Biotechnology Techniques Received 26th September

Vo1 4 NO 6

403-403

(1990)

THE USE OF A COULTER COUNTER TO INVESTIGATE A MIXED CULTURE OF PICHIA STIPlTIS D.R.J. Luyben. Department of Biochemical Engineering, Delft University of Technology, AND SACCHARONYCES CEREYISIAE. Grootjen, M.G.A. Windmeijer, R.G.J.M. van der Lans and K.Ch.A.M.

Jul ianalaan
SUMMARY In mixed description S. cerevisiae.

67, 2628 BC Delft,

The Netherlands.

cultures of the

the

biomass cells that

distribution differ

has to Counter in size, is

be known for a fast

a good and

system.

The Coulter

and accurate

method to distinguish

such as P. stipitis

INTRODUCTION Natural laboratory are better exclusively systems seldom consist Practical of waste water 1982), of mono-cultures. use of mixed yogurt However, these is cultures (Driessen, with most almost

investigations controllable. the domain

mono-cultures

are used because treatment e.g. (Salmon

cultures

and Bull,

and food processing cheese production. (Harrison, reported any special that,

(Steinkraus,

1984) 1981) and

Harrison without another

The use of mixed cultures resistance in some cases, a mixed culture cultures mixed

can have certain to contamination. could cultures stay give

advantages

1987) such as the better precautions. Mixed

aseptic higher

can also conversion the second

yields. stipitis, of this is 3 rates time than xylose,

In the conversion of mixed substrates advantage. In the case of glucose/xylose

diauxic diauxic only g/l are conversion growth converted (Grootjen needed to using occurs (Kilian is and conversion when the glucose et al., achieve a mixed culture. a complete culture that

can offer by Pichia

and van Uden, 1988). The result substrate, concentration conversion is reduced cultures of this to less

199Oc). In continuous
may lead

low dilution mixed in residence

substrate. cerevisiae

However

to a reduction

in a continuous

With a mixed culture

of Saccharomyces

403

and P. stipitis,
xylose In order necessary distribution

the glucose

can be converted and describe distribution the control

by S. cerevisiae these in the mixed

while it

the is

is converted to adequately to

by P. stipitis. monitor biomass needed for cultures system. The biomass

know the is also

of such a system. distribution and P. stipitis as the carbon is the two source as the plates numbers. directly colony by means for a fast can the and be

A common method for plate-count different sole carbon are P. stipitis The disadvantage available. counting Mountney of estimation reliable culture 1986). the and the other method. kinds with

the determination

of the biomass glucose

In the case of S. cerevisiae

are made, one with S. cerevisiae plates is to that the colonies the xylose.

of plates

can not grow on xylose formed on the xylose give the results colonies total are cell not and the counting counting is

source, of

consequently this take method

cells.

The glucose two days

It will
is also and O'Malley

grow the

time-consuming. (1966) is have shown that more the differ reliable electronic than The Coulter in of the plate

a Coulter method

Counter to

of bacterial count ratio

populations. quantities

Counter different size

number of cells in diameter cells a Coulter

a sample.

In a mixed
cells

and the Counter

measured when the cells The Coulter so the volumes

(Davison the cell

and Stephanopoulos, distribution,

can also determine

of the different we have used

can be calculated. Counter to distinguish culture. the two

In this
yeasts,

study

P. stipitis

and S. cerevisiae,

in a continuous

MATERIALS AND METHODS Microorganisms Pichia stipitis CBS 5773 extract and Saccharomyces peptone glucose for cerevisiae agar slants. CBS 8086 Precultures were for maintained continuous Fermentations Fermentations volume according silicone of were 1 1. carried Medium et al. out in a 2 litre and 1990b). fermenter reactor with a working were composition et al., configuration on yeast experiments

were incubated

16-24 h at 30C.

to Grootjen

(1990a). Oxygen was supplied

by means of a

membrane (Grootjen

404

Coulter Coulter yeast

Counter Counter that sample was used to with determine with this the tube ml. biomass composition. The range Counter cells was equipped a tube with Counter an aperture could of 30 pm. The to take

A Coul ter

can be measured volume with of 0.05

have to be in the be regulated with

of 0 to 15 pm. The pump of the Coulter a constant before diameter the measurement of 2.09 pm.

or 0.5

The device

was calibrated a known mean

a suspension

of particles

RESULTS AND DISCUSSION It the depends Coulter on the difference Counter is in size for of the microorganisms the distinction used whether between the two

suitable

microorganisms.

