Enrichment of mixed methanotrophic cultures producing polyhydroxyalkanoates (PHAs) from various environmental sources.

Methanotrophic bacteria can use atmospheric methane (CH4) as a sole carbon source for the growth and production of polyhydroxyalkanoates (PHA). The development of CH4 bioconversion processes relies heavily on the selection of an efficient methanotrophic culture. This research assessed the effect of selected growth conditions, such as nitrogen sources on the enrichment of methanotrophic cultures from various environments for PHA accumulation. Nitrate-based medium favoured the culture growth and selection for PHA-producing methanotrophic cultures with Methylocystis sp. as a major genus and accumulation of up to 27 % polyhydroxybutyrate (PHB) in the biomass. Three PHB-producing cultures: enriched from waste activated sludge (AS), peat bog soil (PB) and landfill biocover soil (LB) were then tested for their ability to produce PHA copolymer at different CH4:O2 ratios. All enriched cultures were able to utilise valeric acid as a cosubstrate for the accumulation of PHA with a 3-hydroxyvaleric (3HV) fraction of 21-41 mol% depending on the inoculum source and CH4 concentration. The process performance of selected cultures was evaluated and compared to the culture of reference strain Methylocystis hirsuta DSM 18500. All mixed cultures irrespective of their inoculum source had similar levels of 3HV fraction in the PHA (38 ± 2 mol%). The highest poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production was observed for AS culture at 10 % CH4 with an accumulation of 27 ± 3 % of dry cell weight (DCW), 3HV fraction of 39 ± 2 mol% and yield of 0.42 ± 0.02 g-PHA/g-substrate.

Simultaneous medium chain carboxylic acids and 1,3-propanediol production in a bioaugmented lactate-based chain elongation induced with glycerol.

The objective was to investigate the impact of the bioaugmentation on chain elongation process using glycerol, lactate and lactose as substrates in an open culture fermentation. In the batch trials the highest selectivity for chain elongation product, i.e. caproate, was observed in trials inoculated with co-culture of Megasphaera elsdenii and Eubacterium limosum grown on glycerol (28.6%), and in non-bioaugmented open culture run on lactose + lactate (14.8%). The results showed that E. limosum, out of two bioaugmented strains, was able to survive in the open culture. A continuous open culture fermentation of glycerol led to caproate and 1,3-propanediol (1,3-PDO) formation, while lactate addition led to 1,3-PDO and short chain carboxylates production. Moving the process into batch mode triggered even-carbon chain elongation. Presence of E. limosum promoted odd-carbon chain elongation and valerate production. Imaging flow cytometry combined with machine learning enabled the discrimination of Eubacterium cells from other microbial strains during the process.

Influence of lactate to acetate ratio on biological production of medium chain carboxylates via open culture fermentation.

Waste valorisation via biological production of widely used in the industry medium chain carboxylates (MCCs) via open culture fermentation (OCF) could be a promising alternative to the commonly used anaerobic digestion. Lactate-rich waste streams are considered as valuable substrates for carboxylate chain elongation (CE), however, there are certain limitations related to the production efficiency. Acetate produced and accumulated in the acetogenesis plays an important role in CE, i.e. acetate is elongated to butyrate and then to caproate which is most popular MCC. Henceforth, it was investigated whether the ratio of lactate to acetate (L:A) affected carboxylates yields and product distribution in the lactate-based CE in OCF. The tested L:A ratios influenced carboxylates selectivity in batch trials. In the ones with lactate as the sole carbon source, propionate production was predominant but when a higher relative acetate concentration was used, the production of butyrate and CE to caproate was favored. The co-utilization of lactate and acetate in a continuous process increased the production of butyrate and caproate compared to the phase with lactate as the sole carbon source, however, controlling the relative concentration of lactate and acetate during co-utilization was not an effective strategy for increasing caproate production. 16S rRNA gene amplicon reads mapping to Caproiciproducens were the most abundant in samples collected throughout the continuous processes regardless of the L:A ratios.

