Polyhydroxyalkanoates (PHA) production from fermented crude glycerol: Study on the conversion of 1,3-propanediol to PHA in mixed microbial consortia

Abstract

Crude glycerol, a by-product from the biodiesel industry, can be converted by mixed microbial consortia into 1,3-propanediol (1,3-PDO) and volatile fatty acids. In this study, further conversion of these main products into polyhydroxyalkanoates (PHA) was investigated with the focus on 1,3-PDO. Two different approaches for the enrichment of PHA accumulating microbial consortia using an aerobic dynamic feeding strategy were applied. With the first approach, where nitrogen was present during the whole cycle, no net production of PHA from 1,3-PDO was observed in the fermented effluent, not even in a nitrogen-limited PHA accumulation assay. Nevertheless, experiments in synthetic substrates revealed that the conversion of 1,3-PDO to PHA was possible under nitrogen limiting conditions. Thus, a different enrichment strategy was formulated where nitrogen was limited during the feast phase to stimulate the storage response. Nitrogen was still supplied during the famine phase. With the latter strategy, a net production of PHA from 1,3-PDO was observed at a yield of 0.24 C PHA/C 1,3-PDO. The overall yield from the fermented effluent was 0.42 C PHA/C substrate. Overall, the PHA yield from 1,3-PDO seemed to be limited, similarly to when using glycerol as a substrate, by a decarboxylation step and accumulation of other storage polymers such as glycogen, and possibly, lipid inclusions.

Polyhydroxyalkanoate (PHA) purification through dilute aqueous ammonia digestion at elevated temperatures

“…Chloroform extraction (65 °C 4 h) followed by methanol precipitation was performed as previously described …”

“…For all digestion methods, the total solids recovered were assessed by weighing pre‐tared tubes before and after the digestion and freeze‐drying. PHA purity (expressed as g PHA/100 g solids) was measured by gas chromatography (GC) after hydrochloric acid (HCl)‐mediated propanolysis in 1,2‐dichloroethane as previously described . Methyl‐3‐hydroxybutyrate (99%, J&K Scientific®) and methyl‐3‐hydroxyvalerate (≥98%, Sigma‐Aldrich®, St Louis, MO, USA) were used as standards for the calibration of 3‐hydroxybutyrate (3HB) and 3HV monomers, given that the standard described in the referenced work (P(3HB‐ co ‐3HV) with 12 mol% 3HV from Sigma‐Aldrich®) led to an average overestimation of 10% of the PHA purity (Supporting Information Appendix F).…”

“…Chloroform extraction (65°C 4 h) followed by methanol precipitation was performed as previously described. 22 Analyses on the material recovered after digestion experiments For all digestion methods, the total solids recovered were assessed by weighing pre-tared tubes before and after the digestion and freeze-drying. PHA purity (expressed as g PHA/100 g solids) was measured by gas chromatography (GC) after hydrochloric acid (HCl)-mediated propanolysis in 1,2-dichloroethane as previously described.…”

“…PHA purity (expressed as g PHA/100 g solids) was measured by gas chromatography (GC) after hydrochloric acid (HCl)-mediated propanolysis in 1,2-dichloroethane as previously described. 22 Methyl-3-hydroxybutyrate (99%, J&K Scientific®) and methyl-3-hydroxyvalerate (≥98%, Sigma-Aldrich®, St Louis, MO, USA) were used as standards for the calibration of 3-hydroxybutyrate (3HB) and 3HV monomers, given that the standard described in the referenced work (P(3HB-co-3HV) with 12 mol% 3HV from Sigma-Aldrich®) led to an average overestimation of 10% of the PHA purity (Supporting Information Appendix F). A secondary calibration curve was built with methyl-esters not submitted to the transesterification reaction dissolved in 1,2-dichloroethane, in order to take into account the extent of incomplete transesterification in the standard samples.…”

Effect of hydraulic retention time on the modelling and optimization of joint 1,3 PDO and BuA production from 2G glycerol in a chemostat process

“…In a previous study the authors have selected different MMC and tested their stability in continuous processes [6,7]. The overall idea was to develop a two-stage process in which glycerol fermentation would be coupled to the production of polyhydroxyalkanoates (PHA) through the selective consumption of butyric acid, while leaving intact the 1,3 PDO, thus facilitating its recovery [50] (or eventually through the conversion of 1,3 PDO and butyric acid to PHA [51]). Therefore, the joint production of 1,3 PDO and butyric acid is of particular interest for the full exploitation of the crude glycerol as a carbon source (in fact butyric acid is considered as a preferred substrate for PHA production [52]), leading to the production of two high-value compounds.…”