Microbial Electrochemical Device of Acidithiobacillus ferrooxidans
As a typical variable-valence metal element, iron plays a core role in the life metabolism of Acidithiobacillus ferrooxidans, especially in the construction and functional realization of the electron transport chain. In view of this, this study used A. ferrooxidans as the chassis microorganism to construct a new type of microbial electrochemical system (MES), aiming to explore the application potential of this strain in the field of electrochemistry [1].
The MES constructed in this study is an electricity-driven bioelectrolytic cell, and its core design concept is to use Fe2+ as an electron transfer carrier and couple electric energy input to achieve carbon dioxide fixation and biomass synthesis. A. ferrooxidans takes up electrons from the extracellular environment such as ferrous iron (Fe²⁺). This inward electron flow enables the organism to use electrical energy—supplied via a cathode—to drive intracellular metabolic processes. In this system (Figure 1), electrons provided from the cathode reduce extracellular ferric iron (Fe³⁺) to ferrous iron (Fe²⁺) [2]. A. ferrooxidans, acting as the cathode biocatalyst, then re-oxidizes Fe²⁺ back to Fe³⁺, capturing the electrons in the process. These electrons are utilized to support carbon dioxide fixation and biomass synthesis, effectively converting electrical energy into storable chemical energy. Through this cyclic iron-mediated electron transfer, the system integrates CO₂ reduction and bio-production in a single electrochemical platform.
Figure 1 Microbial electrochemical synthesis system in our project [3]
Figure 2 Schematic Diagram of Microbial Electrochemical Reactor Based on A. ferrooxidans
Current-time curves were recorded, and the current density was calculated by normalizing the current to the working electrode area (1 cm × 3 cm=3 cm2). The resulting current density-time profile is presented in Figure 3. To further investigate the role of iron as an electron shuttle, we also examined the system's behavior under varying initial concentrations of ferrous ions, as summarized in the same figure.
Figure 3 (A) Current density-time curves of wild-type A. ferrooxidans measured at an initial ferrous iron concentration of 159 mM (equal to the ferrous iron concentration in normal 9k-FeSO4 medium) added to the device (B) Current density-time curves of wild-type A. ferrooxidans measured at an initial ferrous iron concentration of 48 mM added to the device (C) Current density-time curves of wild-type A. ferrooxidans obtained by adding different concentrations of ferrous iron.
In this study, a microbial electrochemical device with A. ferrooxidans as the core material was successfully constructed, providing a reference for research in the field of environmental electrochemistry. This achievement is expected to inspire iGEM teams in the design and implementation of A. ferrooxidans-based electrochemical modules. We would be deeply honored if it could provide technical support or ideological reference for subsequent iGEM researchers.
Basic part
AFE_0053 (BBa_K5323000)
To quantitatively evaluate the performance of part AFE_0053 (BBa_K5323000), this study calculated the current density by real-time monitoring and recording the current-time curve of the system, combined with the effective area of the working electrode (1 cm × 3 cm = 3 cm²). This current density was used as a key indicator to characterize the electron utilization efficiency of the strain.
Figure 4 Current density-time profiles were recorded for engineered A. ferrooxidans strains harboring either the AFE_0053 gene plasmid or the empty vector pYDT. The electrochemical characterization was performed under standardized conditions with an initial ferrous iron concentration of 159 mM, equivalent to the ferrous iron content in conventional 9K-FeSO4 medium.
Results from the long-term operation experiment showed that both the decay rate and decay magnitude of the current density in the engineered strain p0053 group were significantly higher than those in the pYDT group. This phenomenon indicates that the p0053 strain exhibits superior efficiency in the processes of electron capture, transfer, and metabolic utilization, and it may possess stronger electron utilization capacity—providing an efficient electron transfer basis for subsequent CO₂ fixation and biomass synthesis.
Figure 5 （A）box plot (math.) of Current Density Comparison (***p < 0.001) （B） P-value curve for each point of Current Density Comparison.