Effect of external acetate on lactate-based carboxylate platform: Shifted lactate overloading limit and hydrogen co-production.

Chain elongation is an anaerobic biotechnological process that converts short chain carboxylates and an electron donor (e.g. ethanol, lactate) into more valuable medium chain carboxylates. Caproate production in lactate-based chain elongation is gaining popularity, however, the relation between lactate (electron donor) and acetate (electron acceptor) has not yet been fully elucidated. Herein, for the first time, the effect of an external acetate on the lactate-based chain elongation in a continuously-fed bioreactor was tested to verify how the external acetate would affect the product spectrum, gas production, as well as stability and efficiency of carboxylates production. Periodic fluctuations in caproate production were observed in bioreactor continuously fed with lactate as a sole carbon source due to the lack of an electron acceptor (acetate) and low chain elongation performance. The recovery of stable caproate production (68.9 ± 2.2 mmol C/L/d), total lactate consumption, and high hydrogen co-production (748 ± 76 mLH2/d) was observed as an effect of the addition of an external acetate. The lactate conversion with the external acetate in the second bioreactor ensured stable and dominant caproate production from the beginning of the process. Moreover, despite the continuous lactate overloading in the process with external acetate, stable caproate production was achieved (71.7 ± 2.4 mmol C/L/d) and previously unobserved hydrogen production occurred (213 ± 30 mLH2/d). Thus, external electron acceptor addition (i.e. acetate) was proposed as an effective method for stable lactate-based caproate production. Microbiological analysis showed the dominance of microbes closely related to Ruminococcaceae bacterium CPB6 and Acinetobacter throughout the process. Co-occurrence networks based on taxon abundances and process parameters revealed microbial sub-networks responding to lactate concentrations.

Mass Spectrometry Reveals Complexing Properties of Modified PNP-Lariat Ether Containing Benzyl Derivative of (S)-Prolinamine.

In the investigation presented here the synthesis of new lariat ether derivative obtained from the modification of tetrapyrrolidinyl-PNP-crown ether macrocycle is described. The polyheterotopic molecular coreceptor consisted of the replacement of chlorine atoms with an optically active (S)-(1-benzylpyrrolidin-2-yl) methanamine. The structure was confirmed by using elemental analysis, mass spectrometry, and NMR spectroscopy. This work covers results concerning the complexing properties of the new ligand towards Ag+, Cu2+, Co2+, Ni2+, and Zn2+ ions. The formation of non-covalent complexes of 1:1 stoichiometry with the Cu2+, Co2+, Ni2+, and Zn2+ ions have been confirmed by mass spectrometry. Due to the previous work and application possibilities, a large emphasis was put on the investigation of the complexation ability of lariat ether with silver (I) cation to determine stability constants by direct potentiometric method. In this case, the formation of four different forms of complexes AgL, Ag2L, Ag3L, and Ag4L has been proved. The observed unusual binding through the nitrogen atoms from the exocyclic substituents may provide the structural unit to build a new coordination polymers.

Tandem mass spectroscopy as a tool for investigation of complexes of PNP-lariat ether derivative with metal ions.

The novel PNP-lariat ether L with cyclotriphosphazene ring incorporated in the macrocyclic structure was synthesized and checked by the electrospray mass spectrometry (ESI-MS) method for the ability to bind different types of ions Ag+ , Ca2+ , Cd2+ , Cu2+ , and Pb2+ . Furthermore, the stability constants of the abovementioned ion complexes with the investigated ligand have been determined by direct and competitive potentiometric methods. To evaluate the stability of various complex types and to confirm the way of metal cation binding, the tandem mass spectra of the investigated ligand and its complexes were taken. As a result, we obtained quite a good relationship between the number and main types of complex species observed in ESI-MS experiments and the forms of complexes for which the stabilization constants were determined by potentiometric methods. Moreover, we also concluded that in case of big discrepancies of stability constants, ESI-MS experiments could provide information about the most stable form of the complexes, but they fail when the differences between the strength of the coordination binding are slightly different.