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Fermentation Technology

Optimasi medium dan kinetics


MK : Biotechnology
Fakultas Farmasi

Alwani Hamad, ST, MSc


Program Studi Teknik Kimia
Fakultas Teknik
Universitas Muhammadiyah Purwokerto

Course content

Persyaratan proses fermentasi


Kondisi dan variable fermentasi
Fermenter/ bioreaktor
Media dan optimasi media untuk fermentasi
Kinetika pertumbuhan bakteri untuk menghitung
hasil produk fermentasi
Downstream process hasil fermentasi

Evaluasi : Ujian (closed book)


Buku rujukan:
G Rao.2007. Introduction to Biochemical Engineering. Tata Mc Graw- Hill
Publishing Company Limited
Stanbury, Whitaker and Hall, 2003. Principles of Fermentation Technology
Butterworth

Quantification of Microbial Rates using


optimum medium

Microbial rates of consumption or production


H2O
H+

C, N, P, S source

biomass
CO2

O2

product
heat
4

Medium yang dibutuhkan oleh


mikroorganisme
Carbon :molasses, grain, starch, maize, potatos dan cassava,
sucrose, glucose, dan lactoce
Nitrogen : protein dan asam amino (corn steep liquor, soya
beans, fish meal, yeast extract, dll)
Energy source : chemo-organotrophs( carbon source),
heterotrophilic (senyawa organik)
Minerals : K,S, Ca, Fe,Zn, dll
Other nutrient like vitamin, calcium pantothenate untuk vinegar
fermentasi
Oxygen/air for aerobic process:
Water (submerged fermentation)

Persyaratan pemilihan medium


untuk fermentasi skala besar
Murah, udah didapat dan harga dan kualitas
konsisten
Mempunyai produktifitas yang tinggi : dapat
memproduksi jumlah produk maksimum per
substrat yang dikonsumsi
Pertumbuhan pembentukan produk harus
besar
Harus dapat meminimalkan produk samping

Medium formulation
Dalam fermentasi medium digunakan untuk :
Optimal growth of the cells
Product formation
Medium formulation dalam skala scale up
Economic viability
membutuhkan teknik yang tepat agar fermentasi dapat
berjalan dengan baik
Cells maintances untuk biosynthesis membentuk produk

Minimum medium dalam


pembentukan alpha amilase

Soluble starch 20 g/l


Glucose 5 g/l
Peptone 5 g/l
Yeast extract 2,5 g/l
K2HPO4 2 g/l
MgSO4. 7H2O 1 g/l
pH 7
Distilled water 100 ml
8

Aplikasi penggunaan medium


dalam industri

Substrate
Sumber
Malt
Molasses
Carbon
Starch dan dextrin
Carbon
Cellulose
Carbon
Whey
Carbon
Vegetable oil soy, palm Carbon
Methanol
Carbon
Corn steep liqour
Nitrogen
Soya meal
nitrogen

Industri
industri beer
Alkohol, butanol, asam asetat
Fermentasi ethanol
alkohol, nutanol, acetone dan isopropanol
xanthan gum, 2,3 butadienol, asam lactat
fermentasi antibiotic
asam glutamat, serin dan vitamin B12
Asam laktat
antibiotik

Design optimasi medium


menggunakan statistik method
Placket- Burman screening design method
Digunakan untuk menyeleksi medium yang paling
berpengaruh dalam pertumbuhan cell
Sehingga dapat diperoleh medium medium yang
paling dibutuhkan mikroorganisme

Optimasi medium menggunakan respon


surface method
Diperoleh komposisi medium yang digunakan
untuk menghasilkan produk secara optimal

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Design eksperimen Respon Surface method

metode statistik untuk perancangan percobaan, pemodelan matematik, optimasi


dan analisis statistik dalam penelitian
persamaan polinomial kuadratik dikembangkan untuk memperkirakan hasil
percobaan sebagai fungsi dari interaksi antara variabel bebas

Type RSM yang dipakai yaitu Central Composite design


Contoh design optimasi pembuatan nata de coco
Range dan level

Variabel bebas
Star point (-) Low level (-1)
Konsentrasi gula (%w/v)

Center level
(0)

High Level
(+1)

Star point
( + )

0.41%

1%

10%

20%

21.5%

Lama fermentasi (hari)

10

14

16

pH

4.5

Tempuhan berdasarkan design eksperiment menggunakan Central


Composite Design
Run
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

Konsentrasi
gula
-1
-1
-1
-1
+1
+1
+1
+1
-1
+1
0
0
0
0
0
0

Lama
fermentasi
-1
-1
+1
+1
-1
-1
+1
+1
0
0
-
+
0
0
0
0

pH
-1
+
-1
+1
-1
+1
-1
+1
0
0
0
0
-
+
0
0

Kinetic of microbial growth for


fermentation product

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What are the value of rates?


