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Lignin aromatics valorization by Pseudomonas putida : novel strategies to increase the production of mcl-polyhydroxyalkanoate and cis,cis-muconic acid

(2021)
Author
Promoter
Lars M. Blank, (UGent) and Miriam Agler-Rosenbaum
Organization
Abstract
Lignin, a major component of lignocellulosic biomass, is the most abundant aromatic polymer on the planet. Thus, it represents a renewable and non-fossil source of aromatics, promising to positively impact the transition to a circular bioeconomy. Despite being a chemical treasure box, lignin is still widely underutilized. In the last years, there is an emerging paradigm change towards implementing new biorefineries based on chemo- and/or biocatalytic upgrading of lignin aromatics into valuable products. Especially, in the biocatalytic conversion, the native aromatic catabolic ability of many Pseudomomas promises to ease the inherent aromatic heterogeneity of lignin depolymerized mixtures. Simultaneously, some Pseudomomas strains can synthesize valuable molecules for the polymer industry like medium chain length polyhydroxyalkanoate (mcl-PHA) and cis,cis-muconic acid. While enormous progress is underway in metabolic engineering towards improving conversion of aromatics into mcl-PHA, there is a significant gap in detailed bioprocess understanding, especially from mixtures of lignin-derived aromatics. Analogously, despite important advances on strain engineering and bioprocess development, limitations related to product accumulation at toxic levels still prevents reaching industrially-relevant productivities of cis,cis-muconic acid from lignin substrates. Thus, through understanding the key factors for successful bioconversion and applying this knowledge to implement innovative bioprocess strategies, this thesis intended to increase production of mcl-PHA and cis,cis-muconic acid from lignin aromatics. After performing step-wise screenings and parallel cultivations of several known strains of the Pseudomonas family, this work clearly confirmed Pseudomonas putida KT24440 as the most robust, versatile, and well performing biocatalyst for mcl-PHA accumulation from a defined lignin aromatic mixture. In a next step, the detailed resolution of aromatics funneling and microbial metabolic activity was revealed during online measurements of the oxygen consumption. From this experience, oxygen and nitrogen availability were identified as the key factors for a successful biocatalytic upgrading. Overall, the accumulation of mcl-PHA was improved in the wild type strain under technically relevant conditions by up to 43% (polymer content in cell biomass, mg mcl-PHA mg-1 CDW). At this stage, the highest mcl-PHA concentration (582 mg L-1) was obtained for a C/N ratio of 60 for oxygen-unlimited conditions (oxygen transfer rate ≥ 20 mmol L-1 h-1). In contrast, aromatic intermediates accumulated under oxygen-limited conditions at oxygen transfer rates below 10 mmol L-1 h-1. Through this bioprocess characterization, the performance of the biocatalytic funneling in P. putida KT2440 became predictable and thus, the experimental conditions were scalable into a 1-L stirred tank bioreactor based on the oxygen transfer rate. Finally, the benefits of implementing simultaneous biocatalyst and bioprocess optimization strategies were demonstrated. A 1.9- fold increment in mcl-PHA concentration and a 1.5-fold increment in mcl-PHA content were obtained from lignin aromatics. To alleviate the negative effect of product accumulation at toxic levels, this thesis also implemented an innovative solution establishing an in-line extraction of cis,cis-muconate produced by a metabolically engineered strain of P. putida KT2240. The implementation followed a systematic approach of process development. First, an electrolytic extraction of cis,cis-muconate was characterized in terms of flux and Coulombic efficiency from a synthetic media. In parallel, cis,cis-muconic acid bioproduction rates were defined in a conventional process and the negative effect of cis,cis muconic acid on the culture was confirmed at a threshold concentration of 195 mM (27.7 g L-1). In the middle time, long term electrolytic extractions were performed at a higher scale using fermentation broths as approximation of the final process. This experience served to identify and solve relevant technical drawbacks related to muconic acid precipitation in the anodic chamber. The solution consisted of pH control at 3.5, which was a suitable pH where higher amounts of the muconate isomeric forms cis,cis- and cis,trans- were found in solution (i.e., not as precipitated form). This was essential to enable higher muconate accumulation in the anolyte. From all the process parameters (i.e., bioproduction rates and extractive electrolytic performance) obtained separately, a final integration was carried out. Through implementing the in-line extraction from a bioreactor, the volumetric productivity of cis,cis-muconate was increased 15.6 % in respect to a conventional bioprocess run in parallel. Overall, this study contributed to deepening our understanding of the biocatalytic capability of the promising bacterium P. putida KT2440 towards efficient conversions of lignin aromatics for future biorefinery applications. Additionally, the potential of selective removal of cis,cismuconic acid was demonstrated for the first time in a microbial cultivation, contributing as an important step towards implementing a cost-effective bioproduction system.
Keywords
Lignin, Pseudomonas putida KT2440, PHA, biopolymer, muconic acid, Lignin valorization, Biological funneling, Biorefining, Bioprocess development, Lignin aromatics, circular bioeconomy