We further performed a Student's t-test using R to statistically evaluate these differences. The results of the t-test are presented in Figure 5 (A). Additionally, a corresponding p-value plot was generated and is shown in Figure 5 (B). As shown in Figure 5 (A), a statistically significant difference in current density was observed between the engineered p0053 group and the pYDT control group (denoted by, *** p < 0.001). Specifically, the p0053 group exhibited an average current density of -7.53 ± 7.01 A/m², compared to -5.40 ± 4.50 A/m² for the pYDT group. The lower (more negative) mean current density, along with greater variability (standard deviation) in the p0053 group, indicates not only a significant divergence in current output between the two groups but also higher data dispersion within the engineered strain.
From the perspective of temporal dynamics, in the early stage of the experiment (approximately 0~250 h), the -log10 (p-values) of the two groups were close to 0, indicating that there was no significant difference in current density between the two groups at this stage ( * p > 0.05). However, after the cultivation time exceeded 250 h, the p-value decreased sharply, and the corresponding -log10 (p-value) increased significantly. Moreover, the p-value was much less than 0.001, reaching a highly significant level.
This result is highly consistent with the current density decay curve and trend analysis results, further verifying that the difference in current density between the p0053 group and the pYDT group is time-dependent. Such a difference gradually emerges in the later stage of the experiment and reaches a statistically significant level, which rules out the interference of random errors on the results.
Through the optimization of bacterial strain engineering modification, this study finally developed a high-efficiency electron transfer module. This system not only provides a new technical approach for microbial electrochemical CO₂ fixation, but its design concept and experimental methods also hold significant value. Furthermore, it offers a reference for research in the field of synthetic biology. We would be deeply honored if it could provide technical support or ideological reference for subsequent iGEM researchers.
ACIR131C_RS0121050 (BBa_25B5EL76)
To enhance the carbon fixation capacity of the CBB cycle in A. ferrooxidans, we constructed a module to overexpress phosphoribulokinase (PRK), a key enzyme in this cycle. The specific function of PRK is to phosphorylate ribulose-5-phosphate (Ru5P) to generate ribulose-1,5-bisphosphate (RuBP). E. coli does not rely on the Calvin cycle for survival. Instead, it decomposes organic matter to obtain energy and carbon sources through pathways such as glycolysis and the tricarboxylic acid (TCA) cycle. However, under conventional culture conditions, the expression of PRK in E. coli is repressed. After artificial overexpression of PRK, a series of cascade reactions are theoretically triggered, including a significant increase in Calvin cycle flux and enhanced CO₂ fixation. When cells can adapt to the metabolic state following PRK overexpression, they will theoretically acquire a stronger CO₂ fixation capacity. This holds great significance for synthetic biology and metabolic engineering. ACIR131C_RS0121050 is a gene sequence derived from the cyanobacterium Dolichospermum circinale AWQC131C, which encodes PRK. We designed the expression vector ‘pYDT-Ptac-RS0121050’ in E. coli BL21 and A. ferrooxidans to characterize its function.
Figure 6 The gene circuit of expression vector ‘pYDT-Ptac-RS0121050’
We examined the role of RS0121050 by comparing the growth rates of the engineered strain and the wild-type strain, as well as their growth under induction with different concentrations of IPTG.
Figure 7 Growth curves of E. coli BL21 in LB medium under different IPTG concentrations. (A) The growth curve at 0 mM IPTG. (B) The growth curve at 0.1 mM IPTG. (C) The growth curve at 0.5 mM IPTG. (D) The growth curve at 1 mM IPTG. (E)Comparison of pYDT at different IPTG concentrations. (F) Comparison of pRS0121050 at different IPTG concentrations. Experiments were conducted in triplicate and the error bar represent SD.
In contrast, pRS0121050 showed optimal growth at 0 mM IPTG, with a gradual decline in growth as the IPTG concentration increased. This suggests that higher IPTG concentrations lead to elevated PRK expression levels, which in turn imposes a metabolic burden on the cells and consequently inhibits bacterial growth. This study may provide insights for other future iGEM teams in terms of carbon fixation, and we are pleased if it can be of any help to other future iGEMers.
PCC6301pg_RS05100 (BBa_255XTY0G)
Figure 8 The gene circuit of expression vector ‘pYDT-Ptac-RS05100’
We examined the role of RS05100 by comparing the growth rates of the engineered strain and the wild-type strain, as well as their growth under induction with different concentrations of IPTG.