Rates of consumption or production are obtained from
mass balance over reactors

Mass balance over reactors


Transport + conversion = accumulation
(in out) + (production consumption) = accumulation
Batch: transport in = transport out = 0
Chemostat: accumulation = 0, steady state

Fed batch: transport out = 0


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Continuous Reactors

Chemostat - CSTR - continuous stirred tank


reactor for the cultivation of cells.
mixing supplied by impellers and rising gas bubbles
assume complete mixing - composition of any phases
do not vary with position
liquid effluent has the same composition as the reactor
contents

Chemostat with Recycle


FR, XR
F,
X0

V,
X1

F - nutrient flow rate


V - reactor volume
X1 - x concentration in reactor
X2 - X concentration in effluent
XR - X concentration in recycle
FR - recycle flow rate

F,
X2
F+FR,
X1

Chemostat with Recycle Cell


mass equation
Acc = in - out + gen
F X0 + FR XR - (F+ FR) X1 + VmX1 = V

dX 1
dt

FR, XR
F, X0

F, X2
V,
X1

F+FR,
X1

How are rates defined?


Rate (ri) = amount i per hour / volume of reactor

kg .i / hour
m3 reactor

Biomass specific rate (qi)


qi = amount per hour / amount of organism in reactor

Thus:

kg .i / hour
kg . X

ri = qi CX

Substrate (-rS) = (-qS)CX

Biomass

rX = mCX

Product

rP = qPCX

Oxygen (-rO2) = (-qO2)CX

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Yield = ratio of rates


Yij =

rj
q jC X
qj
rate. j

rate.i
ri
qiC X
qi

YSX = rate of biomass production / rate of substrate


consumption [g biomass/g substrate]
YOX = rate of biomass production / rate of oxygen
consumption [g biomass/g oxygen]

19

Introduction
Cell growth and product formation are complex processes
reflecting the overall kinetics and stoichiometry of the
thousands of intracellular reactions that can be observed within
a cell.
Thermodynamic limit is important for process optimization.
The complexity of the reactions can be represented by a simple
pseudochemical equation.
Several definitions have to be well understood before studying
this chapter, for example: YSXmax, YATP X, YOX, maintenance
coefficient based on substrate (ms).

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Composition of biomass
Molecules
Protein 30-60 %
Carbohydrate 5-30 %
Lipid 5-10 %
DNA 1 %
RNA 5-15 %
Ash (P, K+, Mg2+, etc)

Elements
C
40-50 %
H
7-10 %
O
20-30 %
N
5-10 %
P
1-3 %
Ash
3-10%

Typical composition biomass formula: C1H1.8O0.5N0.2

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Log10 of # of CFU/ml

STATIONARY

LOG

DEATH

LAG

SENESCENCE
Figure 6.14

Monod Equation
Growth equation (Monod equation)

m = mmax S/ (Ks +S)


Specific
growth
rate

Substrate conc (S)

Stationary Phase
Growth equations with substrate exhaustion:

Cells: (rate of change of cell concentration)


dX/dt = mX = mmaxS/(Ks + S) * X
Substrate: (rate of change of substrate conc)
dS/dt = -1/Yx/S dX/dt = -1/Yx/S mmaxS/(Ks + S) X

Substrate depletion in batch


culture

Batch Reactor Kinetics Real


data
100

2.5

2
10

OD

1
1
0.1
0.5

0.01
0:00:00

6:00:00

12:00:00

18:00:00

24:00:00

Time (hr:min:sec)

30:00:00

0
36:00:00

Glucose (g/L)

1.5

Batch Reactor Kinetics


100

12
10

OD @ 600 nm

8
1

6
4

0.1

2
0.01
0
0.001

-2
0

10

15

Time (hr)

20

25

Glucose conc (g/L)

10

Series1
Series2

Antibiotic production
There are over 10 000 different antibiotics
known, but only about 200 in commercial
use, since most new antibiotics are no
better than existing ones.
There is a constant search for new
antibiotics. Antibiotics are the mostprescribed drugs and are big business.
Finding a new antibiotic and getting it on
to the market is a very long process and
can take 15 years.

Antibiotic Production Methods


Antibiotics are produced on an industrial scale
using a variety of fungi and bacteria.
Penicillin is produced by the fungus Penicillium
chrysogenum which requires lactose, other
sugars, and a source of nitrogen (in this case a
yeast extract) in the medium to grow well.
Like all antibiotics, penicillin is a secondary
metabolite, so is only produced in the
stationary phase.
What sort of fermenter does it require?
It requires a batch fermenter, and a fed batch
process is normally used to prolong the
stationary period and so increase production.

Further reading
G Rao.2007. Introduction to Biochemical
Engineering , Chapter 6 dan seterusnya

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