26 w 20 1.5 I 1.0 05 0.0 2

:: : A
: ... .a.* :.... ._ - 1.. . ,. ,, ... : 4 diameter

10 Lun 40 f B
20 0

2.6 103 20 lb : 1.0

60 .-:: ;;.; . : :. : : :. ;: :. .. = * l@ -40

-20

m8

4 diameter

~718

A
Figure

1. Coulter Counter measurements (A) and !i. cerevisiae (B) ( 1A and B the measurements cerevisiae the deviation some overlap this between culture overlap was started are are 3.5 pm for show that although of P. stipitis

of pure cultures of P. stipitis , number; , volume). cultures The of Pichia stipitis equivalent The in

In figure and diameters figures the is good

of pure shown.

Saccharomyces

mean volume

and 5.0 pm for between in diameter the feasibility

S. cerevisiae. is more severe these

from the mean diameter will a dividing to proof occur mixed

case of S. cerevisiae.

The separation

microorganisms cultures. be As a to chosen

consequence differentiate A continuous

can

the microorganisms. of this method

405

for the

the

determination

of the The

biomass

distribution. would culture was will

The conditions P. stipitis with culture overgrow cerevisiae. inoculated gradually for Figure 5.

were under P. was P. In

chosen stipitis.

in such a way that After with because a steady the

S. cerevisiae continuous state

overgrow

circumstances.

was reached S. cerevisiae are composition diameter. after

the P. stipitis

inoculated stipitis figure of cells

.S. cerevisiae. conditions with visible

favourable

2 the change of the biomass inoculation is at first as a function of the cell

of the continuous 2a gives graph In this

culture the number of volumes

is shown after S. cerevisiae are calculated

5. cerevisiae. 20 h. size.

the growth 2b the

In figure

as a function

of the cell

A
Figure

4 diameter

4 a,rneter 6 wn
culture after B. Calculated

2. Continuous culture experiments with a P. stipitis inoculation with S. cerevisiae. A. Cell numbers, cell volumes as a function of the cell diameter. figure composition the will is the S. cerevisiae from P. stipitis biomass the composition more essential related

In the latter in biomass in an earlier For monitoring 2b will Figure

peak more pronounced. to S. cerevisiae both figures

A change is known. be useful. the dry than to the

can be detected will

stage of the process provide

when the volume distribution information because

mass of the cells number of cells. To verify composition The results

be more closely

to the volume

the Coulter

Counter

method for are given

the determination with in table

of the biomass method. diameter

two samples

were also

analyzed

the plate-count 1. The dividing

of both methods

406

between high

P. stipitis

and S. cerevisiae found deviation as a fast

for

the Coulter the

Counter

experiments method has a 1966), between the S.

was empirically relative can agreement Counter cerevisiae Table 1.

to be 4.5 pm, Since two methods is

plate-count and O'Malley, well. discriminate

standard between be the used

(25%, Mountney reasonably to method

So the

Coulter

and P. stipitis. Comparison of the Coulter Counter method method in a continuous culture experiment. Coulter Counter S.cerevisiae 1o'O 1 P.stipitis 1 and the plate-count

Plate-count S.cerevisiae 1o'O 1

time h 21

P.stipitis

1oO 1 3.0 2.3

lOi

33

3.3 4.2

5.3 2.3

2.0 3.0

CONCLUSION The Coulter 5. cerevisiae. the biomass cell volumes Counter distribution is necessary can be used to discriminate fast in a continuous for a good insight culture. between P. stipitis and of the

The method is relatively

and can be used to monitor The calculation in the biomass distribution.

REFERENCES Davison 1137. Driessen culture Grootjen Grootjen Grootjen Harrison F.M., B.H., G. Stephanopoulos, 1986a, Bushel1 der der der for Lans Lans Lans Biotechnol.Bioeng., and J.H. Slater 28, (eds.) Luyben, Luyben, Luyben, 1127Mixed 1990a, 1990b, 199Oc,

1981,

In:

M.E. van van van

fermentations,

99-120.
and K.Ch.A.M. and K.Ch.A.M. and K.Ch.A.M. 24, 12, 20-23. 4, 149-154. publication.

D.R.J.,
D.R.J., D.R.J., D.E.F.,

R.G.J.M. R.G.J.M. R.G.J.M.

Enzyme Microb.Technol., Biotechnol.Techniques, Enzyme Microb.Technol.,

submitted

1987, Adv.Appl.Microbiol.,

129-164.

4Q7

Kilian 548. Mountney

S.G. and N. van Uden, G.J., Bull K.H., J. O'Malley, A-T., 1982, 1984, in microbial

1988, Appl.Microbiol.Biotechnol., 1966, Appl.Microbiology, In: M.J. Bull ~01.1, Klug Proc.3rd and C.A.

27, 545-

14, 845-849. Reddy (eds.) 656-662. (eds.) Microbial Current

Salmon I., perspectives Steinkraus interactions

ecology,

Int.Symp., Slater

In: A.T.

and J.H. 407-442.

and communications,

408