Containment in industrial

1
biotechnology within waste water
treatment plants
J.A.C. Noordover, J.J.M. Hofmeester, J.P. van der Burg, A. de Leeuw,
P.W.M. van Dijck, R.G.M. Luiten, G.S.P. Groot, A.B. van Putten
Summary
Physical and biological containment are essential for industrial Good Industrial Large-Scale Practice (GILSP)
processes using genetically modified organisms (GMOs). In the event of an accidental discharge in the production
plant, a large amount of organisms is released into the wastewater treatment (WWT) system. Therefore, the WWT is
considered as a part of the containment. An accurate hydrodynamic and microbiological model for the containment
aspects of industrial WWT plants (verified by measurements using industrial hosts) shows that drainage of an
enormous amount of micro organisms is reduced in the WWT into an environmental effect of a discharge of a small-
scale fermenter. Microcosm experiments correlate well with the survival rates in the WWT and therefore can be of use
to predict the behaviour of GMOs in this environment.

Introduction Hydrodynamic model

The safety of GILSP industrial large-scale processes A basic set of equations was set up describing a single
using GMOs is based on physical and biological continuously stirred tank reactor (CSTR), two CSTR’s in
containment. Physical containment will ensure that living series, and two CSTR’s in parallel. Subsequently the best
organisms are kept within a defined physical boundary. model for each WWT reactor separately has been
Biological containment ensures that the chance of determined by the concentration profiles of the tracer
survival, the dissemination of the GMOs and the transfer (lithium) during the actual drainage experiment. (figure 2)
of the newly introduced DNA to other organisms is
limited.
As small releases of GMOs are allowed, organisms used
in fermentations under GILSP conditions have to fulfil the
demands related to biological containment. The present
study describes the results of the survival of industrial
(G)MOs in both microcosm experiments and in a
simulated accidental discharge at the Delft production site
of DSM using non-GMO strains, which serve as a parent
for industrial GMOs.
DSM Anti-infectives

Microcosm survival studies

Small samples of a relevant environment are inoculated
with the studied micro organisms. The survival rate and
competitiveness is observed.
The experiments with B. subtilis indicated that within a
DSM Gist, P.O. Box 1, 2600 MA Delft, The Netherlands

few days, and both in sterile and in non-sterile

Figure 2: Modelling of lithium profiles.
environments (soil, river water, wastewater) the titre of O Buffertank;
viable Bacillus dropped to below the detection limit. ,D Anaerobic and aerobic reactor street 1;
For K. lactis in non-sterile environments a slow reduction *, Anaerobic and aerobic reactor street 2
in surviving yeast cells is observed with titres falling below
the detection limit within 40 days. In sterile environments
K.lactis grows to levels of 108 CFU/ml. (figure 1) Microbiological observations
Fitting the concentration profiles of B. subtilis gives death
rates (kd) of 3.8 h-1 and 2.1 h-1 for anaerobic reactors and
aerobic reactors, respectively.
CFU/ml The death rate of the in the anaerobic reactors is about 1
1.00E+10 to 1.4 h-1. In the aerobic reactors appears to reflect
survival at approximately the same CFU level.
1.00E+08 With the model and the fitted death rates a reduction of
the micro organisms drained from the pilot plant to the
1.00E+06 WWT by a factor of 104 and 103 is calculated for B.
subtilis and K. lactis, respectively.
1.00E+04 Conclusions
Based on the data of the tracer and the recovery of living
1.00E+02 micro organisms, combined with a model describing the
dominant hydrodynamic effects, the containment level of
1.00E+00 the wastewater treatment system was established.
0 5 10 15 20 25 The use of lithium as a tracer is essential to determine the
Time (d) correct hydrodynamic model for the reactors in the WWT,
Figure 1: Survival of K.lactis in microcosm waste water. resulting in a description of residence time, distribution,
Variations are sterilized (closed symbols) versus untreated
and mixing.
(open symbols) waste water; incubations at 8°C (red
Overall it is concluded that the WWT is an extension of
triangles) or 25°C (blue squares).
the containment of micro organisms in fermentation
industry. The effect of a large accidental discharge is
reduced in the WWT to a laboratory scale volume.

Published: J. of ind. microbiol. and biotech., 2002, vol. 28, iss. 2, pag. 65-69