
batch cultures.
Continuous culture is very suitable for the production of cell biomass and
products, if it is excreted into the medium. It is widely used for the
production of single-cell protein from liquid effluents as a byproduct of the
waste treatment. The organic waste present in the effluent is converted
into microbial biomass, which is known as single-cell proteins.

KULTUR KONTINYU...
Ada 2 cara :
KEMOSTAT :
Menambahkan nutrien dalam suatu tangki sehingga komposisi nutrien
di dalam fermentor tempat kultivasi mikroba selalu dalam keadaan tetap.
TURBIDOSTAT
Menambahkan nutrien secara kontinyu sehingga kerapatan sel selalu
dalam keadaan tetap. Dalam turbidostat aliran medium diatur berdasarkan
kerapatan optik kultur mikrobia.

APLIKASI KULTUR KONTINYU

Digunakan untuk penelitian fisiologi dan biokimia
mikroba, laju pertumbuhan dapat diatur oleh laju alir
dan laju pertumbuhan dibatasi oleh konsentrasi
substrat pembatas
Untuk isolasi dan seleksi mikroba penghasil enzim
menggunakan media diperkaya
Untuk produksi biomassa, contoh ICI (Imperial
Chemical Industries, kapasitas bioreaktor 3000 m3,
substrat metanol)
Untuk produksi bir menggunakan bioreaktor menara
(tower bioreactor)

KELEBIHAN KULTUR KONTINYU

Produktivitas lebih tinggi, laju pertumbuhan & konsentrasi sel dapat
dikontrol, pemasokan oksigen dan pembuangan panas dapat diatur.
Dapat dijalankan pada waktu yang lama.
Cocok untuk proses yang kontaminasnya rendah dan produk yang
berasosiasi dengan pertumbuhan.
Pemantauan dan pengendalian proses lebih sederhana.
Tidak ada akumulasi produk yang menghambat

KELEMAHAN KULTUR KONTINYU

Aliran umpan yang lama, resiko kontaminasi besar (operasi harus hatihati & desain peralatan lebih baik).
Peralatan untuk operasi dan pengendalian proses harus biasa tetap
bekerja baik untuk waktu yang lama.
Memerlukan mikroba dengan kestabilan genetik tinggi, karena akan
digunkan pada waktu yang lama (Irianto, 2007).

CHEMOSTATS
Pada chemostat, kecepatan aliran dari reservoir
medium pertumbuhan diatur pada nilai tertentu
dan kecepatan pertumbuhan biakan mengatur
kecepatan alirannya.
Karena produk akhir tidak diakumulasi dan
nutrient tidak seluruhnya dipergunakan bakteri
tidak pernah mencapai tahap stationer.
1.
2.
3.
4.
5.
6.
7.
8.

Reservoir medium
Flow rate regulator
Air inlet
Air filter
Passage innoculation
Siphon and overflow
Growth chamber
Respectacle

Bakteri dalam chemostats tumbuh dalam nutrisi

yang terus disuplai dan produk yang terus
dipindahkan.

Kemostat untuk Bioreaktor sel kontinyu

FERMENTOR
S
These are
bioreactors used
for the cultivation
of microbial cells
on large scale
under controlled
conditions for
industrial
purposes.

FERMENTOR KULTUR CAIR

Prinsip kultivasi mikroba dalam sistem cair
Mikroba berada dalam cairan yang mengandung nutrien sebagai substrat
untuk tumbuh dan berkembang bercampur dengan produk-produk yang
dihasilkan termasuk limbah. Nutrien dan oksigen yang diperlukan untuk
pertumbuhan optimal mikroba harus tercampur merata (homogen) pada
semua bagian fermenter.
Untuk mendapatkan sistem fermentasi yang optimum, maka fermenter
harus memenuhi syarat sebagai berikut:
1. Terbebas dari kontaminan
2. Volume kultur relatif konstan (tidak bocor atau menguap)
3. Kadar oksigen terlarut harus memenuhi standar
4. Kondisi lingkungan seperti: suhu, pH harus terkontrol. Stirred tank reactor
system model yang banyak dipakai.

Contoh
bioreaktor kultur
cair yang
digunakan untuk
kultur kontinyu:
Fermentor skala
laboratorium

PENGGANDAA
N SKALA
PRODUKSI
MIKROBAALU
R PROSES
PEMBUATAN
INOKULUM
BAKTERI
SECARA
INDUSTRI

 The benefit of using YEp plasmids is the high gene copy number of up to
70 copies per cell [52] resulting in high expression levels of the desired
proteins, although their high segregational instability often results in
plasmid loss especially in rich medium [53,54].
However, the stability of YEp-type vectors can be improved by
autoselection systems, such as the fur1 ura3 system [55], where the
deletion of FUR1 together with the use of a plasmid containing the URA3
marker results in stable plasmid expression even in continuous culture
[56].
Without such autoselection systems it is necessary to use a selective
medium to overcome the instability of YEp plasmids, which may pose a
limitation to the industrial use of such strains especially with lowcost
products.