Enhancing isobutyric acid production from engineered Acidithiobacillus ferrooxidans cells via media optimization

Abstract

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has previously been genetically modified to produce isobutyric acid (IBA) from carbon dioxide while obtaining energy from the oxidation of ferrous iron. Here, a combinatorial approach was used to explore the influence of medium composition in both batch and chemostat cultures in order to improve IBA yields (g IBA/mol Fe(2+)) and productivities (g IBA/L/d). Medium pH, ferrous concentration (Fe(2+)), and inclusion of iron chelators all had positive impact on the IBA yield. In batch experiments, gluconate was found to be a superior iron chelator because its use resulted in smaller excursions in pH. In batch cultures, IBA yields decreased linearly with increases in the final effective Fe(3+) concentrations. Chemostat cultures followed similar trends as observed in batch cultures. Specific cellular productivities were found to be a function of the steady state ORP (Oxidation-reduction potential) of the growth medium, which is primarily determined by the Fe(3+) to Fe(2+) ratio. By operating at low ORP, chemostat cultures were able to achieve volumetric productivities as high as 3.8 ± 0.2 mg IBA/L/d which is a 14-fold increase over the previously reported value.

Development of reactor configurations for an electrofuels platform utilizing genetically modified iron oxidizing bacteria for the reduction of CO2 to biochemicals

“…Higher IBA yields (~2-fold increase) and Gibbs free energy efficiencies (also ~2-fold) were observed in the two loop system as compared to the single loop system. These results are consistent with previous batch results showing the impact of Fe 2+ /Fe 3+ on efficiency and yield [10]. The current versus time graphs for GM cells in both the single loop and two-loop systems are shown in Figures S1 and S2.…”

“…We have been developing an alternative electrofuels process which utilizes iron as an electron mediator and the bacterium Acidithiobacillus ferrooxidans as the production host [5,9,8,10]. A.…”

Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans

“…Previous plasmid-based expression studies included the overexpression of native iron oxidation genes rus, cyc2, and tetH (13)(14)(15). The superfolder green fluorescent protein (sfGFP) gene has been introduced to the cells, and to explore the biosynthetic capabilities of this organism, the 2-keto decarboxylase (KDC) gene from Lactococcus lactis was expressed to enable the production of isobutyric acid (IBA) from precursors in the valine biosynthesis pathway (6,16). In addition, two genes (encoding acyl-acyl carrier protein [acyl-ACP] reductase and aldehyde deformylating oxygenase) from Synechococcus elongatus were expressed to enable the production of the long-chain hydrocarbon heptadecane (6).…”

“…These transient transformations were made using modifications to the broad-host-range pJRD215 plasmid (17). The wild-type (WT) strain and the genetically modified strains have been investigated using a variety of medium compositions, including the addition of metal chelators, to improve growth rates and maximize chemical yields in batch and continuous cultures (16,18,19). Although several constitutive promoters of different strengths have been characterized with respect to driving gene expression, the need for inducible control of gene expression within this organism led to the identification of endogenous promoter sequences compatible with the growth conditions for A. ferrooxidans (12,20).…”

Metals and minerals as a biotechnology feedstock: engineering biomining microbiology for bioenergy applications

“…The efficiency of conversion of this energy by the microorganisms and the titers of biochemicals produced are important factors which will impact the commercial feasibility of these co-generation activities. To date, the production of isobutyric acid by genetically engineered A. ferrooxidans has only been characterized using Fe 2+ as an energy source, and the Gibbs free energy efficiencies for incorporation of this energy into the isoburyric acid product are on the order of 1% [11,56,59]. In order to develop a commercially viable processes, these efficiencies will need to be increased when using mining process streams as feedstocks.…”