Acta Biotechnol.

12 (1992) 4, 281 -291 Akademie Verlag

A Contribution to the Mechanical Disrupture of Microorganisms

on a Small Technical Scale

LUTHER,H., SCHUSTER,
E.

FZB Biotechnik GmbH

Alt Stralau 62
0-1017 Berlin, F.R.G.

Summary
The mechanical disrupture is described with the examples of selected yeasts, fungi and bacteria under
practical circumstances. A particularly high degree of disrupture while simultaneously avoiding a
strong destruction of the cell wall, and a trouble-free continuous operation are considered.
With the aid of laboratory studies and the physicochemical data of the biomass, suitable equipment
for the small technical and industrial scale can be chosen.

Introduction
The use of intracellular enzymes requires their release from the cells. There are numerous
possibilities of cell disrupture, as listed in Tab. 1.
On an industrial scale, the high pressure homogenizer and the ball mill are generally used
[I, 21.
Compared with other principles the main disadvantage of the method of mechanical
disruption is that a certain amount of destruction of the cell walls occurs [3, 41. This leads
to problems during the separation of cell debris by means of centrifuges and separators.
Tab. 1. Feasibilities of cell disrupture

Principle Method

Chemical Acidic disrupture

Alkaline disrupture
Enzymatic disrupture
Solvent extraction
Physical Freeze-thaw
Heat treatment
Osmotic destruction
Decompression
Ultrasonic treatment
Mechanical Wet milling
Pulverizing
Pressure extrusion
Freeze-pressing
282 Acta Biotechnologica 12 (1992) 4

Therefore disrupture conditions should be optimized concerning both the achievement of

a high disrupture amount and the least possible cell wall destruction [5].
The advantages and disadvantages of the mill and the homogenizer can be seen, and from
the results general guidelines for the disruption of microorganisms can be derived.
In order to achieve this, different biomasses are included in the experiments:
- isolation of protein from yeasts
- purification of uratoxidase from Penicillium spec.
- release of penicillin G acylase from E. cofi.

Material and Methods

On a “ml-scale”, the influence of variables on the degree of disruption, and on the separating behaviour
of the cell wall debris, were investigated. On a “I-scale”, the results had to be confirmed and the
technical aspects (rheological properties) had to be considered. The degree of disrupture was estimated
by measuring the released protein [6] or enzymatic activity.
The determination of acylase activity was made by means of the “DAB-method” [7] and that of the
uratoxidase by the degradation of uric acid [S].
The ability to deposit, and the separating efficiency, were evaluated by means of the following criteria:
- clouding of the fugate after centrifugation in a laboratory centrifuge under standard conditions
- dry mass of the residual sediment
- composition of particle size in the supernatant after centrifugation under standard conditions
- clouding of the fugate after separation on a small technical scale (SA 1- 175)
- filtration rates in the ultrafiltration and
- viscosity measurements.
Clouding was characterized by measuring the light passage at 600 nm [3].
The loss of dry mass is the difference between the weights of sediments before (lOO%), and after
disruption.
Standardized separating conditions were applied.
For the particle count, the Laborscale PSLl and PSAl (MEDICOR, Budapest) were used [9].
Distributive curves were recorded by an xy-recorder (“endim 620.02”, MESSGERATEWERK
Schlotheim).
By means of a computer programme “Histo”, the “true” particle size distribution was calculated from
the total particle number and the distributive curves. and presented as histograms.
On a small technical scale, the cell debris and proteins are separated by separation (SA 1 - 175,
WESTFALIA) or centrifugation (CEPA, Padberg). The efliciency is pursued by a clouding measure-
ment of the supernatants.
Ultrafiltration was carried out by means both of the flat filters (UFI 1200, Schwerin with membranes
U F 120, ZELLSTOFF- und ZELLWOLLEWERK Wittenberge) and the hollow fibre modules
(KERADENTA-Werk, Radebeul) [lo].
Viscosity was measured at Rheotest I1 (MEDIZIN- und LABORTECHNIK, Freital).
The structural viscosity of the liquids was expressed with the flow behaviour index “n” [I I]. It can
differentiate between zero and one (Newtonic liquid) and is calculated from the power equation
T - To = K.D” (1)
and the CASSON
equation

1/;=Ko+KI1/Z;;
in which
T = shear stress [dyn cm’]
To = yield value [dyn cm2] a the time t = 0
D = shear rate [s-’1
K,KO,K , = constants
LUTHER,H., SCHUSTER,
E., Mechanical Disrupture of Microorganisms 283

Calculation was carried out by means of the computer programme “Visko”.

