Ghasem D. Najafpour. Biochemical Engineering and Biotechnology

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254 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

10.6 KINETICS OF GROWTH AND PRODUCT FORMATION

It has been suggested that fungi grow in filamentous form at an exponential rate with a con-

stant specific growth rate (m) until some substrate becomes growth limiting, according to

the Monod equation:

4,6

(10.6.1)

where, m

max

is the specific growth rate of the organism in h

⫺1

, K

is the saturation constant

in kg⭈m

⫺3

, and S is the concentration of the limiting substrate in kg⭈m

⫺3

. The ethanol fer-

mentation performance and the bioprocess criteria are summarised below:

• High conversion yield

• High ethanol tolerance

• Resistance to inhibitors in hydrolysed product

• No oxygen requirement

• Low fermentation pH

• Broad substrate utilisation range

10.7 PREPARATION OF THE STOCK CULTURE

It is desirable to store organisms in pure culture for a long time without lack of nutrients.

A nutrient broth may not last for weeks. Solid media nutrients with agar in slants are used.

Normally the freeze-dried media obtained from ATCC is hydrated and grown in broth

media, then transferred to a culture tube with slant agar.

Figure 10.1 shows that a stock cul-

ture on slant agar in a sealed tube can be kept in a refrigerator for a minimum duration of

4–6 months.

⫽

⫹

max

FIG. 10.1. Stock culture Rhodospirillum rubrum on slant agar (a hydrogen producer photosynthetic bacterium).

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APPLICATION OF FERMENTATION PROCESSES 255

There are several steps for transferring pure culture from a broth cultured on slants. It

should be an aseptic technique without transfer of any contaminants. An inoculating loop

(Cole-Parmer) is flamed on a Bunsen burner until red hot. It is then cooled off on a corner

of the media before entering the culture broth. A full loop of cultivated culture is taken in

front of the flame and then transferred to the surface of the slanted agar with continuous

streaking; it is incubated at 30 ⬚C until growth is visible as colonies appear. The stock cul-

ture tube can then be kept in refrigerator and is good for 6 months. Renewal is necessary

for a 6-month-old stock culture.

10.8 INOCULUM PREPARATION

To have stock culture in a slant, also find a single isolated colony of yeast on the Petri dish

from which the culture can be transferred to the fermentation media. This technique may

ensure you that the stock culture is not contaminated with other organisms.

Take a loop full of the creamy culture off the agar plate. Dip the loop into a 250 ml flask

containing 100 ml of 5.0 g⭈l

⫺1

glucose and 1 g⭈l

⫺1

yeast extract. Swirl the loop in the nutri-

ent solution to dislodge the selected culture from the loop.

Flame the neck of the flask and the cotton plug before inserting the plug back in the

flask. Also, flame the loop to kill residual microorganisms.

Place the flask in a temperature-controlled shaker at 37 ⬚C. The exponential growth

phase will last from 2 to 24 hours after inoculation. The exact time and duration depend on

the physiological condition of the inoculum. The data in Table 10.1 are plotted and a growth

curve will be obtained for an exponentially growing culture. Figure 10.2 shows the typical

growth curve obtained for a viable organism.

10.9 SEED CULTURE

Seed culture of Saccharomyces cerevisiae (ATCC 24860) is grown in a rich medium com-

prising of 1 g glucose, 0.1 g peptone and yeast extract, 0.33g KH

and 0.03 g Na

HPO

in 100 ml distilled water. The media will be autoclaved at 126 ⬚C and 15 psig for 20 min.

The stock culture from ATCC media is transferred to a prepared seed culture. The pH of

TABLE 10.1. Data sheet for batch fermentation, at constant agitation, experimental run 1

Time, h Absorbance, Cell Concentration of Ethanol

520 nm

concentration, g/l carbohydrates, g/l concentration, g/l

0 0.0 0.0 31.5 —

6 0.68 0.5 27.78 1.85

8 1.0 0.78 21.5 3.8

12 1.4 1.1 14.3 5.8

24 1.9 1.55 11.0 8.9

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256 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

the medium is adjusted to 5.5, using 0.03 M phosphate buffer solution. A pH meter model

WTW 82362 Weilheim, Germany, was used for the pH reading for the experiment in this

chapter. Figure 10.3 shows a typical seed culture prepared for inoculation of batch fermen-

tation. The transformation is carried out in front of a flame with a sterile syringe.

Cell dry weight, mg/l

0.0 0.3 0.6 0.9 1.2 1.5

Absorbancy, 580 nm

0.0

0.4

0.8

1.2

1.6

2.0

2.4

FIG. 10.2. Standard curve for cell density define based on growth of Saccharomyces cerevisiae.

FIG. 10.3. A 50 ml seed culture is used for inoculation of batch fermenter.

