Poster presented at the 11th annual World Congress on Industrial Biotechnology. Studies on GRP were financed by the EU Integrated Project BIOPRODUCTION (contract nº 026515-2) and those on cellulosic hydrolysates are funded by the EU Collaborative Project BUGWORKERS (contract nº 246449). 
C. Almeida, J. Cavalheiro, T. Cesário and F. Ferreira were supported by Fundação para a Ciência e Tecnologia, Portugal (SFRH/BPD/26678/2006, SFRH/BD/45266/2008 and SFRH/BPD/68587/2010, IF/00442/2012). Waste glycerol was kindly provided by Torrejana, Fábrica de Biocombustíveis (Torres Vedras). Lignocellulosic hydrolysates were produced by biorefinery.de GmbH, Germany, in the framework of BUGWORKERS project.Institute for Biotechnology and Bioengineering, Instituto Superior Técnico,
Universidade de Lisboa, Portugal / Biotrend S.A.,
Cantanhede, Portugal / Ecole Europeènne de
Chimie, Polymères et Matériaux, Université de
Strasbourg, Franc

Almeida, Catarina Dias de

Cavalheiro, João

Cesário, Teresa

Ferreira, Bruno

Ferreira, Frederico

Fonseca, M. Manuela R. da

Pollet, Eric

Raposo, Rodrigo

van Keulen, Frederik

English

Repositório Comum

!   Cellulose	   is	   the	   world’s	   most	  abundant	   natural,	   renewable	  and	  biodegradable	  polymer.	  ! Lignocellulosic	   wastes	   are	  pretreated	   and	   then	   subjected	  to	   an	   enzyma<c	   step	   to	   obtain	  an	   hydrolysate	   which	   can	   be	  used	   as	   C	   source	   by	   bacterial	  strains	  [7].	  	  Biodegradable polyesters from agro&industrial by-products  Catarina	  Dias	  de	  Almeida1,2,	  João	  Cavalheiro1,	  Rodrigo	  Raposo1,	  Teresa	  Cesário1,	  Frederico	  Ferreira1,	  Bruno	  Ferreira3,	  Frederik	  van	  Keulen3,	  Eric	  Pollet4,	  M.	  Manuela	  R.	  da	  Fonseca1	  1	  -­‐	  InsJtute	  for	  Biotechnology	  and	  Bioengineering,	  InsJtuto	  Superior	  Técnico,	  Universidade	  de	  Lisboa,	  Portugal,	  	  2	  -­‐	  Centro	  de	  InvesJgação	  Interdisciplinar	  Egas	  Moniz,	  Monte	  de	  Caparica,	  Portugal,	  	  3	  -­‐	  Biotrend	  S.A.,	  Cantanhede,	  Portugal	  	  4	  -­‐	  Ecole	  Europeènne	  de	  Chimie,	  Polymères	  et	  Matériaux,	  Université	  de	  Strasbourg,	  France	  catarina.almeida@tecnico.ulisboa.pt	  Introduction Results and Discussion Methodology Conclusions Objective Conversion of waste glycerol and hydrolyzed straw into biodegradable, biocompatible polyesters through up-scalable, high productivity processes References [1] Lee, S. Y. Biotechnol. Bioeng. 49, 25-30, 1996.  [2] Steinbüchel, A. In Rehm, H.J., Reed, G., Pühler, A., Stadler, P. (eds), Biotechnol. 6, 403-464, 1996.  [3] http://www.ebb-eu.org/biodiesel.php (10-04-2014) [4] Solaiman et al. Appl. Microbiol. Biotechnol. 71, 783 – 789, 2006.  [5] Koller et al. Biomacromol. 6, 561‑565, 2005. [6] Mothes et al., Eng.  Life Sci. 7, 5, 475-479, 2007. [7] Almeida et al., J. Chem. Tech.  Biotechnol., 82, 4, 340–349, 2007.  [8] Cavalheiro et al., Process Biochem. 44, 509-515, 2009.  [9] Cavalheiro et al., Biores. Technol., 111, 391-397, 2012. [10] Lee et al., J. of Biosci. Bioeng, 89 (4), 380-383, 2000. [11] Cavalheiro et al. Biores. Technol., 147, 434–441, 2013. [12] Canadas et al., Int. J. Biol. Macromol., accepted, 2014. [13] Cesário et al., New Biotechnol., 31, 1, 104-113, 2014. [14] Cesário et al. Int. J. Biol. Macromol., accepted, 2014. !   