Influent carbon to phosphorus ratio drives the selection of PHA-storing organisms in a single CSTR

Abstract

Enriching a biomass with a high fraction of polyhydroxyalkanoate-storing organisms (PHA-storers) represents an essential step in the production of PHAs (bioplastics) from municipal wastewater using mixed microbial cultures. A major challenge is however to create selective growth conditions that are favourable to PHA-storers. Our study thus investigates to what extent the influent COD to phosphorus (COD:P) ratio can be used as a tool for the robust selection of PHA-storers in a single continuous-flow stirred-tank reactor (CSTR). Therefore, we operated five CSTRs in parallel, fed with synthetic wastewater (50% acetate - 50% propionate) with different COD:P ratios (200-1000 gCOD gP^-1), and performed a detailed analysis of the microbial communities over long-term (30-70 solid retention times). Our study demonstrates that efficient and robust selection of PHA-storers can be achieved in a single CSTR at high influent COD:P ratios. The selective advantage for PHA-storers increases with the influent COD:P ratio, but only if growth conditions remain limited by both C-substrate and P. In contrast, selection performance deteriorates when COD:P ratios are too high and growth conditions are limited by P only. At an optimal COD:P ratio of 800 gCOD gP^-1, a stable microbial community consisting of >90% PHA-storers and dominated by Pannonibacter sp. was selected in the long-term. Finally, our results suggest that high COD:P ratios provide a selective advantage to microorganisms with low cellular P requirements, explaining why different PHA-storers (i.e., Xanthobacter sp. vs. Pannonibacter sp.) were selected depending on the influent COD:P ratio (i.e., 200 vs. 800 gCOD gP^-1). Overall, our results provide relevant insights for the development of a new approach for selecting PHA-storers, based on the use of a single CSTR and control of the influent COD:P ratio.

Keywords: Biomass PHA content; Cellular phosphorus requirements; Dual carbon and phosphorus limitation; Growth conditions; Long-term selection; Polyhydroxyalkanoates (PHAs).

Graphical abstract

Fig. 1

Microbial community composition over time in the different reactors at the taxonomic level of the genus. Genera are grouped at the order level. The group “Others” contains all taxa with a mean relative abundance <1% and a maximum relative abundance <10% with respect to all samples. The names of the three most abundant taxa were highlighted in bold-red (Pannonibacter, Xanthobacter and Achromobacter).

Fig. 2

(A) Boxplot showing the relative abundance of PHA-storers in the selected microbial community during steady-state based on information gathered on the 19 most abundant genera (mean relative abundance of >2% in at least one of the reactors during steady-state) (see SI Table A7). The black stars in the boxes are the mean values. The symbols on top of the brackets indicate the statistical significance of the difference between mean values according to an Independent Samples t-test: ns (not significant, p>0.05), * (p<0.05), ** (p<0.01), *** (p<0.001) and **** (p<0.0001). (B) Principal Component Analysis (PCA) plot based on Hellinger transformed relative abundances of the bacterial taxa (genus level) present in the different reactors during steady-state. Samples (dots) appearing close to each other can be expected to be similar in terms microbial community composition. The dots corresponding to the onset of steady-state and the end of the experiment are labelled with the corresponding day. The ellipses cover the 90% confidence interval for each reactor, assuming a normal distribution. Thus, the ellipses are a visual indicator of the stability of the microbial community composition in the different reactors. PC3 (explaining 11% of the variance) vs PC1 and vs PC2 plots are provided in Supporting Information A (SI Fig. A7).

Fig. 3

Balloon plot showing the average relative abundance of the 19 most abundant genera (average relative abundance >2% in at least one of the reactors during steady-state) in the COD:P 200-1000 reactors. The taxa are divided into three groups: the known PHA-storers (green), the putative PHA-storers (blue) and the others (red). The corresponding standard deviations and the literature references, based on which the genera were classified, are shown in SI Table A7.

Fig. 4

Boxplots showing the biomass iP (A) and PHA content (B) in the different reactors during steady-state. The black stars are mean values. The symbols on top of the brackets indicate the statistical significance of the differences between mean values: ns (not significant, p>0.05), * (p<0.05), ** (p<0.01), *** (p<0.001) and **** (p<0.0001).

Fig. 5

C-substrate (COD) removal over time in the different reactors. The vertical dashed line indicates the onset of steady-state with respect to the microbial community composition. The horizontal dashed line highlights the 90% removal benchmark. The reason for the sudden drop in COD-removal between day 35 and day 40 in the COD:P 400 reactor is unknown, but not related to operational problems.

Fig. 6

Scatter-pie plot showing the biomass PHA content (in %COD) as a function of the biomass iP and the selection of PHA-storers. The plot represents only average values from the steady-state. The center of the pie-charts marks the intersection of the biomass PHA content and iP. The size of the pie-charts is proportional to the relative abundance of PHA-storers within the selected microbial community. The pie-charts themselves specify the average composition of the PHA-storer community at the genus level. The COD:P 1000 reactor is “whitened” to highlight the partial COD removal. The arrows are intended to help read the plot. Dark-teal arrow: Increasing the COD:P ratio (200−800 gCOD gP⁻¹) enhances the selection. Pink arrow: Increasing the COD:P ratio (800−1000 gCOD gP⁻¹) mitigates the selection.

Fig. 7

Conceptual summary of our results. Relative abundance of PHA-storers, biomass iP and PHA content, and COD-removal are represented on the y-axis in %. The reduction of the iP was expressed as the relative decrease of the biomass iP compared to the iP value measured in the COD:P 200 reactor. For each reactor, only average values are shown. The blue background indicates the range of COD:P ratios resulting in dual C-substrate and P-limited conditions, while the yellow background marks the beginning of the range of COD:P ratios leading to solely P-limited conditions.