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MLA
Ramirez Morales, Juan Esteban. Lignin Aromatics Valorization by Pseudomonas Putida : Novel Strategies to Increase the Production of Mcl-Polyhydroxyalkanoate and Cis,Cis-Muconic Acid. RWTH Aachen University. Faculty of Mathematics, Computer Science and Natural Sciences ; Ghent University. Ghent University. Faculty of Bioscience Engineering, 2021, doi:10.18154/RWTH-2021-11234.
APA
Ramirez Morales, J. E. (2021). Lignin aromatics valorization by Pseudomonas putida : novel strategies to increase the production of mcl-polyhydroxyalkanoate and cis,cis-muconic acid. RWTH Aachen University. Faculty of Mathematics, Computer Science and Natural Sciences ; Ghent University. Ghent University. Faculty of Bioscience Engineering, Aachen, Germany ; Ghent, Belgium. https://doi.org/10.18154/RWTH-2021-11234
Chicago author-date
Ramirez Morales, Juan Esteban. 2021. “Lignin Aromatics Valorization by Pseudomonas Putida : Novel Strategies to Increase the Production of Mcl-Polyhydroxyalkanoate and Cis,Cis-Muconic Acid.” Aachen, Germany ; Ghent, Belgium: RWTH Aachen University. Faculty of Mathematics, Computer Science and Natural Sciences ; Ghent University. Ghent University. Faculty of Bioscience Engineering. https://doi.org/10.18154/RWTH-2021-11234.
Chicago author-date (all authors)
Ramirez Morales, Juan Esteban. 2021. “Lignin Aromatics Valorization by Pseudomonas Putida : Novel Strategies to Increase the Production of Mcl-Polyhydroxyalkanoate and Cis,Cis-Muconic Acid.” Aachen, Germany ; Ghent, Belgium: RWTH Aachen University. Faculty of Mathematics, Computer Science and Natural Sciences ; Ghent University. Ghent University. Faculty of Bioscience Engineering. doi:10.18154/RWTH-2021-11234.
Vancouver
1.
Ramirez Morales JE. Lignin aromatics valorization by Pseudomonas putida : novel strategies to increase the production of mcl-polyhydroxyalkanoate and cis,cis-muconic acid. [Aachen, Germany ; Ghent, Belgium]: RWTH Aachen University. Faculty of Mathematics, Computer Science and Natural Sciences ; Ghent University. Ghent University. Faculty of Bioscience Engineering; 2021.
IEEE
[1]
J. E. Ramirez Morales, “Lignin aromatics valorization by Pseudomonas putida : novel strategies to increase the production of mcl-polyhydroxyalkanoate and cis,cis-muconic acid,” RWTH Aachen University. Faculty of Mathematics, Computer Science and Natural Sciences ; Ghent University. Ghent University. Faculty of Bioscience Engineering, Aachen, Germany ; Ghent, Belgium, 2021.
@phdthesis{8729234,
  abstract     = {{Lignin, a major component of lignocellulosic biomass, is the most abundant aromatic polymer
on the planet. Thus, it represents a renewable and non-fossil source of aromatics, promising
to positively impact the transition to a circular bioeconomy. Despite being a chemical treasure
box, lignin is still widely underutilized. In the last years, there is an emerging paradigm change
towards implementing new biorefineries based on chemo- and/or biocatalytic upgrading of
lignin aromatics into valuable products. Especially, in the biocatalytic conversion, the native
aromatic catabolic ability of many Pseudomomas promises to ease the inherent aromatic
heterogeneity of lignin depolymerized mixtures. Simultaneously, some Pseudomomas strains
can synthesize valuable molecules for the polymer industry like medium chain length
polyhydroxyalkanoate (mcl-PHA) and cis,cis-muconic acid. While enormous progress is
underway in metabolic engineering towards improving conversion of aromatics into mcl-PHA,
there is a significant gap in detailed bioprocess understanding, especially from mixtures of
lignin-derived aromatics. Analogously, despite important advances on strain engineering and
bioprocess development, limitations related to product accumulation at toxic levels still
prevents reaching industrially-relevant productivities of cis,cis-muconic acid from lignin
substrates. Thus, through understanding the key factors for successful bioconversion and
applying this knowledge to implement innovative bioprocess strategies, this thesis intended
to increase production of mcl-PHA and cis,cis-muconic acid from lignin aromatics.