For the cell disrupture various types of equipment were at our disposal:
- ball mill:
Dyno mill KDL (Bachofen, Switzerland) with a total volume of 600 ml (laboratory scale)
self-made device with a volume of 300 ml (Frequently 3 mills were arranged to a cascade.)
ball mill HFlO (NAGEMA, Radebeul) with a total volume of 10 1 (small technical scale)
- high pressure:
Gaulin-Homogenizer 15M-8TA, GAULIN Corporation, EVERETT, USA with a maximum
capacity of 3 l/min
Gaulin-Homogenizer LAB 60, APV SCHRODER GmbH, Liibeck with a maximum capacity of
3 l/min.
The ball mill HFlO and the homogenizer LAB 60 are the smallest types of a serial manufacture.
Therefore, results obtained with these derivatives can be transferred to a larger scale without problems.
The experiments were carried out in the case of uratoxidase on a 100-1 scale, at the penicillin acylase
isolation on a 20-1 scale and with yeast on a 1-1 scale, respectively.

Results
Experiments on a 1-1 (laboratory) scale
The technological parameters of the ball mill were investigated by means of the disrupture
of yeast and E. coli. In Fig. 1, the influence of ball diameter, time of staying and speed rate
u p o n the degree of disrupture of a 10 percentage yeast suspension is shown.

100 -

I
0 2 6 10
Time o f staying C m i n l
Fig. 1. Influence of variables (10 percentage yeast suspension)
Diameter of glass beads Speed rates
[mml b/sI
v 2.0 10
0 0.2 10
A 0.2 15
0 2.0 15
0 0.2 5
284 Acta Biotechnologica 12 (1992) 4

Tab. 2. Disruption of baker's yeast by means of the ball mill (laboratory scale)

Variables Results
1. Speed rate (5 -20 m/sec) < 10 m/sec: complete disrupture impossible
2 20 m/sec: glass breaking
2. Time of staying Increase in temperature,
increased solid content in supernatant
3. Ball diameter small balls: high degree of disrupture
(0.15-0.18; 0.8- 1.9; 1.6-2.4 mm) foam formation
large balls: glass breaking
4. Degree of filling with balls (50-75%) 75%: highest degree of disrupture/time
5. Concentration of suspension pumpable at high concentrations
(Biomass/buffer = 1 :10- 1 :5)
6. Temperature (5 - 20 "C) 20 "C: higher enzyme release
7. Specificgravity of the balls; iron, cavenit,glass iron, cavenit: metal rub-off, high degree of dis-
rupture
In Tab. 2, the results obtained by the ball mill are summarized:
- A complete disruption is impossible at too small speed rates (510 m/sec).
- At too high speed rates (>20 m/sec), the mechanical load causes increased glass
breaking.
- A high degree of disruption is reached with increasing time of staying and at speed
rates of 2 10 m/s. For the isolation of enzymes, intensive cooling is necessary.
Furthermore, the solid content of the supernatant increases.
- The diameter of the glass balls influences the degree of disrupture.
The use of a large ball lead to increased glass destruction. It is therefore preferable to
use small balls. Thus, information from the literature [12] is confirmed.
Material with high specific gravitiy cannot be used, because even at low speed rates,
intensive rub-off takes place.
Similar to the high pressure homogenizer, a n intensive disruption cannot be attained under
less than optimum conditions (too low pressure). Noticeable in Fig. 2, the design of the valve,

0 2 4 6
Number o f q c l e s

Fig. 2. Influence of pressure and design of the valve on the degree of disruption
= Yeast, standard valve, 400 bar
o = Yeast, standard valve, 700 bar
x = Yeast, cell rupture valve, 600 bar
LUTHER,H., SCHUSTER,
E., Mechanical Disrupture of Microorganisms 285

0 2 4
"Unit -/eve/"

Fig. 3. Influence of disruption devices on the separation of cell debris

x = High-pressure Homogenizer
o = Bead mill
0 = Original-sample

including the bump-ring, also influences the degree of disrupture. In the experiments, the
laboratory homogenizer of the type 15M-8TA was used.
In the case of yeasts, the mechanical destruction of the cell walls by means of ball milling
is essentially more complete as compared to the high pressure homogenizer (see Fig. 3).
Therefore, for the isolation of products from yeast cells, using the homogenizer is
advantageous.
The mechanical destructibility of E. cofi was studied by means of a self-made Dyno-Mill.
In this mill, as in the mill HFlO, the use of small glass balls was impossible due to technical
limitations. Through the aid of a statistic experimental plan [ 131, the variables listed in
Tab. 3 were tested.
Tab. 4 shows the results. Within the tested ranges, the following relations apply:
- A long time of staying, the addition of tensids, the maintenance of a high temperature
during the disrupture, and a low extraction temperature lead to high degrees of
disruption.
- Within the tested variables, only the total particle number is significantly increased with
a longer time of staying.
This is in contradiction to the fact that the variables have no influence on the aim parameters
of clouding and loss of dry mass. In the presence of tensids, and at low temperatures of
extraction, the total particle number in the supernatant is probably descreased.