Waste	  glycerol	  was	  successfully	  used	  as	  an	  alterna<ve	  C-­‐source	  to	  produce	  PHAs.	  	  !  	   Integra<ng	   biopolyesters	   and	   biodiesel	   produc<on	   can	   contribute	   to	   (i)	   reduce	   costs	  associated	  with	   the	   C-­‐source	   for	   PHAs	   produc<on	   (ii)	   the	   upgrade	   of	   the	   surplus	   glycerol	  generated	  in	  the	  biodiesel	  plants.	  !  	  P(3HB)	  and	  P(3HB-­‐4HB)	  were	  successfully	  bioproduced	  from	  different	  batches	  of	  wheat	  straw	  hydrolysates.	  !  	  The	  aOained	  P(3HB)	  and	  P(3HB-­‐4HB)	  volumetric	  produc<vi<es	  are	  by	  far	  the	  highest	  ever	  achieved	   on	   agricultural	   waste	   hydrolysates	   and	   further	   op<miza<on	   is	   under	   way	   (see	  poster	  Cesário	  et	  al.	  for	  more	  details!).	  	  !   	   Polyhydroxyalkanoates	   (PHAs)	   are	   biodegradable,	   compostable	   polyesters	  synthesized	  by	  many	  microbial	  strains	  under	  unbalanced	  growth	  condi<ons	  [1]	  [2].	  !  	  Under	  appropriate	  feeding	  strategies,	  co-­‐polymers	  with	  a	  wide	  range	  of	  proper<es	  can	  be	  produced,	  e.g.	  for	  agricultural,	  biomedical	  and	  packaging	  applica<ons.	  !   	   The	   most	   common	   type	   of	   PHA	   is	   the	   homopolymer	   poly(3-­‐hydroxybutyrate)	  (P(3HB)).	   Co-­‐polymers	   such	   as	   P(3HB-­‐co-­‐4HB)	   exhibit	   higher	   elas<city,	   lower	  crystallinity	  and	  present	  more	  adequate	  proper<es	  for	  specific	  uses.	  !   Current	  biodiesel	   produc<on	   is	   based	  on	   transesterifica<on	  of	   vegetable	  oils	   genera<ng	  FAMEs	  and	  a	  surplus	  of	  waste	  glycerol	  (GRP)	  (1	  t	  of	  GRP	  for	  each	  9	  t	  of	  biodiesel).	  !   Today,	  approx.	  120	  plants	  produce	  up	  to	  6	  100	  thousand	  tonnes	  of	  biodiesel	  annually	   in	  the	  EU,	  genera<ng	  600	  thousand	  t	  of	  GRP	  [3].	  	  !   GRP	  can	  be	  used	  as	  the	  major	  C	  source	  for	  PHA	  produc<on	  [4]-­‐[6].	  	  !   Cupriavidus	  necator	  DSM	  545	  produced	  the	  homopolymer	  P(3HB)	  from	  GRP	  at	  a	  produc<vity	  of	  1.1	  gL-­‐1.h-­‐1	  [8].	  	  ! Using	  γ-­‐butyrolactone	  (GBL)	  as	  the	  precursor	  for	  4HB	  monomers,	  C.	  necator	  DSM	  545	  cells	  were	  able	  to	  accumulate	  P(3HB-­‐co-­‐4HB)	  with	  different	  4HB	  to	  3HB	  ra<os	  [9].	  !   Propionic	  acid	  (PA)	  was	  used	  as	  s<mulator	  for	  4HB	  incorpora<on,	  as	  suggested	  by	  Lee	  et	  al.	  2000	  [10].	  PA	  considerably	  increased	  the	  4HB	  ra<o,	  but	  also	  acted	  as	  3-­‐hydroxyvalerate	  (3HV)	  precursor,	  resul<ng	  in	  the	  produc<on	  of	  P(3HB-­‐4HB-­‐3HV).	  	  !   By	  manipula<ng	  the	  dissolved	  oxygen	  concentra<on	  (DOC)	  and	  cul<va<on	  <me,	  4HB	  molar	  percentages	  in	  the	  range	  11.4	  -­‐	  21.5	  were	  aOained.	  Terpolymers	  were	  obtained	  with	  24.8%	  to	  43.6%	  4HB	  and	  5.6%	  to	  9.8%	  3HV	  (see	  Table	  1,	  Figure	  1	  and	  2).	  Results	  indicate	  that	  a	  higher	  DOC	  favors	  PHA	  accumula<on.	  	  Average	  MW	  varied	  between	  5.5	  x	  105	  Da	  and	  1.37	  x	  106	  Da	  with	  a	  PI	  from	  2.6	  to	  4.0.	   Upgrading by-products Waste glycerol Figure	  1.	  