Research Article: Poly(3-hydroxybutyrate) production in an integrated electromicrobial setup: Investigation under stress-inducing conditions

Date Published: April 26, 2018

Publisher: Public Library of Science

Author(s): Israa Salem Al Rowaihi, Alexis Paillier, Shahid Rasul, Ram Karan, Stefan Wolfgang Grötzinger, Kazuhiro Takanabe, Jörg Eppinger, Martin Koller.

http://doi.org/10.1371/journal.pone.0196079

Abstract

Poly(3-hydroxybutyrate) (PHB), a biodegradable polymer, can be produced by different microorganisms. The PHB belongs to the family of polyhydroxyalkanoate (PHA) that mostly accumulates as a granule in the cytoplasm of microorganisms to store carbon and energy. In this study, we established an integrated one-pot electromicrobial setup in which carbon dioxide is reduced to formate electrochemically, followed by sequential microbial conversion into PHB, using the two model strains, Methylobacterium extorquens AM1 and Cupriavidus necator H16. This setup allows to investigate the influence of different stress conditions, such as coexisting electrolysis, relatively high salinity, nutrient limitation, and starvation, on the production of PHB. The overall PHB production efficiency was analyzed in reasonably short reaction cycles typically as short as 8 h. As a result, the PHB formation was detected with C. necator H16 as a biocatalyst only when the electrolysis was operated in the same solution. The specificity of the source of PHB production is discussed, such as salinity, electricity, concurrent hydrogen production, and the possible involvement of reactive oxygen species (ROS).

Partial Text

Biodegradable plastics are gaining wide interest. They are derived from inexpensive biomaterial, hence represent a sustainable and environmentally safe alternative to the synthetic petroleum-based polymers [1, 2], which are introduced in substantial amounts into the ecosystem as residential and industrial waste products [3–5]. The most common biodegradable plastic is poly(3-hydroxybutyrate) (PHB) that belongs to the family of polyhydroxyalkanoate (PHA) [6]. PHB is a highly reduced carbon storage compound that serves as carbon and energy reserve in different microorganisms. In these microorganisms, it is synthesized from acetyl-CoA through the successive action of three enzymes, namely the β-ketoacyl-CoA thiolase (phb A), the acetoacetyl-CoA reductase (phb B) and the PHB polymerase (phb C) that polymerizes acyl coenzyme A (acyl-CoA). The bacteria utilize PHB when nutrients are limited through depolymerization into 3-hydroxybutyric acid (3HB) which is used to produce acyl-CoA and acetyl-CoA, that is metabolized in the tricarboxylic acid (TCA) cycle as a source of carbon and energy [4, 7]. PHB is produced in native and engineered microorganisms by accumulating as granules in the cytoplasm in response to conditions of physiological stress. Cells with high PHB content had enhanced survival and tolerance toward heat challenge and oxidative stress [8–10]. A study using recombinant E. coli reported an increase in heat resistance with PHA production compared with the control strain [11]. Another study using Aeromonas hydrophila suggested the enhancement of the survival ability of the strain by the simultaneous biosynthesis of PHA granules under various stress conditions [12]. A third study that exposed knock out mutants of Burkholderia strains lacking essential genes for PHA production, to nutrient limitations, high osmotic pressure and high temperature showed lower survival compared to the wild-type [12].

The PHB production was attempted using IEMC setup, being placed the microorganisms under comparatively high salinity, which essentially reduces solution resistance for electrolysis for initial formate production. To analyze the effects of different stressors on cell growth and PHB production, the IEMC experiments were conducted at limited carbon source (formate), relatively high salinity, along with nitrogen and trace metals (TM) limitation (I2 medium) or nitrogen and TM deprivation (I1 medium).

A basic amount of salinity was required for electrochemical conversion of CO2. However, this salt addition generally led to a decrease of cell population and formate uptake compared to non-salinity control conditions for C. necator (Fig 4, left most column). This effect could partially be rescued by adding nitrogen and trace metals (Fig 4, media I2), and their absence can severely affect growth (Fig 4, media I1). This reduced decline in cell amount can be explained by providing essential components for production and function of metalloproteins [20]. On the contrary, the addition of both components led to depositions on the cathode and an increased formation of H2 as shown during IEMC conditions (S5 Fig). But the presence of relatively high salinity does not seem to influence PHB production (Fig 4) for C. necator.

The one-pot IEMC setup, a combination of In cathode and C. necator, was attempted to respectively convert CO2 to formate, followed by its transformation to PHB. The microbial PHB production was observed only when electrolysis was conducted in the system (at an In cathode potential of −1.2 V vs. RHE). The cogeneration of H2 was not the cause that can trigger the PHB production, confirmed by the experiment co-feeding H2 as a reactant. The obtained results suggest that ROS induced stress might stimulate PHB production, which is known to have a stress protection function for bioplastic converting bacteria. Increasing the stress conditions for the cells by nitrogen deprivation and a lack of trace metals might further enhance the microbial PHB production.

 

Source:

http://doi.org/10.1371/journal.pone.0196079