Tab. 3. Influence of variables on the disrupture of E. cofi (experimental plan corre-

sponding to [ 131)

Variable Level

+ -

Temperature ("C) 30-40 10

Influence of tensids [%I (SDS) 0.2 0
Extraction time [h] 12 2
Extraction temperature ["C] 40 5
Time of staying [min] 16 7
286 Acta Biotechnologica 12 (1992) 4

Tab. 4. Statistical interpretation of the experimental programme regarding the influence of variables
on the degree of disrupture
~ ~~

Aim parameter Significance of variables [YO]

~ ~~

Time of Time for Tensid Temperature

staying extr. content
disrup. extr.

Degree of disruption 99.9 79 99.3 97 -92.7

Dry mass loss in the < 50 - 84 < 50 57
sediment
Clouding 57 < 50 < 50 -71.2
Total particle number 97.7 - 87.7 < 50 -84

Thus, for the disrupture of E. coli by means of laboratory ball mills, the following parameters
are preferred :
time of staying: 18 min at a flow of 200 ml/min
temperature: 35-40 "C
extraction at: 1 h, 5 "C
Under these conditions, the results given in Tab. 5 are achieved. They can be compared
with those obtained by homogenization.
For the disrupture of E. coli, the following conclusions can be drawn from Tab. 5 :
- The degrees of disruption are comparable.
- The properties concerning separation do not show significant differences.
Because of its mycelium structure, the disrupture of Penicillium spec. by the homogenizer
demands special preliminary treatment (colloidal milling).
Thus, with the ball mill, the influence of time of staying, temperature of disrupture and
through-flow on the release of uratoxidase, and particularly on the properties of debris
regarding separation (clouding, dry mass and particel content in the fugat), was studied.
For achieving a degree of disrupture respective to an extractability of 80-90%, the
following conclusions are valid :
- At a flow of 240 ml/min, a minimum time of staying of 75 sec is necessary
- High temperatures (i.e., 20 "C) are better than lower temperatures (i.e., 5 "C)
- A high flow (460 instead of 240 ml/min) is favourable with respect to extractability.

Tab. 5 . Comparisons between the high pressure homogenizer and the ball mill, regarding
the degree of disrupture of E. coli and the separation properties

Aim parameter Mill High pressure

homogenizer

Degree of disrupture [%] 109* 109 *

Specific activity 150 132
[E/mg pure protein]
Sediment after disrupture [YO] 70 60
Optical density [%I 50 65
* By the means of the measurements of acylase activity of E. coli, the attainment of
degree values of disruption >loo% are possible. The total activity is determined
from the intact cells. However, the degree of disrupture is calculated from the activity
of supernatant after centrifugation.
LUTHER,H., SCHUS'ITR,
E., Mechanical Disrupture of Microorganisms 287

The separation of the course cell debris (CEPA/Padberg) in all experiments, took place
without problems. Further information corresponding to the isolation of uratoxidase can
be obtained from the literature.
The kinetic course of disrupture is shown in Fig. 4.
The graphic deduction of the rate low demonstrates a low of first order for the mechanical
disrupture of microorganisms both in the laboratory, and on a small technical scale by
means of milling and homogenizing. Data of literature [14, 1.51 are thus confirmed. For
the calculated rate constants and half-times, see below.

Experiments on a Small Technical Scale

The disrupture of yeast via high pressure homogenization is preferred to milling because
of the former's better separation of the cell debris.
Therefore, further comparative experiments on a small technical scale were contraindi-
cated.
In order to disrupture mycelium on a 100-1 scale, only the ball mill could be used for the
described reason. Disruption and separation of the cell debris were carried out without
problems. Therefore, a description is not necessary. For the disrupture of bacteria, for
instance E. coli, corresponding to the literature [15], the high pressure homogenizer is
used as a rule. On a laboratory scale, we already had obtained comparable results by means
of the ball mill.
Therefore, the aim was to confirm these advantageous results on a small technical scale.
Because the viscosity is interesting on a industrial scale, rheological data were measured
depending on the degree of disrupture during homogenization (Fig. 5).
Particularly at the beginning, viscosity is strongly decreased with an increasing shearing
gradient. Curves of this kind are characteristic for structure-viscous solution (suspensions).
That means that pumps better transport the biomass, or the partly disrupted biomass, if
their consistency is lowered by mechanical stirring.

/ /.

