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.