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Miniaturization of measurement systems offers several advantages, including reduced sample and reagent volumes, improved control over experimental conditions, and the ability to multiplex complementary measurement modalities, thereby enabling new types of studies in microbial electrochemistry. We present a scalable glass-based microfluidic bioelectrochemical cell (µ-BEC) platform for multiplexed investigations of microbial extracellular electron uptake (EEU). The platform integrates eight independently addressable three-electrode cells in a 2×4 array, with transparent working electrodes that support simultaneous electrochemical analysis and optical imaging. Using Rhodopseudomonas palustris TIE-1 as a model phototroph, we measured EEU activity under light-dark cycling. Microfluidic flow was used to selectively remove planktonic cells, enabling isolation of the electron uptake signal associated with surface attached cells. These results demonstrate the µ-BEC as a robust and adaptable platform for probing microbial electron transfer, with broad potential for high-throughput and multimodal studies. 

ABSTRACT With the rising demand for sustainable renewable resources, microorganisms capable of producing bioproducts such as bioplastics are attractive. While many bioproduction systems are well-studied in model organisms, investigating non-model organisms is essential to expand the field and utilize metabolically versatile strains. This investigation centers onRhodopseudomonas palustrisTIE-1, a purple non-sulfur bacterium capable of producing bioplastics. To increase bioplastic production, genes encoding the putative regulatory protein PhaR and the depolymerase PhaZ of the polyhydroxyalkanoate (PHA) biosynthesis pathway were deleted. Genes associated with pathways that might compete with PHA production, specifically those linked to glycogen production and nitrogen fixation, were deleted. Additionally, RuBisCO form I and II genes were integrated into TIE-1’s genome by a phage integration system, developed in this study. Our results show that deletion ofphaRincreases PHA production when TIE-1 is grown photoheterotrophically with butyrate and ammonium chloride (NH₄Cl). Mutants unable to produce glycogen or fix nitrogen show increased PHA production under photoautotrophic growth with hydrogen and NH₄Cl. The most significant increase in PHA production was observed when RuBisCO form I and form I & II genes were overexpressed, five times under photoheterotrophy with butyrate, two times with hydrogen and NH₄Cl, and two times under photoelectrotrophic growth with N₂. In summary, inserting copies of RuBisCO genes into the TIE-1 genome is a more effective strategy than deleting competing pathways to increase PHA production in TIE-1. The successful use of the phage integration system opens numerous opportunities for synthetic biology in TIE-1.IMPORTANCEOur planet has been burdened by pollution resulting from the extensive use of petroleum-derived plastics for the last few decades. Since the discovery of biodegradable plastic alternatives, concerted efforts have been made to enhance their bioproduction. The versatile microorganismRhodopseudomonas palustrisTIE-1 (TIE-1) stands out as a promising candidate for bioplastic synthesis, owing to its ability to use multiple electron sources, fix the greenhouse gas CO₂, and use light as an energy source. Two categories of strains were meticulously designed from the TIE-1 wild-type to augment the production of polyhydroxyalkanoate (PHA), one such bioplastic produced. The first group includes mutants carrying a deletion of thephaRorphaZgenes in the PHA pathway, and those lacking potential competitive carbon and energy sinks to the PHA pathway (namely, glycogen biosynthesis and nitrogen fixation). The second group comprises TIE-1 strains that overexpress RuBisCO form I or form I & II genes inserted via a phage integration system. By studying numerous metabolic mutants and overexpression strains, we conclude that genetic modifications in the environmental microbe TIE-1 can improve PHA production. When combined with other approaches (such as reactor design, use of microbial consortia, and different feedstocks), genetic and metabolic manipulations of purple nonsulfur bacteria like TIE-1 are essential for replacing petroleum-derived plastics with biodegradable plastics like PHA. 

Abstract Anthropogenic carbon dioxide (CO₂) release in the atmosphere from fossil fuel combustion has inspired scientists to study CO₂to biofuel conversion. Oxygenic phototrophs such as cyanobacteria have been used to produce biofuels using CO₂. However, oxygen generation during oxygenic photosynthesis adversely affects biofuel production efficiency. To producen-butanol (biofuel) from CO₂, here we introduce ann-butanol biosynthesis pathway into an anoxygenic (non-oxygen evolving) photoautotroph,Rhodopseudomonas palustrisTIE-1 (TIE-1). Using different carbon, nitrogen, and electron sources, we achieven-butanol production in wild-type TIE-1 and mutants lacking electron-consuming (nitrogen-fixing) or acetyl-CoA-consuming (polyhydroxybutyrate and glycogen synthesis) pathways. The mutant lacking the nitrogen-fixing pathway produce the highestn-butanol. Coupled with novel hybrid bioelectrochemical platforms, this mutant producesn-butanol using CO₂, solar panel-generated electricity, and light with high electrical energy conversion efficiency. Overall, this approach showcases TIE-1 as an attractive microbial chassis for carbon-neutraln-butanol bioproduction using sustainable, renewable, and abundant resources.