Time of staying C m i n l
I I

0 2 4
Number of cycles

Fig. 4. Kinetic experiments relevant to the mechanical disrupture of microorganisms

A = Yeast, mill - laboratory; 0 = E. coli, mill - laboratory; + = E. coli, HFIO;
0 = Pmccil. spec. HFIO; = E. coli, LAB60
288 Acta Biotechnologica 12 (1992) 4

L I

Shear rote [s-‘I

Fig. 5. Influence of the shearing gradient on viscosity in relation to the degree of

disrupture
0 = original-sample; W = first passage; A second passage; v = third passage; x =
fourth passage; 0 = fifth passage
The flow behaviour index “n” is an expression of structure-viscosity. Depending on the
degree of disrupture, it amounts to about 0.5 (see Tab. 6).
By all appearances, the structure-viscosity increases with a growing degree of disrupture
(for the most part, the cell content consists of proteins distinguished by their structure-
viscous properties).
A drastic increase in viscosity during the first squeezing is noticeable in Fig. 5 and 6. The
biomass comes to resemble pudding. Afterwards, the viscosity decreases and attains its
minimum in the third cycle.
The viscosity measured after the extraction step increases constantly, due to the released
cell content (Fig. 6, curve +).
Tab. 6. Influence of the degree of disrupture on the flow behaviour index “n”

Number of Degree of Flow behaviour

cycles disrupture index “n”

0 0 0.56
I 62 0.51
3 86 0.46
5 94 0.41
LUTHER,H., SCHUSTER,
E., Mechanical Disrupture of Microorganisms 289

0 2 4
Number of cycles

Fig. 6 . Dependence of viscosity on the degree of disrupture

o = measurement directly after disruption, charge 1
x = measurement directly after disruption, charge 2
+ = measurement after extraction

In Fig. 7, the influence of the mechanical disrupture (high pressure homogenizer) on the
particle distribution, or the total particle number/ml supernatant, respectively, is repre-
sented.
Whereas the total particle number clearly increases until the third cycle, the distribution
of particle sizes is only insignificantly changed. From this, it follows that the cell debris
should not be much more separable than intact E. coli cells.
Similar indications can be deduced from the determinations of residual sediment (see
Tab. 7).
For disrupture by means of the ball mill H F 10, biomass from only one fermentation process
was available (about 20 1). This amount is insufficient for the continuous operation of the
imill HF 10.

Tab. 7. Influence of the degree of disrupture (number of squeezing operations) on the

loss of sediment of E. coli

Number of cycles Residual sediment

(mg/dry ma@ g biomass set in)

0 298
1 230
205
188
189
192

20 Acta Biotschnol. 12 (1992) 4

290 Acta Biotechnologica 12 (1992) 4

d, Cpml

Fig. 7. Particle size distribution in supernatants of disrupted biomass

x = original-sample; A = first passage; 0 = second passage; A = third passage;
o = fourth passage; = fifth passage
Therefore, the mill was filled in portions. After disrupture, and during 20 minutes of
moderate cooling (30-40 "C), the suspension was drained off. On a small technical scale
a complete disrupture was also achieved. Here, however, the constant rate only amounts
to half of the value attained on a laboratory scale (see Tab. 8). Further optimizations are
therefore necessary.
The results obtained by means of both the homogenizer and the ball mill, including the
further working up, are summarized here.

Tab. 8. Mechanical disrupture and working up of E. coli - a comparison

( + = see remark in Tab. 5 )

Aim parameter Ball mill High pressure

( H F 10) Homogenizer (LAB 60)

Degree of disrupture [%] 103' 110+

Rate constant [min-'1 0.27
Optical density [%I
purification step 1 65 31
purification step 2 52 54
purification step 3 88 83
purification step 4 98 96
Filtration rate at
ultrafiltration [ l/h . m']
flat filter 15 20
hollow fibre 2.3 2.2
 E., Mechanical Disrupture of Microorganisms
LUTHER,H., SCHUSTER, 29 1
In order to isolate penicillin acylase after the disrupture, a treatment regarding temperature
and pH is carried out for an effective separation of cell debris and proteins. The supernatant
is concentrated via ultrafiltration. From comparison results that also biomass disruptured
on a small technical scale by milling possesses similar properties like that after disrupture
by homogenizing.
Considering the requirements of a
- high degree of disrupture
- rather low destruction of cell walls and
- trouble-free continuous operation
the following recommendations can be summarized :
- Yeasts and bacteria can be disruptured by means of ball mills as well as by high pressure
homogenizers. However, in the case of the high pressure homogenizer, slime forming
microrganisms (for instance E. coli], or mechanical contaminations easily lead to
occlusions. This risk is the primary problematic factor of this device. For continuous
operation (e.g. disrupture of E. coli), the biomass has to be filtered, therefore resulting
in losses of yield and time.
- Mycelium forming microorganisms, as a rule, are to be processed for disrupture by
means of a ball mill.
- Only in the disrupture of yeast could it be established that disrupture by means of the
high pressure homogenizer destroys fewer cells, and thus facilitates the separation of
cell debris.

Received June 3, 1991

Revised November 2 1. I99 1

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