1. A fluorescence-based method for the assesment of PolyHydroxyAlkanoates (PHA) production
Anne ELAIN, Assist. Prof. Dr, IRDL, CNRS FRE 3744
Université Bretagne Sud, FRANCE

2. Presentation Outline:
Bioplastics market data
Basics of PHA
Challenges in PHA production
BluEcoPHA project ( )
Conclusions and Outlook
15th International Pharmaceutical Microbiology and Biotechnology Conference
June 21-23, 2017
London, UK

3. Bioplastics market data
The « Plastic Age »
World production (2015): 322 Mt /year
Most are non-biodegradable

4. Bioplastics market data
1% of the global production
A market growth of 20%/year

5. Global production capacities of bioplastics 2016 (by type)
Bioplastics market data

6. Basics of PHA
Biologically produced and biologically degradable bioplastics
Produced under conditions of:
- excess carbon
- low limiting nutrients (N, P, S, O)
Accumulated inside bacterial cells as carbon
and energy storage
TEM image of microorganisms containing PHA (from Metabolix)

7. Basics of PHA
Large family of polyesters (over 100 naturally occurring monomers known)
PHB
PHBV

8. from elastomeric to resins
Basics of PHA
PHA properties (Sudesh et al. 2000)
from elastomeric to resins

9. Basics of PHA Polymer properties are highly dependent on:
- the producing strain,
- the raw material,
- the fermentation strategy
--> can be tailored to the application:
- packaging materials
- various medical and pharmaceutic purposes
(tissue engineering, drug carrier, therapeutic applications,…)
- Disposable items such as razors, cosmetics containers, shampoo bottles, …

10. Challenges in PHA production
TO REDUCE THE PRODUCTION COST
Decrease raw material cost  Low-cost carbon sources
(molasses, whey, fats and oils,…)
Increase PHA yield  High producing strain
 New feeding strategy
 better recovery and purification

11. BluEcoPHA project ( )
Development of a sustainable process for the production of PHA « Made in Brittany »
Industrial partners:

12. BluEcoPHA project Production of PHA from wastewater:
Abundant and local waste stream
High content of reducing sugars
No pretreatment needed

13. BluEcoPHA project Development of a rapid and reliable method to :
Select powerful PHA-producing strains
Define cultivation conditions that maximize PHA yield
Monitor PHA biosynthesis
Adaptation of the staining method of PHA granules with Nile Red
(Spiekermann et al. Arch. Microbiol, 1999)

14. BluEcoPHA project Nile Red staining :
0.4 % v/v in the culture medium at the
beginning of the production stage
Chemical structure of NR dye.
Granule of P(3HB) inside A. chroococum.
(Lenz & Marchessault, 2005).
TEM image and schematic depiction of a single PHA granule (D. Jendrossek, 2007)

15. Microscopic observations measurements (lex: 5430 nm)
BluEcoPHA project
Cultivation in 4-L fermenteur under a two stage process (24h growth phase + 72h production phase)
Detection of the emitted fluorescence by microscopy or spectroscopy:
Microscopic observations
(lex: nm)
O.D. = 0.7
(PBS)
2 mL of
culture broth
7500 rpm
10 min
Spectroscopic
measurements (lex: 5430 nm)

16. BluEcoPHA project Selection of powerfull PHA- producers :
Emission spectra 1: SF/2006, 2: SF/2003, 3: SF/2066, 4: PHA+, 5: PHA-, 6: SF/2069, 7: SF/2089, after 72h cultivation.

17. IF measured after 72h of cultivation for different C/N ratio.
BluEcoPHA project
Screening of nutritional conditions that optimize PHA production
IF measured after 72h of cultivation for different C/N ratio.

18. BluEcoPHA project Monitoring of the biosynthesis: 19 h 50 h 67 h
biodegradation
50 h
67 h
A. Elain et al., W. J. of Microbiol. and Biotechnol., 2015

19. BluEcoPHA project Production results Medium CDW (g.L-1) [PHA] PHA
content (%)
LPW
2.8
1.56 55
FPW
2.3
1.79 78
Medium MW
(g.mol-1) Tg
(°C)
Composition
LPW -1
PHBHx
FPW -5
PHBHO

20. Conclusions and outlook
Agri-food processing waters are readily available and cost-effective carbon source for the production of PHA,
Regarding the sugar content of the effluent, polymers with distinct properties can be synthesized,
The modified-NR method is effective to identify novel PHA-producers, define an optimized culture medium and monitor the biosynthesis,
The method can be helpful to optimize and control the production of PHA from the laboratory scale to the industrial scale

21. Conclusions and outlook
Cultivations on LPW and FPW are carried-out in 3 m3 fermentor to scale-up the process prior to industrial implementation
Green chemicals are also investigated to provide greater insights into economical and sustainable recovery of PHA from bacterial cells.

22. Aknowledgments
S. BRUZAUD (Pr., Univ. Bretagne Sud) V. LE TILLY (Ass. Prof., Univ. Bretagne Sud) M. LE FELLIC (Ass. Ing., Univ. Bretagne Sud) Y.M. CORRE (Ing., Univ. Bretagne Sud) P. LEMECHKO (Ing., Univ. Bretagne Sud) J.L. AUDIC (Pr., Univ. Rennes 1)