Average	  composiJon	  of	  lignocellulosic	  biomass	  and	  	  major	  derived	  hydrolysis	  products	  [7]	  Figure	  3.	  ProducJon	  of	  P(3HB-­‐4HB-­‐3HV);	  CulJvaJon	  Jme	  course	   for	  the	   producJon	   of	   P(3HB-­‐4HB)	   with	   the	   DOC	   controlled	   at	   20%.	  AccumulaJon	   phase	   –	   2	   gL-­‐1	   of	   propionic	   acid	   were	   added	   at	   the	  beginning	  and	  GRP	  and	  GBL	  were	  conJnuously	  fed.	  Figure	   4.	   PHA	   (%)	   accumulated	   per	   total	   DW,	   by	   cells	   of	   C.	  necator	  DSM	  545	   fed	  with	  GRP	  and	  GBL	   in	  culJvaJons	  with	   the	  DOC	  controlled	  at	  2	  %	  or	  20	  %	   in	   the	  presence	  or	  absence	  of	  2	  gL-­‐1	  of	  PA.	  05101520253035400 10 20 30 40 50 60 70PHA	  %Time	  (h)2	  gL-­‐1	  PA;	  DOC	  20%2	  gL-­‐1	  PA;	  DOC	  2%DOC	  20%DOC	  2%Table	  1.	  PHA	  producJon	  -­‐	  effect	  of	  DOC	  (%),	  PA	  	  and	  culture	  Jme	  on	  PHA	  	  producJvity,	  concentraJon	  and	  composiJon	  [9].	     Values at the time of max volumetric productivity Values at the end of the cultivation Accum. conditions PHA ProdVol gL-1h-1 PHA PHA 4HB 3HV ProdVol gL-1h-1 PHA PHA 4HB 3HV DOC (%) Supplements type [ time (h) ] gL-1 % % % [ time (h) ] gL-1 % % % 20 GBL P(3HB-4HB) 0.33 9.7 25.9 11.4 - 0.17 10.9 36.1 17.6 - [ 29.3 ] [ 64.3 ] 2 GBL P(3HB-4HB) 0.15 4.0 14.8 12.3 - 0.06 4.2 17.9 21.5 - [ 26.0 ] [ 67.5 ] 20 GBL + PA P(3HB-4HB-3HV) 0.29 10.8 29.5 26.2 9.8 0.25 16.7 36.9 43.6 6.0 [ 38.0 ] [ 67.0 ] 2 GBL + PA P(3HB-4HB-3HV) 0.35 11.0 25.0 30.6 6.7 0.21 9.3 18.1 24.8 5.6 [ 31.8 ] [ 44.0 ] 	  !   	   The	   proper<es	   of	   the	   polymers	   considerably	  differ	   from	  those	  of	   the	  homopolymer	  P(3HB),	  even	   at	   low	   percent	   incorpora<on	   of	   4HB	  monomers	  in	  the	  polyester	  chain	  [11].	  !   Evidence	   (obtained	   by	   DSC	   and	   SEC)	  d emon s t r a t e s	   t h e	   b i o s y n t h e s i s	   o f	  heterogeneous	   PHA	   blends	   composed	   by	  var ious	   f rac<ons	   of	   d ifferent	   molar	  composi<on,	   instead	   of	   a	   narrow	   distribu<on	  of	  chains	  with	  similar	  composi<on	  [11].	  	   1 2    3 4 5 1 3 6 5 PHA accumulation – N-limitation or P-limitation as trigger for PHA accumulation C-source quantification – by HPLC PHA recovery – CHCl3 extraction followed by precipitation with C2H5OH PHA and GBL quantification – GC PHA characterization – H1-NMR, SEC, DSC Wastes as C-source Selected Bacterial strains 2L STR fermentation on-line data Lyophilized cells Extracted polymer Nile-blue stained PHA granules inside C. necator cells Figure	  2.	  Bench	  scale	  methodology	  	  Poly(3-­‐hydroxybutyrate)	  (P(3HB))	  H	  HO	  Poly(3HB-­‐co-­‐4HB)	  H	  HO	  02468101214161820051015202530354045500 10 20 30 40 50 60 70PHA,	  3HB,	  4HB,	  3HV	  (gL-­‐1 )Dry	  Weight	  (gL-­‐1)Time	  (h)DWRes	  DWPHA3HB4HB3HVaccumulaJon	  phase	  Figure	  6.	  From	  P(3HB-­‐4HB-­‐3HV)	  to	  scaffolds	  for	  hMSC	  proliferaJon	  [12].	  Acknowledgements Studies on GRP were financed by the EU Integrated Project BIOPRODUCTION (contract nº 026515-2) and those on cellulosic hydrolysates are funded by the EU Collaborative Project BUGWORKERS (contract nº 246449)  C. Almeida, J. Cavalheiro, T. Cesário and F. Ferreira were supported by Fundação para a Ciência e Tecnologia, Portugal (SFRH/BPD/26678/2006, SFRH/BD/45266/2008 and SFRH/BPD/68587/2010, IF/00442/2012). Waste glycerol was  kindly provided by Torrejana, Fábrica de Biocombustíveis (Torres Vedras). Lignocellulosic hydrolysates were produced by biorefinery.de GmbH, Germany, in the framework of BUGWORKERS project.  