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

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 vannielii and Rhodomicrobium 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 N₂ fixation 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 palustris TIE-1. This study illustrates the value of studying non-model organisms like Rhodomicrobium for sustainable PHA production and indicates future directions for exploring PNSB metabolisms.

FIGURE 1

The putative PHA synthesis cycle and genes in R. vannielii and R. udaipurense. (A) The putative PHA synthesis cycle of R. vannielii (Rv) and R. udaipurense (Ru) based on the hypothesized PHA cycle of Rhodopseudomonas palustris TIE‐1. Acetyl‐CoA is produced from organic or inorganic carbon sources followed by condensation of two acetyl‐CoAs into acetoacetyl‐CoA by PhaA. NAD‐dependent acetoacetyl‐CoA reductase (PhaB) reduces acetoacetyl‐CoA, producing 3‐Hydroxybutyryl‐CoA. 3‐Hydroxybutyryl‐CoA is polymerized into PHA granules by PHA polymerase (PhaC). When cells call upon PHA granules for carbon and/or energy reserves, PHA depolymerase (PhaZ) degrades the granules back into 3‐Hydroxybutyrate and then to acetyl‐CoA via multiple enzymatic reactions (depicted as double white lines). PhaR likely represses the expression of PhaC, while phasins (PhaP) dictate size and number of PHA granules. Locus tags for each strains' homologues are listed under their respective genes. (B) The putative genes involved in the PHA synthesis cycle of R. vannielii (Rv) and R. udaipurense (Ru) based on the highest degree of similarity to TIE‐1 homologues. Locus tags corresponding to the genes with the highest similarity to those in TIE‐1 are listed below each gene for Rv and Ru. PHA: Polyhydroxyalkanoate. CoA‐SH: Coenzyme A. NADPH: Nicotinamide adenine dinucleotide phosphate. Adapted from Ranaivoarisoa et al. (2019).

FIGURE 2

PHA production by Rhodomicrobium spp. during photoautotrophic and photoheterotrophic growth. Intracellular PHA production as a percentage of cell dry weight based on total protein (cdw_prot) during photoautotrophic growth with the following electron and carbon sources: Hydrogen (H₂) gas and carbon dioxide (CO₂) (photohydrogenotrophy), ferrous chloride (FeCl₂) and CO₂ (photoferrotrophy), and sodium butyrate (photoheterotrophy). Cdw_prot was calculated as described in the Materials and Methods. N₂: Nitrogen‐fixing growth with dinitrogen gas as sole nitrogen source. NH₄Cl: Non‐nitrogen fixing growth with ammonium chloride as sole nitrogen source. p‐values are calculated between N₂ and NH₄Cl growth conditions. Statistically significant differences are indicated with horizontal bars and p‐values.

FIGURE 3

PHA production by Rhodomicrobium spp. during photoelectrotrophy. (A) Intracellular PHA production as a percentage of cell dry weight based on total protein (cdw_prot) during photoelectrotrophic growth using a carbon felt cathode poised at +100 mV versus standard hydrogen electrode. Cdw_prot was calculated as described in the Materials and Methods. p‐values are calculated between N₂ and NH₄Cl growth conditions. Statistically significant differences are indicated with horizontal bars and p‐values. (B) Current uptake by R. vannielii and R. udaipurense in microbial electrosynthesis reactors. N = 3 for biological MES reactors. N = 2 for abiotic controls. N₂: Nitrogen‐fixing growth with dinitrogen gas as sole nitrogen source. NH₄Cl: Non‐nitrogen fixing growth with ammonium chloride as sole nitrogen source.

FIGURE 4

Carbon and electron yields compared to PHA production. (A) PHA carbon yield (%) and (B) PHA electron yield (%) vs. PHA production as a percentage of cell dry weight (cdw_prot). Samples have been grouped by bisecting the horizontal and vertical axes at one‐half the maximum x and y values, respectively. Trendline and corresponding R ² value reflects all samples. Cdw_prot was calculated as described in the Materials and Methods. Blue, Rv: R. vannielii. Orange, Ru: R. udaipurense. B: Butyrate. H₂: Hydrogen electron donor. Fe: FeCl₂. E: Poised electrode. Triangles, N₂: Nitrogen gas. Circles, NH₄: Ammonium chloride.

FIGURE 5

Rhodomicrobium spp. and TIE‐1 carbon and electron yields compared to normalized PHA production. (A) PHA carbon yield (%) and (B) PHA electron yield (%) vs. PHA production per litre normalized to calculated cell numbers. Samples have been grouped by bisecting the horizontal and vertical axes at one‐half the maximum x and y values, respectively. Black: R. palustris TIE‐1 (Ranaivoarisoa et al., 2019). Blue: R. vannielii. Orange: R. udaipurense. Triangles: Nitrogen gas. Circles: Ammonium chloride.