Figure 9 Growth curves of E. coli BL21 harboring pYDT (E. coli BL21/pYDT) and pRS05100 (E. coli BL21/ pRS05100) in LB medium under induction with varying concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG) are presented as follows: (A) 0 mM IPTG, (B) 0.1 mM IPTG, (C) 0.5 mM IPTG, and (D) 1 mM IPTG. Additionally, growth curves for strains (E) E. coli BL21/pYDT and (F) E. coli BL21/ pRS05100 are compared across the different IPTG concentrations. The data are expressed as the mean of three independent replicates (n=3), with error bars indicating standard deviation (SD).
By comparing, expression of the pRS05100 construct in E.coli BL21 slowed its growth rate. The growth impairment was further exacerbated in a dose-dependentmanner by increasing IPTG concentrations, with higher induction levels leading to slower growth.
Composite part
Ptac+RBS+fps1+RBS+gpd1-gpp2+TT (BBa_25TAEV6S)
Glycerol-3-phosphate dehydrogenase (Gpd1) and glycerol-3-phosphatase (Gpp2) are key genes identified in the Saccharomyces cerevisiae S288C, both of which are involved in the biosynthetic process of glycerol. To achieve efficient glycerol synthesis and extracellular secretion, this study constructed a recombinant expression vector, pYDT-Ptac-fps1-gpd1-gpp2fus, which is driven by the IPTG-inducible Ptac promoter. We engineered gpd1 and gpp2 into a fusion protein to facilitate the continuous intracellular synthesis of glycerol in the engineered bacteria. Subsequently, the fps1 glycerol transport protein gene was introduced, enabling the efficient transport of intracellularly synthesized glycerol to the extracellular environment via channel-mediated facilitated diffusion. This part may inspire other iGEM teams in the community working on carbon fixation and the synthesis of chemical products, and we are pleased if it can be of any help to other future iGEMers.
To verify the successful introduction and functional expression of the target genes, E. coli BL21 transformed with this recombinant vector was used as the model strain. The strain was cultured in M9 minimal medium, and treatment groups with different IPTG induction concentrations (0, 0.1, 0.5, and 1 mM) were set up to dynamically monitor the bacterial growth curve and extracellular glycerol yield.
Figure 10 The gene circuit of expression vector ‘pYDT-Ptac-fps1-gpd1-gpp2fus’
Figure 11 (A) Growth curve when IPTG = 0 mM (B) Growth curve when IPTG = 0.1mM (C) Growth curve when IPTG = 0.5mM (D) Growth curve when IPTG = 1mM Experiments were conducted in triplicate and the error bar represent SD.
Figure 12 (A) Glycerol production at different IPTG concentrations at 11h (B) Glycerol production at different IPTG concentrations at 23h (C) Comparison of glycerol production by pYDT at different concentrations between 11h and 23h (D) Comparison of glycerol production by Fps1at different concentrations between 11h and 23hExperiments were conducted in triplicate and the error bar represent SD.
Figure 13 Glycerol Standard Curve.An R-squared value of 0.9914 indicates a good fit of the curve.
Under IPTG induction, the growth performance of the pYDT control strain was generally superior to that of the pYDT-Ptac-fps1-gpd1-gpp2fus recombinant strain, indicating that IPTG exerted a certain inhibitory effect on the growth of the engineered bacteria, and this inhibitory effect was enhanced with increasing IPTG concentration.
A comprehensive analysis of growth and expression efficiency revealed that an IPTG concentration of 0.5 mM could not only effectively induce the expression of the target genes but also maintain a good growth state of the engineered bacteria, thus being identified as the optimal induction condition.
Based on the glycerol standard curve shown in Figure 13, the study quantitatively analyzed the glycerol yields under different IPTG concentrations [4]. The experimental results in Figure 12 demonstrated that when the IPTG concentration was 0.5 mM, the glycerol yield of the engineered bacteria reached the highest level, which was significantly higher than that of the empty vector control strain ( * p<0.05 ).Glycerol synthesis mainly occurred in the late logarithmic growth phase, and the yield at 11 hours of cultivation was higher than that at 23 hours, suggesting that prolonging the cultivation time did not significantly increase the total glycerol yield.
