1. Genetically Modified Organisms for Bulk Chemical Production Leo van Overbeek

2. Outline presentation Introduction Risk evaluations ‘White’ and ‘Green’ biotechnology for bulk chemical production Bulk chemical production in the future Conclusions

3. Background Research at Plant Research International, Wageningen Construction of genetically modified plants: disease suppression, qualitative aspects, optimization (marker-free GM plants) GMO acceptance (reports, discussions) Soil biology (GMO impact analysis)

4. Introduction Bulk chemical production E.g. Polyhydroxyalkanoate (PHA) Production by making use of Genetically Modified Organisms (GMOs) Optimal yield Chemical modification ‘White’ Biotechnology (contained use) and ‘Green’ Biotechnology (GM plants in open fields)

5. Goal Overview of prospects and limitations in the application of GMOs for bulk chemical production Emphasis on ‘White’ Biotechnology Effects on nature and food chains Knowledge gaps for future (large quantity) production

6. Risk evaluation and public perception Release of GMO will always occur What are the events after GMO release In order of severity:  Effect (neutral)  Hazard (negative consequence)  Risk (impact) Risk assessment: Risk = chance of hazard x exposure (volume/ time) Public perception on modern biotechnology (occasionally no rational arguments used in discussions)

7. Non-rational arguments Field experiment with a GM potato line Aimed to establish possible effects on the indigenous soil and plant- associated microflora Field destroyed by activists

8. From literature Field release studies with GM bacteria and plants GM plants and micro-organisms are constructed to demonstrate an effect (worst case) No effects observed Or only transient effects observed No obvious hazards could be find in literature!

9. Use of GMOs for bulk chemical production Effect on food chains PHA is non-toxic and non-allergenic Effects on natural environments PHA is biodegradable No impact on consumption goods and natural environments expected!

10. GMO effect after release EffectMeasure Recombinant gene expressionControlled regulation of recombinant gene construct GMO survival and spreadPhysiologically impaired host (e.g. auxotrophic strains) Containment genes Gene transferRecombinant DNA insertion in non-mobile constructs Gene type  Genes whose products do not have obvious effects on other organisms  Assessment for genes whose products have an effect

11. Limitations to evaluate consequences of GMO releases Analytical tools Technical limitations for detection Environmental impact Where to compare with? Natural fluctuations are large and not always understood Ecology Not all organisms are described (soil) Not all interactions are clear

12. ‘White’ Biotechnology Contained use of micro-organisms (or biotechnological derivatives) for production of e.g. enzymes and bulk chemicals Use of renewable raw materials and advanced enzyme systems, replacing fossil raw materials bio-energy biomaterials bulk chemicals Direct: e.g. bulk chemicals like PHA Indirect: production of enzymes required for bulk chemical production Realistic for industry

13. PHA production in closed systems ConstructReference Ralstonia eutropha with phaC from Aeromonas punctata Fukui and Doi 1997 and 1998. Aeromonas hydrophila with yafH from E. coli Lu et al., 2004 A. Hydrophila with phaPCJ genes from A. punctata Han et al., 2004 Arxula adeninivorans with phbABC genes from R. eutropha Terentiev et al., 2004

14. Recommendations for ‘white’ biotechnology Microbial host Suitable for optimization (growth properties, nutrient requirements) Containment (loss of viability after release) Recombinant gene Possibilities for modification of the product Control on gene regulation Containment genes (killing of host after accidental release) Waste Other applications Eradication of living GMOs in waste products

15. ‘Green’ Biotechnology Genetically modified plants in fields Open production facilities Possibility of free exchange of GM materials with the environment and food chains Coexistence between agricultural systems (controversy organic – conventional farming) Lower emphasis for industry

16. PHA production by plants ConstructReference Flax (Linum usitatissimum) with phbABC genes from R. eutropha Wróbel et al., 2004 Tobacco (Nicotiana tabacum) with phbABC genes from R. eutropha Arai et al., 2004

17. Requirements for ‘Green’ biotechnology Plant host Choice of best performing crops for bulk chemical production Preference for non-food crops Recombinant gene Marker-free constructs Restrictions on sexual exchange of rec DNA (e.g. plastid transformation) Logistics to keep GMO seeds separated from non-GMO seeds

18. Seed logistics White Biotechnology (contained use of GM micro- Organisms) Green Biotechnology (Growth of GM plants in open fields) Other applications (viability of GMO) waste Crop wastes (GMO still viable) Waste after processing (nonviable GMO material)

19. Bulk chemical production Application of GM microbes for bulk chemical production under contained conditions is realistic Safe production Containment guaranteed Applications of GM plants in open fields is uncertain and thus less realistic Containment in open fields is difficult to maintain Post harvest measures are required (transport, storage, raw material treatments)

20. Prospects ‘White’ biotechnology will become important for bulk chemical production Production with GM micro-organisms in closed reactors will largely increase Risk assessment must be adapted for larger- scale production facilities Processing of fermentation waste products will become important

21. time White Biotechnology Expected scale enlargement Environmental-friendly production Adaptations: Production facilities Biological containment Wastes

22. Consequences Increased biotechnological production means: Less chemicals and energy required Less toxic wastes produced More emphasis on containment Infrastructure (input raw materials, processing) Biological containment (facilities and constructs) Increased organic waste from reactors Concern for living GMOs in products made out of waste

23. Solutions Technical improvement of production facilities, circumstances and GMO constructs Alternative use of waste from fermentation reactors Agricultural use; e.g. by composting and heat inactivation or recycling of waste compounds

24. Conclusions Only temporal effects have been observed in small-scale GMO release studies GM constructs for bulk chemical production must be qualified as ‘low in risk’ No effect can be expected with the application of GM microbes for bulk chemical production in ‘white’ biotechnology Uncertainties exist with increased scale and long-term production with GM plants Waste products from fermentation reactors must be processed and free of living GMOs

25. Knowledge gaps Present analytical tools may be too limited to detect effects by increased-scale and long-term production; special emphasis on GM plant production Ecological baseline knowledge to discriminate GMO from non-GMO effects Relevant information on ecological interactions between species (e.g. what can be the effect of elevated levels of PHA on different populations)