Engineering Microbial Consortia for High-Performance Cellulosic Hydrolyzates-Fed Microbial Fuel Cells

Microbial fuel cells (MFCs) are eco-friendly bio-electrochemical reactors that use exoelectrogens as biocatalyst for electricity harvest from organic biomass, which could also be used as biosensors for long-term environmental monitoring. Glucose and xylose, as the primary ingredients from cellulose hydrolyzates, is an appealing substrate for MFC. Nevertheless, neither xylose nor glucose can be utilized as carbon source by well-studied exoelectrogens such as Shewanella oneidensis. In this study, to harvest the electricity by rapidly harnessing xylose and glucose from corn stalk hydrolysate, we herein firstly designed glucose and xylose co-fed engineered Klebsiella pneumoniae-S. oneidensis microbial consortium, in which K. pneumoniae as the fermenter converted glucose and xylose into lactate to feed the exoelectrogens (S. oneidensis). To produce more lactate in K. pneumoniae, we eliminated the ethanol and acetate pathway via deleting pta (phosphotransacetylase gene) and adhE (alcohol dehydrogenase gene) and further constructed a synthesis and delivery system through expressing ldhD (lactate dehydrogenase gene) and lldP (lactate transporter gene). To facilitate extracellular electron transfer (EET) of S. oneidensis, a biosynthetic flavins pathway from Bacillus subtilis was expressed in a highly hydrophobic S. oneidensis CP-S1, which not only improved direct-contacted EET via enhancing S. oneidensis adhesion to the carbon electrode but also accelerated the flavins-mediated EET via increasing flavins synthesis. Furthermore, we optimized the ratio of glucose and xylose concentration to provide a stable carbon source supply in MFCs for higher power density. The glucose and xylose co-fed MFC inoculated with the recombinant consortium generated a maximum power density of 104.7 ± 10.0 mW/m2, which was 7.2-folds higher than that of the wild-type consortium (12.7 ± 8.0 mW/m2). Lastly, we used this synthetic microbial consortium in the corn straw hydrolyzates-fed MFC, obtaining a power density 23.5 ± 6.0 mW/m2

Engineering Shewanella oneidensis enables xylose-fed microbial fuel cell

Abstract Background The microbial fuel cell (MFC) is a green and sustainable technology for electricity energy harvest from biomass, in which exoelectrogens use metabolism and extracellular electron transfer pathways for the conversion of chemical energy into electricity. However, Shewanella oneidensis MR-1, one of the most well-known exoelectrogens, could not use xylose (a key pentose derived from hydrolysis of lignocellulosic biomass) for cell growth and power generation, which limited greatly its practical applications. Results Herein, to enable S. oneidensis to directly utilize xylose as the sole carbon source for bioelectricity production in MFCs, we used synthetic biology strategies to successfully construct four genetically engineered S. oneidensis (namely XE, GE, XS, and GS) by assembling one of the xylose transporters (from Candida intermedia and Clostridium acetobutylicum) with one of intracellular xylose metabolic pathways (the isomerase pathway from Escherichia coli and the oxidoreductase pathway from Scheffersomyces stipites), respectively. We found that among these engineered S. oneidensis strains, the strain GS (i.e. harbouring Gxf1 gene encoding the xylose facilitator from C. intermedi, and XYL1, XYL2, and XKS1 genes encoding the xylose oxidoreductase pathway from S. stipites) was able to generate the highest power density, enabling a maximum electricity power density of 2.1 ± 0.1 mW/m2. Conclusion To the best of our knowledge, this was the first report on the rationally designed Shewanella that could use xylose as the sole carbon source and electron donor to produce electricity. The synthetic biology strategies developed in this study could be further extended to rationally engineer other exoelectrogens for lignocellulosic biomass utilization to generate electricity power

Adaptive Evolution of Sphingobium hydrophobicum C1T in Electronic Waste Contaminated River Sediment.

Electronic waste (e-waste) has caused a severe worldwide pollution problem. Despite increasing isolation of degradative microorganisms from e-waste contaminated environments, the mechanisms underlying their adaptive evolution in such habitats remain unclear. Sphingomonads generally have xenobiotic-degrading ability and may play important roles in bioremediation. Sphingobium hydrophobicum C1T, characterized with superior cell surface hydrophobicity, was recently isolated from e-waste contaminated river sediment. To dissect the mechanisms driving its adaptive evolution, we evaluated its stress resistance, sequenced its genome and performed comparative genomic analysis with 19 other Sphingobium strains. Strain C1T can feed on several kinds of e-waste-derived xenobiotics, exhibits a great resistance to heavy metals and possesses a high colonization ability. It harbors abundant genes involved in environmental adaptation, some of which are intrinsic prior to experiencing e-waste contamination. The extensive genomic variations between strain C1T and other Sphingobium strains, numerous C1T-unique genes, massive mobile elements and frequent genome rearrangements reflect a high genome plasticity. Positive selection, gene duplication, and especially horizontal gene transfer drive the adaptive evolution of strain C1T. Moreover, presence of type IV secretion systems may allow strain C1T to be a source of beneficial genes for surrounding microorganisms. This study provides new insights into the adaptive evolution of sphingomonads, and potentially guides bioremediation strategies