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APPLICATION OF FERMENTATION PROCESSES 257

10.10 ANALYTICAL METHOD FOR SUGAR ANALYSIS

10.10.1 Quantitative Analysis

After the experiment is concluded, for each sample, measure the glucose concentration

with the DNS reagent. DNS solution as a reducing sugar was prepared by dissolving 10 g

3,5-dinitrosalicylic acid in 200ml of 2 M NaOH. A separate solution of 300 g sodium potas-

sium tartrate was prepared in 500 ml of distilled water; mixing and heating were required.

The hot salt solution was added to the DNS. The volume of the solution was made up

to 1 litre by adding distilled water. A standard glucose solution (2 g⭈l

⫺1

) is prepared in a

day advance for any structural deformation or changes. A Calibration curve is prepared.

A straight line can easily be obtained in the range 200–1600 mg⭈l

⫺1

glucose.

10.11 ANALYTICAL METHOD DEVELOPED FOR

ETHANOL ANALYSIS

Ethanol concentration in the fermentation broth is determined by using gas chromatogra-

phy (HP 5890 series II with HP Chemstation data processing software, Hewlett-Packard,

Avondale, PA) with a Poropak Q Column, and a Hewlett-Packard model 3380A integrator.

A flame ionisation detector (FID) is used to determine ethanol. The oven temperature is

maintained at 180 ⬚C, and the injector and detector temperature are maintained at 240 ⬚C.

The sample taken from the fermentation media has to be filtered and any internal standard

must be added for analysis based on internal standard methods; otherwise, the area under

the peak must be compared with known standard samples for calculation based on external

standard methods.

10.12 REFRACTIVE INDEX DETERMINATION

Refractive Index is used to analyse the sample. It can also be used to help determine the

percentage of a chemical (such as ethanol) in an aqueous solution. Refractive index (RI) is

always reported to four decimal places. An example of the RI scale is shown in Figure 10.4.

The correct reading from the RI of the sample would be 1.3764.

10.13 MEASURING THE CELL DRY WEIGHT

About 5 ml of sample is withdrawn for every 4–6 hours. The absorbance reading of the

sample at 580 nm was measured using a Hitachi U-2000 spectrophotometer. The sample is

filtered in a vacuum through Whatman filter paper with a pore size of 2.5 ␮m and diameter

of 47 mm. The dry weight of cells is measured to monitoring microbial cell population and

cell density. A plot of optical density reading from the spectrophotometer against cell dry

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258 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

weight can be obtained. The cell density, a standard calibration curve for S. cerevisiae at

wavelength 580 nm, has been experimentally determined, which was shown earlier.

10.14 YIELD CALCULATION

To calculate percentage yield, one needs to know the theoretical value that can be obtained,

based on material balance, and if 100% of the reactant(s) is converted to product. After

obtaining the experimental value (actual yield), divide it by the theoretical value, then

multiply by 100 to get percentage yield.

(10.14.1)

10.15 BATCH FERMENTATION EXPERIMENT

It is very simple to perform batch fermentation in a small flask with a volume of say 200 ml.

Now our target is to use a 2 litre B. Braun fermenter. All accessories are shown in Figure 10.5.

The fermentation vessel only, as shown in Figure 10.6, with about 250ml of media without

any accessories but with some silicon tubing attached with a filter for ventilation is auto-

claved at a 131 ⬚C for 10 minutes at 15 psig.

After that, the system is handled with special

care and all accessories attached. Media is separately sterilised and pumped into the vessel.

Inoculum is transferred and the batch experiment is started right after the inoculation of

seed culture. An initial sample is withdrawn for analysis.

10.16 CONTINUOUS FERMENTATION EXPERIMENT

The experiment is accomplished with a 2 litre B. Braun fermenter biostat (Germany)

equipped with DO and pH meters. Temperature and level controllers are very sensitive, with

highly accurate response from the sensors installed in the vessel. Figure 10.7 shows a perfect

continuous fermentation set up used in photosynthetic production. A small modification of

%Yield

Experimental value

Theoretical value

⫽⫻100

FIG. 10.4. Refractive index reading for ethanol solution.

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APPLICATION OF FERMENTATION PROCESSES 259

the sampling port is necessary to change the sample jar with a stainless steel tube sample port

connected with a rubber septum, which is gas tight. A simple syringe needle is used to take

liquid samples aseptically. The original fabricated sampling jar must be attached to on-line

steam to prevent any contamination while sampling liquid is withdrawn. The fermentation

Motor

Effluent

Gas analysis

(GC)

Base

Acid

Media

Gas

Temp. control

Level control

pH control

FIG

. 10.5. Continuous stirred tank fermenter, experimental setup with instrumentations and controllers, effluent.

