Addition of citrate to Acidithiobacillus ferrooxidans cultures enables precipitate‐free growth at elevated pH and reduces ferric inhibition

Abstract

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is important in biomining and other biotechnological operations. The cells are able to oxidize inorganic iron, but the insolubility and product inhibition by Fe(3+) complicates characterization of these cultures. Here we explore the growth kinetics of A. ferrooxidans in iron-based medium in a pH range from 1.6 to 2.2. It was found that as the pH was increased from 1.6 to 2.0, the maintenance coefficient decreased while both the growth kinetics and maximum cell yield increased in the precipitate-free, low Fe(2+) concentration medium. In higher iron media a similar trend was observed at low pH, but the formation of precipitates at higher pH (2.0) hampered cell growth and lowered the specific growth rate and maximum cell yield. In order to eliminate ferric precipitates, chelating agents were introduced into the medium. Citric acid was found to be relatively non-toxic and did not appear to interfere with iron oxidation at a maximum concentration of 70 mM. Inclusion of citric acid prevented precipitation and A. ferrooxidans growth parameters resumed their trends as a function of pH. The addition of citrate also decreased the apparent substrate saturation constant (KS ) indicating a reduction in the competitive inhibition of growth by ferric ions. These results indicate that continuous cultures of A. ferrooxidans in the presence of citrate at elevated pH will enable enhanced cell yields and productivities. This will be critical as these cells are used in the development of new biotechnological applications such as electrofuel production.

Engineering the iron‐oxidizing chemolithoautotroph Acidithiobacillus ferrooxidans for biochemical production

“…The growth kinetics of wild type A. ferrooxidans have been investigated in continuous cultures (Li et al, 2014b). It was found that increased pH reduced the maintenance energy, raised the maximum growth rate and cell yield on iron.…”

“…ferrooxidans cultures were grown in shake flasks (225 RPM) at 30 C in filter sterilized ATCC 2039 media (AFM media) containing: 0.8 g/L (NH 4 ) 2 SO 4 , 2 g/L MgSO 4 • 7H 2 O, 0.4 g/L K 2 HPO 4 , 5 mL/L MD-TMS TM trace mineral solution and at least 10 g/L (36 mM) of FeSO 4 • 7H 2 O adjusted to pH 1.8 with concentrated sulfuric acid (Li et al, 2014b). ferrooxidans cultures were grown in shake flasks (225 RPM) at 30 C in filter sterilized ATCC 2039 media (AFM media) containing: 0.8 g/L (NH 4 ) 2 SO 4 , 2 g/L MgSO 4 • 7H 2 O, 0.4 g/L K 2 HPO 4 , 5 mL/L MD-TMS TM trace mineral solution and at least 10 g/L (36 mM) of FeSO 4 • 7H 2 O adjusted to pH 1.8 with concentrated sulfuric acid (Li et al, 2014b).…”

“…The total cell number was determined from OD 600 measurements, where OD 600 of 1.0 was equal to a total cell density of 8.3 Â 10 9 cells/mL, which is equivalent to a dry cell weight density of 0.00053 g/mL (Li et al, 2014b). 1 mL samples were centrifuged and suspended in iron-free AFM medium.…”

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

“…The configuration and operation of the continuous bioreactor was described previously (Li et al, 2014b …”

“…Cells were propagated and maintained in batches with ATCC 2039 A. ferrooxidans media as previously described (Li et al, 2014b). The growth medium contains 0.8 g/L (NH 4 ) 2 SO 4 , 2 g/L MgSO 4 Â 7H 2 O, 0.4 g/L K 2 HPO 4 , 5 mL/L Wolfe's Mineral Solution and 20 g/L (72 mM) of FeSO 4 Â 7H 2 O with an initial pH of 1.8 adjusted using concentrated sulfuric acid.…”