After performing step-wise screenings and parallel cultivations of several known strains of the
Pseudomonas family, this work clearly confirmed Pseudomonas putida KT24440 as the most
robust, versatile, and well performing biocatalyst for mcl-PHA accumulation from a defined
lignin aromatic mixture. In a next step, the detailed resolution of aromatics funneling and
microbial metabolic activity was revealed during online measurements of the oxygen
consumption. From this experience, oxygen and nitrogen availability were identified as the
key factors for a successful biocatalytic upgrading. Overall, the accumulation of mcl-PHA was
improved in the wild type strain under technically relevant conditions by up to 43% (polymer
content in cell biomass, mg mcl-PHA mg-1 CDW). At this stage, the highest mcl-PHA
concentration (582 mg L-1) was obtained for a C/N ratio of 60 for oxygen-unlimited conditions
(oxygen transfer rate ≥ 20 mmol L-1 h-1). In contrast, aromatic intermediates accumulated
under oxygen-limited conditions at oxygen transfer rates below 10 mmol L-1 h-1. Through this
bioprocess characterization, the performance of the biocatalytic funneling in P. putida KT2440
became predictable and thus, the experimental conditions were scalable into a 1-L stirred
tank bioreactor based on the oxygen transfer rate. Finally, the benefits of implementing
simultaneous biocatalyst and bioprocess optimization strategies were demonstrated. A 1.9-
fold increment in mcl-PHA concentration and a 1.5-fold increment in mcl-PHA content were
obtained from lignin aromatics.

To alleviate the negative effect of product accumulation at toxic levels, this thesis also
implemented an innovative solution establishing an in-line extraction of cis,cis-muconate
produced by a metabolically engineered strain of P. putida KT2240. The implementation
followed a systematic approach of process development. First, an electrolytic extraction of
cis,cis-muconate was characterized in terms of flux and Coulombic efficiency from a synthetic
media. In parallel, cis,cis-muconic acid bioproduction rates were defined in a conventional
process and the negative effect of cis,cis muconic acid on the culture was confirmed at a
threshold concentration of 195 mM (27.7 g L-1). In the middle time, long term electrolytic
extractions were performed at a higher scale using fermentation broths as approximation of
the final process. This experience served to identify and solve relevant technical drawbacks
related to muconic acid precipitation in the anodic chamber. The solution consisted of pH
control at 3.5, which was a suitable pH where higher amounts of the muconate isomeric forms
cis,cis- and cis,trans- were found in solution (i.e., not as precipitated form). This was essential
to enable higher muconate accumulation in the anolyte. From all the process parameters (i.e.,
bioproduction rates and extractive electrolytic performance) obtained separately, a final
integration was carried out. Through implementing the in-line extraction from a bioreactor,
the volumetric productivity of cis,cis-muconate was increased 15.6 % in respect to a
conventional bioprocess run in parallel.

Overall, this study contributed to deepening our understanding of the biocatalytic capability
of the promising bacterium P. putida KT2440 towards efficient conversions of lignin aromatics
for future biorefinery applications. Additionally, the potential of selective removal of cis,cismuconic
acid was demonstrated for the first time in a microbial cultivation, contributing as
an important step towards implementing a cost-effective bioproduction system.}},
  author       = {{Ramirez Morales, Juan Esteban}},
  keywords     = {{Lignin,Pseudomonas putida KT2440,PHA,biopolymer,muconic acid,Lignin valorization,Biological funneling,Biorefining,Bioprocess development,Lignin aromatics,circular bioeconomy}},
  language     = {{eng}},
  pages        = {{XIV, 169}},
  publisher    = {{RWTH Aachen University. Faculty of Mathematics, Computer Science and Natural Sciences ; Ghent University. Ghent University. Faculty of Bioscience Engineering}},
  school       = {{Ghent University}},
  title        = {{Lignin aromatics valorization by Pseudomonas putida : novel strategies to increase the production of mcl-polyhydroxyalkanoate and cis,cis-muconic acid}},
  url          = {{http://dx.doi.org/10.18154/RWTH-2021-11234}},
  year         = {{2021}},
}

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