120 mm

Gas in

50 mm

120 mm

10 mm

80 mm

13 mm

30 mm

FIG. 10.6. Geometrical dimensions of B. Braun fermentation vessel.

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260 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

vessel is jacketed, and an external heating and cooling water bath is provided with the

fermentation unit to control the temperature, set at 32 ⬚C. Since ethanol fermentation does

not require any oxygen, neither air nor pure oxygen is supplied. The CO

is vented from the

carboy by installing a sterilised air filter.

Draw a sample at the time intervals proposed in Table 10.1. The fermentation should last

for approximately 24 hours before the culture enters the stationary phase.

• A 5 ml sample is adequate to analyse optical density, glucose/sucrose concentration and

ethanol concentration. For sugar analysis you may dilute 1ml of sample and 9 ml of dis-

tilled water to have a suitable concentration range for DNS analysis.

• Save a drop of the sample on a slide for later microscopic examination of the purity of

the culture.

• Initially, when the cell density is still low, the optical density of the sample can be measured

without dilution with water. Perform this step quickly with a spectrophotometer at 580 nm.

Then, filter out the cells from the sample. After the optical density is over, save 1ml of the

sample by pipetting it into a test tube for optical density measurement. Force the remaining

sample through a filter. Normally a sterile syringe is used to withdraw the sample. A suitable

filter case fitted on the tip of syringe is used for removing cells. The filtered sample is used

for analysis by gas chromatography (GC). Ethanol is determined by GC. Otherwise, the

refractive index method is easy to use but it may not be very accurate for research purposes.

• The clear filtrate is collected in a tightly capped sampling vial for later analysis. Freezing

the filtrate will better preserve the existing condition.

• If a 1 ml sample is saved for the optical density measurement, dilute the sample with 5 ml

of water. Record the optical density.

FIG. 10.7. A complete laboratory set-up of biostat, B. Braun fermenter with external feed pumps and product

reservoirs.

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APPLICATION OF FERMENTATION PROCESSES 261

10.17 MEDIA STERILISATION

Next, the prepared nutrient must be sterilised. Usually, this is done by autoclaving.

However, autoclaving is not a practical sterilisation method for the formulation used in this

experiment. First, the heat of autoclaving may caramelise the sugar and darken the nutrient

to a brown colour. Secondly, vitamins will be destroyed by the heat. Furthermore, the loss

of liquid due to boiling during autoclaving will change the concentration of various nutri-

ent components, including the rate-limiting carbon source. Evaporation loss is especially

severe when ethanol is the designated carbon source. Even with all the disadvantages of an

autoclave, it is still customary to use autoclave routine equipment with some modification

such as for a fast programme or to sterilise sugar and media separately.

Instead, membrane filtration may be used to sterilise the nutrient in this experiment. This

can be accomplished by drawing the nutrient from a mixing jar and forcing it through an

in-line filter (0.2␮m pore size) either by gravity or with a peristaltic pump. The sterilised

medium is fed into an autoclaved nutrient jar with a rubber stopper fitted with a filtered vent

and a hooded sampling port.

For each run, calculate and plot the cell biomass concentration, glucose concentration,

ethanol concentration, and pH as a function of time. Identify the major phases in batch

fermentation: lag, exponential, stationary and death.

10.18 BATCH EXPERIMENT

10.18.1 Optical Cell Density, Ethanol and Carbohydrate Concentration

Measure the optical cell density of S. cerevisiae at a wavelength of 520 nm. Try to collect

data based on information required in Table 10.1. Draw a growth curve based on incuba-

tion time and cell dry weight. The cell concentration is an indication of microorganism

growth. A standard calibration curve is needed before any actual experiment.

10.18.2 Continuous Ethanol Fermentation Experiment

The batch experiment had neither incoming fresh media nor any product stream leaving

the fermentation vessel. A complete experimental set up with a B. Braun Biostat, is shown

in the above laboratory experimental set up. The continuous flow of media requires a feed

tank and product reservoir. The batch process has many disadvantages such as substrate and

product inhibition, whereas in the continuous process the fresh nutrients may remove any

toxic by-product formed.

10.19 EXPECTED RESULTS

1. The parameters of the Monod cell growth model are needed: i.e. the maximum specific

growth rate and the Michaelis–Menten constant are required for a suitable rate equa-

tion. Based on the data presented in Tables 10.1 and 10.2, obtain kinetic parameters for

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262 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

the experimental data in ethanol production. It is customary to have a linear model and

plot a double reciprocal model known as the Lineweaver–Burk plot.

