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1. Sustainable n-butanol production by a metabolically versatile anoxygenic phototrophic bacterium

   Bai W, Ranaivoarisoa TO (January 2020, bioRxiv)

   Anthropogenic carbon dioxide (CO2) release in the atmosphere from fossil fuel combustion has inspired scientists to study CO2 to fuel conversion. Oxygenic phototrophs such as cyanobacteria have been used to produce biofuels using CO2. However, oxygen generation during oxygenic photosynthesis affects biofuel production efficiency. To produce n-butanol (biofuel) from CO2, here we introduced an n-butanol biosynthesis pathway into an anoxygenic (non-oxygen evolving) photoautotroph, Rhodopseudomonas palustris TIE-1 (TIE-1). Using different carbon, nitrogen, and electron sources, we achieved n-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 produced highest n-butanol. Coupled with novel hybrid bioelectrochemical platforms, this mutant produced nbutanol 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-neutral n-butanol bioproduction using sustainable, renewable, and abundant resources. 

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2. Overexpression of RuBisCO form I and II genes in Rhodopseudomonas palustris TIE-1 augments polyhydroxyalkanoate production heterotrophically and autotrophically

   https://doi.org/10.1128/aem.01438-24  

   Ranaivoarisoa, Tahina Onina; Bai, Wei; Karthikeyan, Rengasamy; Steele, Hope; Silberman, Miriam; Olabode, Jennifer; Conners, Eric; Gallagher, Brian; Bose, Arpita (September 2024, Applied and Environmental Microbiology)

   Reguera, Gemma (Ed.)

   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. 

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3. Evaluation of argon‐induced hydrogen production as a method to measure nitrogen fixation by cyanobacteria

   https://doi.org/10.1111/jpy.13129  

   Wilson, Samuel T.; Caffin, Mathieu; White, Angelicque E.; Karl, David M.; Palenik, ed., B. (April 2021, Journal of Phycology)

   The production of dihydrogen (H₂) is an enigmatic yet obligate component of biological dinitrogen (N₂) fixation. This study investigates the effect on H₂production by N₂fixing cyanobacteria when they are exposed to either air or a gas mixture consisting of argon, oxygen, and carbon dioxide (Ar:O₂:CO₂). In the absence of N₂, nitrogenase diverts the flow of electrons to the production of H₂, which becomes a measure of Total Nitrogenase Activity (TNA). This method of argon‐induced hydrogen production (AIHP) is much less commonly used to infer rates of N₂fixation than the acetylene reduction (AR) assay. We provide here a full evaluation of the AIHP method and demonstrate its ability to achieve high‐resolution measurements of TNA in a gas exchange flow‐through system. Complete diel profiles of H₂production were obtained for N₂fixing cyanobacteria despite the absence of N₂that broadly reproduced the temporal patterns observed by the AR assay. Comparison of H₂production under air versus Ar:O₂:CO₂revealed the efficiency of electron usage during N₂fixation and place these findings in the broader context of cell metabolism. Ultimately, AIHP is demonstrated to be a viable alternative to the AR assay with several additional merits that provide an insight into cell physiology and promise for successful field application. 

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4. The phototrophic purple non‐sulfur bacteria Rhodomicrobium spp. are novel chassis for bioplastic production

   https://doi.org/10.1111/1751-7915.14552  

   Conners, Eric_M; Rengasamy, Karthikeyan; Ranaivoarisoa, Tahina; Bose, Arpita (August 2024, Microbial Biotechnology)

   Abstract Petroleum‐based plastics levy significant environmental and economic costs that can be alleviated with sustainably sourced, biodegradable, and bio‐based polymers such as polyhydroxyalkanoates (PHAs). However, industrial‐scale production of PHAs faces barriers stemming from insufficient product yields and high costs. To address these challenges, we must look beyond the current suite of microbes for PHA production and investigate non‐model organisms with versatile metabolisms. In that vein, we assessed PHA production by the photosynthetic purple non‐sulfur bacteria (PNSB)Rhodomicrobium vannieliiandRhodomicrobium udaipurense.We show that both species accumulate PHA across photo‐heterotrophic, photo‐hydrogenotrophic, photo‐ferrotrophic, and photo‐electrotrophic growth conditions, with either ammonium chloride (NH₄Cl) or dinitrogen gas (N₂) as nitrogen sources. Our data indicate that nitrogen source plays a significant role in dictating PHA synthesis, with N₂fixation promoting PHA production during photoheterotrophy and photoelectrotrophy but inhibiting production during photohydrogenotrophy and photoferrotrophy. We observed the highest PHA titres (up to 44.08 mg/L, or 43.61% cell dry weight) when cells were grown photoheterotrophically on sodium butyrate with N₂, while production was at its lowest during photoelectrotrophy (as low as 0.04 mg/L, or 0.16% cell dry weight). We also find that photohydrogenotrophically grown cells supplemented with NH₄Cl exhibit the highest electron yields – up to 58.89% – while photoheterotrophy demonstrated the lowest (0.27%–1.39%). Finally, we highlight superior electron conversion and PHA production compared to a related PNSB,Rhodopseudomonas palustrisTIE‐1. This study illustrates the value of studying non‐model organisms likeRhodomicrobiumfor sustainable PHA production and indicates future directions for exploring PNSB metabolisms. 

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5. No‐till establishment improves the climate benefit of bioenergy crops on marginal grasslands

   https://doi.org/10.1002/saj2.20082  

   Ruan, Leilei; Robertson, G_Philip (June 2020, Soil Science Society of America Journal)

   Abstract Expanding biofuel production is expected to accelerate the conversion of unmanaged marginal lands to meet biomass feedstock needs. Greenhouse gas production during conversion jeopardizes the ensuing climate benefits, but most research to date has focused only on conversion to annual crops and only following tillage. Here we report the global warming impact of converting USDA Conservation Reserve Program (CRP) grasslands to three types of bioenergy crops using no‐till (NT) vs. conventional tillage (CT). We established replicated NT and CT plots in three CRP fields planted to continuous corn, switchgrass, or restored prairie. For the 2 yr following an initial soybean year in all fields, we found that, on average, NT conversion reduced nitrous oxide (N₂O) emissions by 50% and CO₂emissions by 20% compared with CT conversion. Differences were higher in Year 1 than in Year 2 in the continuous corn field, and in the two perennial systems the differences disappeared after Year 1. In all fields net CO₂emissions (as measured by eddy covariance) were positive for the first 2 yr following CT establishment, but following NT establishment net CO₂emissions were close to zero or negative, indicating net C sequestration. Overall, NT improved the global warming impact of biofuel crop establishment following CRP conversion by over 20‐fold compared with CT (−6.01 Mg CO₂e ha⁻¹ yr⁻¹for NT vs. −0.25 Mg CO₂e ha⁻¹ yr⁻¹for CT, on average). We also found that Intergovernmental Panel on Climate Change estimates of N₂O emissions (as measured by static chambers) greatly underestimated actual emissions for converted fields regardless of tillage. Policies should encourage adoption of NT for converting marginal grasslands to perennial bioenergy crops to reduce C debt and maximize climate benefits. 

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