Sustainable autotrophic production of polyhydroxybutyrate (PHB) from CO2 using a two-stage cultivation system
Graphical abstract
Introduction
Polyhydroxybutyrate (PHB) is a biodegradable and bio-based plastic, synthesized by a variety of organisms as an intracellular storage material from renewable resources. Although it has the potential to substitute conventional fossil fuel based plastics for a wide range of applications, PHB is still commercially behind the petroleum based synthetic plastics. The major drawback is the high production cost which is dominated for approximately 50% by the raw material costs [1]. To attain bulk commercial viability and to further improve the sustainability profile of PHB production, it is desirable to use waste and surplus materials for PHB biosynthesis. In addition, the conversion of waste materials to PHB is advantageous for waste management. Both liquid (such as crude glycerol) and gaseous substrates (using CO2 as feedstock) have been studied for PHB production [2], [3], [4].
Cupriavidus necator is a metabolically versatile organism capable of shifting between heterotrophic growth (utilizing organic compounds as carbon and energy source) and autotrophic growth (utilizing CO2 as carbon source and H2 or formate as energy source). In addition, the bacterium can accumulate PHB up to 80% of the dry cell weight in a non-growth-associated manner [5]. Two cultivation methods exist to utilize CO2 for PHB production by C. necator. The most frequently applied cultivation method uses a gas mixture of CO2, H2 and O2 for both cell mass growth and PHB accumulation according to Eq. (1) [6] and Eq. (2) respectively [7]21.36 H2 + 6.21 O2 + 4.09 CO2 + 0.76 NH3 → C4.09H7.13O1.89N0.76 + 18.7 H2O33 H2 + 12 O2 + 4 CO2 → C4H6O2 + 30 H2O
A gas composition ratio of H2:O2:CO2 = 7:2:1 is needed to attain sufficient cell growth by avoiding gas-limited conditions [8], but lies within the gas-explosion range. Several solutions have been proposed to solve the explosion risk problem. The cultivation could be carried out by applying other terminal electron acceptors such as nitrate than O2. This results however in an extreme reduction in the cell yield and growth [9]. A better strategy is to reduce the O2 content in the gas phase below the explosion limit. Depending on the method used [10], the lower explosion limit (LEL) of O2 in H2 has been estimated to range from 4.0 vol% [11], 5 vol% [12] to 6.9 vol% [13]. By reducing the O2 concentration below its LEL, the driving force for mass transfer of O2 decreases, increasing the risk for mass transfer limitation. As a result, a lower cell mass concentration is attained at the end of cell mass growth phase, yielding a lower final PHB concentration and productivity.
In the second cultivation method for PHB production from CO2, the formation of cell mass occurs under heterotrophic conditions [14], followed by PHB accumulation using a gas mixture of CO2, H2 and O2 (Eq. (2)). Similar to the first cultivation method, the O2 concentration in the mixture of substrate gases needs to be maintained below LEL to avoid gas detonation. The advantage of this cultivation system is that a high cell mass concentration and thus productivity can be obtained during the cell mass growth phase as O2 can be supplied under non-limiting conditions, while in the second autotrophic phase PHB biosynthesis will be triggered when the O2 concentration is below its critical value which is reported to be 3% [15].
The objective of this study was to evaluate the technical feasibility of C. necator DSM 545 for sustainable autotrophic PHB production from a gas mixture (CO2, H2, O2) that followed heterotrophic cell mass growth from an organic substrate. To ensure that test conditions were relevant for later industrial application, a safety marge of 2.0 vol% below the LEL of 5 vol% O2 was taken into account during autotrophic cultivation [16]. The influence of the organic carbon source on the formation of key enzymes of autotrophic metabolism was evaluated in terms of PHB accumulation by using two different organic substrates, glucose and waste glycerol, as carbon source for cell mass growth. PHB biosynthesis was induced under imbalanced growth conditions by limiting nitrogen and O2 at different cell mass concentrations. Furthermore, the biopolymers were characterized with different techniques and compared with polymers synthesized on solely organic carbon sources and a commercial polymer to evaluate the influence of the fermentation mode and substrates on the properties of the biopolymers.
Section snippets
Organism, media and inoculum preparation
The microorganism, culture media and inoculum preparation used in this study were the same as previously reported [17].
Set-up
The set-up for heterotrophic–autotrophic production of PHB, consisted of a bioreactor, online gas analysis system and gas control system. A schematic representation is shown in Fig. 1.
Results and discussion
This study aimed to evaluate autotrophic PHB production from a gas mixture (CO2, H2, O2) following heterotrophic cell mass growth on an organic substrate. The choice of organic carbon source is essential from technological and economical point of view as the substrate influences the growth rate, the degree of synthesis of key enzymes for chemolithoautotrophic metabolism [19], [20] and raw material cost. This concept has already been applied using either fructose or acetic acid as organic carbon
Conclusions
- •
PHB was produced from CO2 using a heterotrophic–autotrophic cultivation system at an oxygen concentration below the LEL for two organic substrates, namely glucose and waste glycerol, by limiting nitrogen and oxygen at three cell mass concentrations.
- •
PHB production from CO2 on waste-glycerol grown cell mass under conditions relevant for industrial application resulted in the highest reported PHB concentration synthesized from CO2 so far.
- •
Independent of the organic carbon source, the fermentation
Acknowledgments
The authors gratefully acknowledge the Environmental and Energy Technology Innovation Platform (MIP) for financial support. Silvia Vangeel, Helmut Elslander and Filip Vanhoof are acknowledged for their technical support and Metin Bulut for the graphical abstract.
References (40)
- et al.
Bioresour. Technol.
(2009) - et al.
J. Ferment. Bioeng.
(1991) - et al.
J. Ferment. Bioeng.
(1996) - et al.
Bioresour. Technol.
(2014) - et al.
Process Biochem.
(2014) - et al.
J. Ferment. Bioeng.
(1994) - et al.
Process Biochem.
(2011) New Biotechnol.
(2013)- et al.
Biotechnol. Adv.
(2009) - et al.
Renew. Sust. Energy Rev.
(2011)
Bioresour. Technol.
Res. Microbiol.
Prog. Polym. Sci.
Int. J. Biol. Macromol.
Process Biochem.
Appl. Microbiol. Biotechnol.
J. Chem. Technol. Biotechnol.
Food Technol. Biotechnol.
Nat. Biotechnol.
Appl. Microbiol. Biotechnol.
Cited by (72)
Polyhydroxyalkanoates bioproduction from bench to industry: Thirty years of development towards sustainability
2024, Bioresource TechnologyBiotransforming CO<inf>2</inf> into valuable chemicals
2024, Journal of Cleaner ProductionPolyhydroxybutyrate (PHB) accumulation by a mangrove isolated cyanobacteria Limnothrix planktonica using fruit waste
2023, International Journal of Biological Macromolecules