Sustainable autotrophic production of polyhydroxybutyrate (PHB) from CO2 using a two-stage cultivation system

doi:10.1016/j.cattod.2014.05.025

Catalysis Today

Volume 257, Part 2, 15 November 2015, Pages 237-245

https://doi.org/10.1016/j.cattod.2014.05.025 Get rights and content

Highlights

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Sustainable PHB production from CO₂ using two-stage culture system was demonstrated.
•
PHB concentration obtained from CO₂ in glycerol-grown cells is highest reported one.
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Oxygen mass transfer was found as rate limiting for PHB production from CO₂.
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PHB synthesized from CO₂ presented similar properties as commercial PHB.

Abstract

The technical feasibility of Cupriavidus necator DSM 545 for sustainable autotrophic polyhydroxybutyrate (PHB) production from CO₂ using a two-stage cultivation system was evaluated. In this cultivation method, cell mass growth occurred under heterotrophic conditions using two different organic substrates, namely glucose and waste glycerol. In both cases, PHB biosynthesis was triggered by applying nitrogen and oxygen limitation at three different cell mass concentrations under autotrophic conditions using a gas mixture of H₂, O₂ and CO₂. To ensure that the test conditions were relevant for later industrial application, O₂ concentration was kept below the safety value during autotrophic PHB production. PHB production from CO₂ on waste-glycerol grown cell mass resulted in a PHB concentration of 28 g/L, which is the highest reported value in literature for PHB synthesized from CO₂ at an O₂ concentration below the lower explosion limit of 5 vol%. The fermentation performance decreased when nutrient limitation was delayed at higher cell mass concentrations. Furthermore, it was shown that PHB production from CO₂ at high cell mass concentration is metabolically feasible, but under the tested conditions the mass transfer of O₂ was limiting PHB accumulation. Characterization of the produced polymers showed that the organic carbon source affected the properties of PHB and that the cultivation method developed in this study provided PHB with properties similar to a commercial PHB and PHB typically found in literature. It can be concluded that heterotrophic–autotrophic production of PHB by C. necator is a promising cultivation method to reduce the overall production cost of PHB. In order to compete with the current heterotrophic cultivation system, the oxygen transfer rate must however be enhanced to achieve a higher PHB productivity.

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 → C_4.09H_7.13O_1.89N_0.76 + 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

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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.
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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)

L.R. Castilho et al.
Bioresour. Technol.
(2009)
A. Ishizaki et al.
J. Ferment. Bioeng.
(1991)
T. Takeshita et al.
J. Ferment. Bioeng.
(1996)
M.S.I. Mozumder et al.
Bioresour. Technol.
(2014)
M.S.I. Mozumder et al.
Process Biochem.
(2014)
K. Tanaka et al.
J. Ferment. Bioeng.
(1994)
J.A. Posada et al.
Process Biochem.
(2011)
N. Berezina
New Biotechnol.
(2013)
F. Garcia-Ochoa et al.
Biotechnol. Adv.
(2009)
M. Mohammadi et al.
Renew. Sust. Energy Rev.
(2011)

F.C. Oliveira et al.

Bioresour. Technol.

(2007)

T.G. Volova et al.

Res. Microbiol.

(2013)

K. Sudesh et al.

Prog. Polym. Sci.

(2000)

L.A. Madden et al.

Int. J. Biol. Macromol.

(1999)

S. Khanna et al.

Process Biochem.

(2005)

J. Choi et al.

Appl. Microbiol. Biotechnol.

(1999)

E. Akaraonye et al.

J. Chem. Technol. Biotechnol.

(2010)

M. Koller et al.

Food Technol. Biotechnol.

(2010)

A. Pohlmann et al.

Nat. Biotechnol.

(2006)

A. Ishizaki et al.

Appl. Microbiol. Biotechnol.

(2001)

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View full text

Sustainable autotrophic production of polyhydroxybutyrate (PHB) from CO2 using a two-stage cultivation system

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Organism, media and inoculum preparation

Set-up

Results and discussion

Conclusions

Acknowledgments

Bioresour. Technol.

J. Ferment. Bioeng.

J. Ferment. Bioeng.

Bioresour. Technol.

Process Biochem.

J. Ferment. Bioeng.

Process Biochem.

New Biotechnol.

Biotechnol. Adv.

Renew. Sust. Energy Rev.

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.

Sustainable autotrophic production of polyhydroxybutyrate (PHB) from CO₂ using a two-stage cultivation system