Modular Engineering Intracellular NADH Regeneration Boosts Extracellular Electron Transfer of <i>Shewanella oneidensis</i> MR‑1

Efficient extracellular electron transfer (EET) of exoelectrogens is essentially for practical applications of versatile bioelectrochemical systems. Intracellular electrons flow from NADH to extracellular electron acceptors <i>via</i> EET pathways. However, it was yet established how the manipulation of intracellular NADH impacted the EET efficiency. Strengthening NADH regeneration from NAD⁺, as a feasible approach for cofactor engineering, has been used in regulating the intracellular NADH pool and the redox state (NADH/NAD⁺ ratio) of cells. Herein, we first adopted a modular metabolic engineering strategy to engineer and drive the metabolic flux toward the enhancement of intracellular NADH regeneration. We systematically studied 16 genes related to the NAD⁺-dependent oxidation reactions for strengthening NADH regeneration in the four metabolic modules of <i>S. oneidensis</i> MR-1, <i>i.e.</i>, glycolysis, C1 metabolism, pyruvate fermentation, and tricarboxylic acid cycle. Among them, three endogenous genes mostly responsible for increasing NADH regeneration were identified, namely <i>gapA2</i> encoding a NAD⁺-dependent glyceraldehyde-3-phosphate dehydrogenase in the glycolysis module, <i>mdh</i> encoding a NAD⁺-dependent malate dehydrogenase in the TCA cycle, and <i>pflB</i> encoding a pyruvate-formate lyase that converted pyruvate to formate in the pyruvate fermentation module. An exogenous gene <i>fdh</i>* from <i>Candida boidinii</i> encoding a NAD⁺-dependent formate dehydrogenase to increase NADH regeneration in the pyruvate fermentation module was further identified. Upon assembling these four genes in <i>S. oneidens</i>is MR-1, ∼4.3-fold increase in NADH/NAD⁺ ratio, and ∼1.2-fold increase in intracellular NADH pool were obtained under anaerobic conditions without discharge, which elicited ∼3.0-fold increase in the maximum power output in microbial fuel cells, from 26.2 ± 2.8 (wild-type) to 105.8 ± 4.1 mW/m² (recombinant <i>S. oneidensis</i>), suggesting a boost in the EET efficiency. This modular engineering method in controlling the intracellular reducing equivalents would be a general approach in tuning the EET efficiency of exoelectrogens

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Additional file 2: Table S1. Summary of the reported energy output of Xylose-Fed MFCs. Table S2. Genes used in this study. Table S3. Synthesized sequences of genes in this study. Table S4. Strains and plasmids used in this study. Table S5. Main constituents for S. oneidensis basal medium (SBM). Table S6. Main constituents for M9 buffer

MOESM1 of Engineering Shewanella oneidensis enables xylose-fed microbial fuel cell

Additional file 1: Figure S1. Construction of synthetic xylose metabolic pathways in Shewanella oneidensis MR-1. (A) Schematic of the plasmid with a synthesized functional fragment of genes. The restriction sites EcoRI and XbaI with the ribosome binding site (RBS) are located upstream of each codon-optimized gene sequence, while the restrictions SpeI and PstI are located downstream of the gene. (B) Four plasmid constructs with xylose utilization pathways. To construct the multigene assembly in S. oneidensis, a Biobrick compatible expression vector pYYDT was adopted, which was previously constructed in our laboratory. Layout of the four plasmid constructs containing gene components in the xylose pathway examined in this study. Figure S2. Xylose consumption rate by E. coil (BL21) and by the recombinant S. oneidensis strain. The error bars were calculated from triplicate experiments. Figure S3. Metabolic pathway of riboflavin synthesis from xylose fermentation in S. oneidensis. A synthetic intracellular xylose metabolic pathway, i.e. the oxidoreductase pathway including genes XYL1, XYL2 and XKS1 from S. stipites, is incorporated into S. oneidensis MR-1 to enable the direct utilization of xylose. Xylulose 5-phosphate, as a metabolite in the oxidoreductase pathway, was converted to ribulose-5-P by ribulose-phosphate 3-epimerase (encoded by the rpe gene) in the pentose phosphate pathway, which was a crucial precursor for the biosynthesis of riboflavin via the riboflavin synthesis pathway. Figure S4. Xylose consumption under anaerobic conditions with 10 mM and 50 mM fumarate. The error bars were calculated from triplicate experiments