Discussion
Acidithiobacillus ferrooxidans has been shown to exhibit diauxic
growth where the cells prefer to oxidize ferrous iron before sulfur when
both substrates are available (Ponce et al., 2012). In this study, we
show that ferrous iron oxidation precedes the oxidation of untreated
elemental sulfur largely due to the saturation of the sulfur surface
available for cell adsorption. At high cell loadings, exceeding the
adsorption capacity of the sulfur particle surface, sufficient cells
remain planktonic so that ferrous iron is consumed rapidly, long before
sulfur oxidation is apparent. However, at lower cell loadings, the
adsorption process dominates the macroscopic behavior. The oxidation of
ferrous iron is delayed until there are sufficient planktonic cells.
Therefore, this observed diauxic behavior can be explained through the
separation of the total cell population into planktonic and
non-planktonic parts. Our experiments altering iron concentration with
c-sulfur suggest that iron could affect acid tolerance in these cells.
The behavior observed where lower concentrations of iron led to an
earlier slowdown in the acidification of the media in late exponential
phase could be influenced by the ferric uptake (fur) regulator which has
been found to affect extreme acid resistance in A. caldus ,
warranting further studies on the effects of fur in A.
ferrooxidans as well (Chen et al., 2020). As ferric iron reduction was
associated with cultures in which the media acidified the quickest, the
redox state of iron could be affecting the acid stress response in these
bacteria.
We then examined the role of the hydrophobicity of the sulfur particles
on the bioavailability of this energy source. For A.
ferrooxidans , the consumption of elemental sulfur is limited by the
need to make sulfur less hydrophobic for oxidation via the deposition of
EPS and possibly other secreted molecules (Gehrke et al., 1998). We
discovered that sulfur likely formulated for agricultural purposes,
d-sulfur, avoids the toxicity issues encountered with other surfactants
and allows A. ferrooxidans to display a new phenotype where
sulfur oxidation largely precedes that of iron oxidation. A simple
method to pre-disperse sulfur particles into a dry formulation was used
to demonstrate that that this newly found growth pattern could be
reliably recapitulated. In this study, we found that calcium
lignosulfonate to function as the best surfactant at 20 wt %. The
lignosulfonate could be acting surfactant and forming micelles around
sulfur particles to allow them to mix with aqueous solution (Qiu, Kong,
Zhou, & Yang, 2010). Furthermore, the use of lignosulfonate has the
added benefit of being an inexpensive and underutilized material should
the rapid growth of A. ferrooxidans be needed at the industrial
scale, as only 1-2% of the lignin produced globally is used for
value-added products (Aro & Fatehi, 2017). As a wide variety of
chemical compounds were shown to improve sulfur utilization, these
results suggest biocompatibility of the dispersant with the bacteria to
be important. As cell lysate could also disperse sulfur to enable quick
sulfur oxidation, this result provides an explanation for why some
sulfur oxidation occurred quickly resulting in the lower maximum pH
values measured for the cultures containing both ferrous iron and
c-sulfur at the high initial cell density. While cell lysate would
likely be most biocompatible for sulfur dispersion, this formulation is
inefficient to produce, ruling out its use on at larger scales. Thus,
the chemical method was sufficient to replace the secreted moleculesA. ferrooxidans produces to activate sulfur particles. Overall,
by dispersing the sulfur into an aqueous mixture and reducing its
hydrophobic properties to prevent phase separation, the sulfur
formulation becomes more bioavailable for A. ferrooxidans to
oxidize quickly.
Increasing amounts of d-sulfur or lig-sulfur in the medium resulted in
acidification below which ferrous iron oxidation was possible. Under
conditions where the pH fell below the critical point of roughly 1.3,
additional sulfur oxidation was seemingly accompanied by the aerobic
reduction of ferric iron. Furthermore, re-oxidation of ferrous iron
initially produced above this critical pH seemed to be negligible with
the use of d-sulfur (Fig. 3). The mesophilic Acidithiobacillusspecies has been shown before to reduce small amounts of ferric iron to
ferrous iron when cells are cultured aerobically below pH of 1.3 with
untreated sulfur, similar in properties to c-sulfur, over the time span
of months (Johnson, Hedrich, & Pakostova, 2017; Sand, 1989). This
reduction reaction had also been observed before under anaerobic
conditions where ferric iron serves as the terminal electron acceptor
for sulfur oxidation (Ohmura, Sasaki, Matsumoto, & Saiki, 2002; Osorio
et al., 2013; Pronk, De Bruyn, Bos, & Kuenen, 1992). By improving the
bioavailability of sulfur enabled the aerobic ferric iron reduction
phenotype to be detected rapidly. Using available information about the
enzymes involved with the iron and sulfur pathways under anaerobic
growth, we can hypothesize a similar enzymatic pathway to explain the
observed ferric iron reduction behavior (Kucera et al., 2020). In
extremely acidic conditions where the ferrous iron oxidation pathway is
inhibited, some of the electrons obtained from sulfur oxidation flow
downhill through the forward-acting bc1 complex to
ferric iron as a terminal electron acceptor (Chao, Wang, Xiao, & Liu,
2008; Kucera, Pakostova, Lochman, Janiczek, & Mandl, 2016; Osorio et
al., 2013). Since A. ferrooxidans is unable to oxidize ferrous
iron below pH 1.3, but can continue to oxidize sulfur, either the
cytochrome c4 (cycA2) or the terminal
aa3 oxidase responsible for oxygen reduction is
inhibited by extremely low pH. As A. ferrooxidans is considered a
generalist bacterium in the environment, the reduction of ferric iron
could be an adaptation to relieve inhibition by high redox potentials
and to improve growth conditions for oxidizing sulfur (Esparza,
Jedlicki, González, Dopson, & Holmes, 2019; Kawabe, Inoue, Suto, &
Chida, 2003; Li, West, & Banta, 2016; Smith & Johnson, 2018).
Therefore, ferric iron reduction capability seems to be driven by pH as
well as oxygen availability.
Given this holistic understanding of A. ferrooxidans , we are able
synthesize a unified scheme that can describe the population behavior of
the cells when provided ferrous iron and sulfur in the context of pH,
percentage of planktonic cells in relation to the total population, and
the hydrophobicity of the sulfur particles which we adjusted in this
study (Fig. 6). The percentage of planktonic cells is dependent on the
cell density and concentration of sulfur in the medium, as we have
demonstrated that the cells tend to become non-planktonic given
available adsorption sites on sulfur. The hydrophobicity of the sulfur
particles can be controlled through the extent of dispersion using
surfactants compatible with A. ferrooxidans .
Finally, we demonstrate that lig-sulfur is compatible with the genetic
systems we have developed previously (Inaba et al., 2018). The use of
lig-sulfur at higher pH allows for rapid growth of cells while
maintaining selective pressure as we find that AF-GFP cells containing
kanamycin resistance had a growth advantage over WT cells so that the
average MFI returned to that of the pure AF-GFP culture within a few
passages. This is a stark comparison to the lack of growth inhibition
when these antibiotics are placed with high concentrations of iron
typical for A. ferrooxidans cultivation. The average MFIs in the
LSM4 media were found to be much higher than that in AFM1(K) media
indicating that sulfur is more energetically rich and has a higher
reduction potential favorable for cellular metabolism than ferrous iron
alone (Bird et al., 2011; Brasseur et al., 2004).
As such, the lig-sulfur formulation demonstrated here is suitable for
future synthetic biology applications with these bacteria. This
discovery enabling sulfur oxidation before ferrous iron oxidation will
allow for improved control of the inorganic reactions that A.
ferrooxidans catalyzes.