1. Poly-β-hydroxybutyrate (PHB) Production by Cyanobacteria
ECB Krakow, Poland
July 3-6, 2016
 Poly-β-hydroxybutyrate (PHB) Production by Cyanobacteria
Roberta Carpinea, Giuseppe Olivieria, Klaas Hellingwerfb, Antonino Pollioc, Gabriele Pintoc , Antonio Marzocchellaa
a Dipartimento di Ingegneria Chimica, dei Materali e della Produzione Industriale - Università degli Studi di Napoli “Federico II” – P.le V. Tecchio, Napoli, Italy
b Laboratory for Microbiology, Swammerdam Institute for Life Sciences - Science Park XH Amsterdam, The Netherlands
c Biology Department - Università degli Studi di Napoli “Federico II” - Via Foria, Napoli, Italy

2. BIOPLASTICS
Plastics may be classified in four groups depending on source/biodegradability features
Biobased or partially biobased non-biodegradable plastics 
Plastics that are both biobased and biodegradable
Plastics that are based on fossil resources and are biodegradable
Plastics that are based on fossil resources and are non-biodegradable
European Biolastics,
ECB Krakow, Poland
July 3-6, 2016

3. The BIOPLASTIC market is growing at a rate of more than 20 % per year.
BIOPLASTICS MARKET
The BIOPLASTIC market is growing at a rate of more than 20 % per year.
PHA: 2.1% ˜ 34 ktons
Europe: 280ktons
European Biolastics,
ECB Krakow, Poland
July 3-6, 2016

4. Polyhydroxybutyrate (PHB)
Polyhydroxyalkanoates (PHAs) are a group of biodegradable polymers of biological origin [1].
Polyhydroxybutyrate (PHB) is the most widespread and completely characterized PHA found in bacteria [1].
PHB is accumulated as a carbon and/or energy storage material in various microorganisms usually under limiting nutritional conditions [1].
PHB Properties [2]: Thermoplastic processibility, hydrophobicity, complete biodegradability and biocompatibility.
Currently, PHB is produced by bacteria grown on different feedstocks.
The challenge is to find autotrophic microorganisms to produce PHB.
[1]Panda et al. , (2006). Bioresource Technology 97:
[2]Balaji et al. , (2013). Algal Research, 2:
ECB Krakow, Poland
July 3-6, 2016

5. WHY CYANOBACTERIA? PHB Butanol Protein … ECB 2016 - Krakow, Poland
Gram negative bacteria.
Photoautotrophic prokaryotes that perform oxygenic photosynthesis.
Fast growing with simple nutritional demands.
Production of different product of interest: protein and chemical products.
Accumulation of PHB under photoautotrophic conditions.
Synechocystis PCC6803 is an extensively investigated cyanobacterium.
Gupta et al. , (2013). Algal Reserch, 2:
PHB
Butanol
Protein …
ECB Krakow, Poland
July 3-6, 2016

6. AIMS PHB production using cyanobacteria, light and CO2.
Culture media optimization changing nitrate concentration to find the best contition for PHB production.
Screening of cyanobacteria strains to find the best one for PHB production
ECB Krakow, Poland
July 3-6, 2016

7. Microorganisms and medium
Synechocystis fuscopigmentosa
Synechocystis aquatilis
Synechococcus nidulans
Chlorogloeopsis fritshii
Synechocystis PCC6803
Università di Napoli Federico II
Amsterdam University
BG11
BG0
BG1/4
BG1/2
NaNO3
1.5
0.375
0.75
K2HPO4
0.04
MgSO4
0.075
CaCl2*2H2O
0.036
Citric acid
0.006
Ammonium ferric citrate
Na2EDTA*2H2O
0.001
NaCO3
0.02
HBO3
2.86
Mn Cl2 * 4H2O
1.81
ZnSO4 * 7H2O
0.22
Na2MoO4 *2 H2O
0.39
Co(NO3)2
0.05
Medium
BG11
BG0
BG1/4
BG1/2
Nitrate optimal concentration
No nitrate
Nitrate concentration is one fourth of the optimal
Nitrate concentration is half of the optimal
ECB Krakow, Poland
July 3-6, 2016

8. Apparatus PRECULTURES Rectangular shape PBRs
For cultures to be used to inoculate photobioreactors
Rectangular shape PBRs
For intensive cyanobacterial cultures
[Gargano et al. , (2013). Chemical Engineering Transaction. 32:
ECB Krakow, Poland
July 3-6, 2016

9. Cyanobacteria Screening
Experimental procedures
PARAMETERS
TEST TIPOLOGY
Media optimization
Cyanobacteria Screening
Light/dark (h)
18/6
I (μE/m2s)
150
CO2 (%) 2
Reactor configuration
Rectangular shape
Media
BG0
BG1/4
BG1/2
BG11
Strain
S. PCC6803
S. fuscopigmentosa
S. aquatilis
S. nidulans
C. fritshii
ECB Krakow, Poland
July 3-6, 2016

10. 1. Medium optimization results
BG1/2 ~ 8%
BG1/2 was selected as best medium for PHB production by S. PCC6803
BG1/2 ~ 7g/m3d
ECB Krakow, Poland
July 3-6, 2016

11. 2. Cyanobacteria screening results
SA & SP ~ 8%
S. PCC6803 and S. aquatils were selected as best strains for PHB production
SA & SP ~ 7g/m3d
ECB Krakow, Poland
July 3-6, 2016

12. Final remarks 1. Media Optimiziation 2. Cyanobacteria screening
BG1/2 medium is the best medium for PHB production in terms of PHB fraction and productivity: PHB content of about 8% and PHB productivity of about 7 g/m3d.
2. Cyanobacteria screening
S.PCC6803 and S. aquatilis are the best strains for PHB production in terms of PHB fraction and productivity:
i) S. aquatilis produced PHB since the beginning of the growth. PHBproduction continued even after nitrogen starvation;
ii) S.PCC6803 began to produce PHB at the nitrogen starvation onset.
ECB Krakow, Poland
July 3-6, 2016

13. Next steps ECB 2016 - Krakow, Poland July 3-6, 2016
Genetic engineering of S.PCC6803 to improve PHB production
Development of model for growth and PHB production
Screeing of recovery techniques for intracellular PHB
ECB Krakow, Poland
July 3-6, 2016

14. Acknowledgement
ECB Krakow, Poland
July 3-6, 2016

15. THANKS FOR THE ATTENTION
ECB Krakow, Poland
July 3-6, 2016
Poly-β-hydroxybutyrate (PHB) Production by Cyanobacteria
THANKS FOR THE ATTENTION
Roberta Carpine
Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industriale, Dipartimento di Biologia Vegetale
Università degli studi di Napoli - Federico II
University of Amsterdam
PhD Biotechnology