PRODUCTIONbio	  Figure	  5.	  The	  effect	  of	  (4HB	  +	  3HV)	  molar	  %	  on	  the	  terpolymer	  mechanical	  properJes:	  (a)	  and	  (b)	  elongaJon	  at	  break	  ploled	  on	  two	  different	  scales,	  (c)	  Young’s	  modulus	  and	  (d)	  tensile	  strength.	  	  Polyhydroxyalkanoates Wheat straw lignocellulosic hydrolysates !   	  A	  standardized	  fermenta<on	  protocol	  was	  successfully	  developed	  allowing	  for	  the	  tes<ng	  of	  the	  supplied	  hydrolysates,	  as	  well	  as	  for	  further	  scale	  up	  of	  the	  process.	  !   Feedback	   from	   bench	   scale	   assays	   allowed	   for	   hydrolysates	   improvement	   by	   biorefinery.de	  GmbH.	  !   Feeding	  strategy	  for	  mul<ple	  carbon	  source	  consump<on	  successfully	  implemented	  [13].	  !   Cell	  density	  and	  P(3HB)	  produc<vity	  were	  similar	  to	  those	  reached	   in	  control	  cul<va<ons	  with	  mixtures	  of	  commercial	  sugars	  [13].	  	  !   Cell	  density	  and	  P(3HB-­‐4HB)	  produc<vity	  using	  hydrolysates	  were	  lower	  than	  those	  reached	  in	  P(3HB)	  produc<on	  cul<va<ons	  due	  to	  inhibi<on	  caused	  by	  the	  precursor	  (GBL)	  [14].	  Table	  2.	  Preliminary	  strain	  selecJon.	  Table	   4.	   PHA	   producJon	   from	   wheat	   straw	   hydrolysates	   (hydrolysate	   composiJon:	  465	  gL-­‐1	  glucose,	  146	  gL-­‐1	  xylose,	  42	  gL-­‐1	  	  arabinose).	  Biomass (milled wheat straw) Concentrated hydrolysate  AFEX	   ENZYMATIC	  HYDROLYSIS	  WATER	  EVAPORATION	  C-­‐source Y	  P	  (3HB)/S	  Prod	  vol	  (gL-­‐1.h-­‐1) P	  (3HB)	  (%) Dw	  	  (gL-­‐1) t	  	  (h) Sugar	  mix 0.18 1.7 51 138 36.5 Hydrolysate 0.20 1.7 55 150 38.5 Table	   3.	   P(3HB)	   producJon	   from	   a	   sugar	  mixture	   simulaJng	   the	   hydrolysate	   (as	   control)	   and	  from	   	   a	   real	   hydrolysate	   (hydrolysate	   composiJon:	   563	   gL-­‐1	   glucose,	   284	   gL-­‐1	   xylose,	   46	   gL-­‐1	  arabinose).	  	  	  	  	  	  	  	  Polymer CDW (gL-1) PHA (gL-1) PHA	  (%) Prodvol	  (gL-1.h-1) P(3HB)	  40h	   125	   71	   57	   1.5	  P	  (3HB-­‐co-­‐	  6%	  4HB)	  49h	   88	   24	   27	   0.5	  Figure	  7.	  Main	  steps	  	  involved	  in	  	  hydrolysate	  producJon	  at	  biorefinery.de	  GmbH	  	  Strain Criteria Comments glucose xylose PHA Risk Alcaligenes latus DSM 1122 + - + 1 Aggregates during the first stages of growth Bacillus sp MA3.3  + + + 1 Not available Burkholderia cepacia ATCC 17759 + + + 2 Burkholderia  sacchari DSM 17165 + + + 1 High growth rates Cupriavidus necator DSM 545 + - + 1 High growth rates Haloferax mediterranei + - + 1  Costly growth medium and corrosion problems. Methylobacterium extorquens + - + 1 Low concentration of the optimal C sources in the hydrolysates Sphingopyxis macrogoltabida LMG 17324  + - + 1 Preferential uptake of oligosaccharides 

Biodegradable polyesters from agro&industrial by-products

https://comum.rcaap.pt/bitstream/10400.26/8118/1/Poster01_ProfCatarinaAlmeida.pdf

Biodegradable polyesters from agro&industrial by-products

Abstract

Similar works

Full text

Available Versions

Repositório Comum