2. Another parameter for living organisms is the media pH, which may change during the

course of the fermentation. The cause of change in pH is due to product formation and

metabolites from the cells being liberated. Here, the question is: did the change in pH

coincide with the different batch growth phases? The answer may not be so clear, as it

depends on the activity of the microorganisms and the media absorbing the metabo-

lites.

3. Identification of the major elements of life for living organisms is a necessary step to

have a deep understanding of the growth pattern, i.e. nutrient requirement, in a typical

yeast cell. Trace metals and minerals act as cofactors in enzyme activities, so the

amount of each element such as Zn, Cu, Co, Mg, Mn and Mo in a typical yeast cell

may have major role. In fact, these essential elements or trace metals may need to be

represented in very small amounts, even micrograms, in our synthetic medium formu-

lation. From the relative ratios of the elements present in the media, we may be able to

identify the limiting substrate. It is well known that the carbon source is the limiting

substrate in application of kinetic models like the Monod rate model.

REFERENCES

1. Demain, A.L. and Solomon, A.N., Sci. Am. 245, 67 (1981).

2. Phaff, H.J., Sci. Am. 245, 77 (1981).

3. Aiba, S., Humphrey, A.E. and Millis, N.F., “Biochemical Engineering”, 2nd edn. Academic Press, New York,

1973.

4. Baily, J.E. and Ollis, D.F., “Biochemical Engineering Fundamentals”, 2nd edn. McGraw-Hill, New York, 1986.

5. Blanch, H.W. and Clark, S.D., “Biochemical Engineering”. Marcel Dekker, New York, 1996.

6. Doran, P.M., “Bioprocess Engineering Principles”. Academic Press, New York, 1995.

7. Pelczar, M.J., Chan, E.C.S. and Krieg, N.R., “Microbiology”. McGraw-Hill, New York, 1986.

8. Scragg, A.H., “Bioreactors in Biotechnology, A Practical Approach”. Ellis Horwood Series in Biochemistry

and Biotechnology, New York, 1991.

9. Shuler, M.L. and Kargi, F., “Bioprocess Engineering, Basic Concepts”. Prentice-Hall, New Jersey, 1992.

TABLE 10.2. Data sheet for continuous ethanol fermentation, experimental run 2, S

⫽35 g/l

Media Retention Cell Substrate 1/S, l/g ⫺r

, Rate 1/⫺r

Ethanol

flow rate, time, t, h Density, concentration of substrate concentration,

ml/h g/l (S), g/l uptake, g/l.h g/l

83 24 2.30 8.3 0.12 1.11 0.90 8.9

125 16 1.95 9.5 0.105 1.59 0.63 6.9

167 12 1.45 13.0 0.077 1.83 0.55 5.2

250 8 1.03 20.5 0.049 1.81 0.55 3.8

500 4 0.38 27.5 0.036 1.88 0.53 1.5

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CHAPTER 11

Production of Antibiotics

11.1 INTRODUCTION

Antibiotics are produced by fermentation. The process may take a few days to obtain an

extractable amount of product. Antibiotic production is done by the batch process. Oxygen

transport is the major concern; therefore sufficient polymeric sugar and protein with a trace

amount of elemental growth factors are used to enhance production. An anti-biogram test

is used to observe the amount of antimicrobial agent in the fermentation broth. A bioassay

determines the activity unit of the bactericides.

11.2 HERBAL MEDICINES AND CHEMICAL AGENTS

Herbal medicine was used in ancient treatment. Natural chemicals extracted from herbs

were used. For seventeen centuries, natural quinine extracted from the bark of a particular

tree known as “cinchona” was used to treat malaria. American Indians and southern Asians

were able to fight malaria by chewing the bark of cinchona trees. Gradually, chemical

agents were identified and used for treating patients. Toxic compounds, such as mercury,

were used to treat syphilis for eighteen centuries. Even arsenic compounds were used

to cure many diseases without great danger to the patient. Arsphenamine and neoar-

sphenamine were also used to treat patients with syphilis. Investigation of chemotherapy

expanded into a wide range of compounds. Chemotherapy is known as treatment of a dis-

ease with chemicals called chemotherapy agents. Treatment with drugs has been practised

for many centuries. In mid-1950s, the first generation of sulphonamide was successfully

used against certain bacteria, which was a great victory in field of medicine. Antibiotics as

chemotherapy agents were discovered. The action of drugs or chemical agents was to

destroy germs and to control the growth of microorganisms, or to prevent microbial growth

where the aim was to treat a patient. The chemical substances possessed selective toxicity,

but over-dosage created complications for the host. The side-effects of chemicals were

monitored based on observed symptoms during and after treatment. The drug targeted spe-

cific germs and parasites. In general, germicides were not selective in their mechanisms,

and often interfered with the immune system. Inactivation of germs and parasites with anti-

gen and antibody mechanisms played a major role in the development of germicides and

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