Fed-Batch Cultivations of Rhodospirillum rubrum Under Multiple Nutrient-Limited Growth Conditions on Syngas as a Novel Option to Produce Poly(3-Hydroxybutyrate) (PHB).

Syngas from gasified organic waste materials is a promising feedstock for the biotechnological synthesis of the bioplastic poly([R]-3-hydroxybutyrate) (PHB) with Rhodospirillum rubrum. In a first approach, growth studies were carried out with this strain in gas-tight serum vials. When syngas (40% CO, 40% H2, 10% CO2, and 10% N2 v/v) was diluted with N2 to 60%, a 4-fold higher biomass production was detected compared to samples grown on 100% syngas, thus indicating a growth inhibitory effect. The best performing syngas-mixture was then used for C-, C,N-, and C,P-limited fed-batch fermentations in a bioreactor with continuous syngas and acetate supply. It was found that C,P-limited PHB productivity was 5 times higher than for only C-limited growth and reached a maximal PHB content of 30% w/w. Surprisingly, growth and PHB production stopped when N, as a second nutrient, became growth-limiting. Finally, it was concluded that a minimal supply of 0.2 g CO g-1 biomass h-1 has to be guaranteed in order to cover the cellular maintenance energy.

The Bistable Behaviour of Pseudomonas putida KT2440 during PHA Depolymerization under Carbon Limitation.

Poly(hydroxyalkanoates) (PHAs) are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor PHA accumulation, whereas carbon limitation triggers PHA depolymerization when all other essential nutrients are present in excess. We studied the population dynamics of Pseudomonas putida KT2440 at the single cell level during different physiological conditions, favoring first PHA polymerization during growth on octanoic acid, and then PHA depolymerization during carbon limitation. PHAs accumulate intracellularly in granules, and were proposed to separate preferentially together with nucleic acids, leading to two daughter cells containing approximately equal amounts of PHA. However, we could show that such P. putida KT2440 cells show bistable behavior when exposed to carbon limitation, and separate into two subpopulations: one with high and one with low PHA. This suggests an asymmetric PHA distribution during cell division under carbon limitation, which has a significant influence on our understanding of PHA mobilization.

Tailor-made PAT platform for safe syngas fermentations in batch, fed-batch and chemostat mode with Rhodospirillum rubrum.

Recently, syngas has gained significant interest as renewable and sustainable feedstock, in particular for the biotechnological production of poly([R]-3-hydroxybutyrate) (PHB). PHB is a biodegradable, biocompatible polyester produced by some bacteria growing on the principal component of syngas, CO. However, working with syngas is challenging because of the CO toxicity and the explosion danger of H2 , another main component of syngas. In addition, the bioprocess control needs specific monitoring tools and analytical methods that differ from standard fermentations. Here, we present a syngas fermentation platform with a focus on safety installations and process analytical technology (PAT) that serves as a basis to assess the physiology of the PHB-producing bacterium Rhodospirillum rubrum. The platform includes (i) off-gas analysis with an online quadrupole mass spectrometer to measure CO consumption and production rates of H2 and CO2 , (ii) an at-line flow cytometer to determine the total cell count and the intracellular PHB content and (iii) different online sensors, notably a redox sensor that is important to confirm that the culture conditions are suitable for the CO metabolization of R. rubrum. Furthermore, we present as first applications of the platform a fed-batch and a chemostat process with R. rubrum for PHB production from syngas.

Robust at-line quantification of poly(3-hydroxyalkanoate) biosynthesis by flow cytometry using a BODIPY 493/503-SYTO 62 double-staining.

Poly(3-hydroxyalkanoates) (PHAs) are bio-based and biodegradable polyesters which have been considered as a promising alternative to petrol-based plastics. Their bacterial production is a dynamic process in which intracellular polymerization and depolymerization are closely linked and depend on the availability of carbon substrates and other nutrients. These dynamics require a fast and quantitative method to determine the optimal harvest-time of PHA containing cells or to adjust carbon supply. In principle, flow cytometry (FCM) is an ideal tool that suits these requirements and, in addition, provides data on the PHA content of different cell populations. However, FCM-based PHA quantification methods have often relied on laborious sample preparation including washing steps and long incubation times. Here, we introduce a fast method based on double-staining using BODIPY 493/503 for PHA staining and SYTO 62 for DNA that allows acquiring reliable fluorescence and cell count data in <10min. Finally, fed-batch experiments with Pseudomonas putida KT2440 and Rhodospirillum rubrum S1 revealed that the method was robust and independent of the strain and type of PHA (medium-chain-length [mcl-] and short-chain-length [scl-] PHA, respectively). Interestingly, the specific PHA fluorescence was in case of mcl-PHA larger than for scl-PHA, probably reflecting the different material properties (e.g., specific density, hydrophilicity and crystallinity).