Real-time monitoring of biomass during Escherichia coli high-cell-density cultivations by in-line photon density wave spectroscopy.

An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µs ' with CDW was observed in the range of 5-69 g L-1 (R2 = 0.98). The growth rates calculated based on µs ' were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µs ' as a function of the absorption coefficient µa allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.

Continuous feeding strategy for polyhydroxyalkanoate production from solid waste animal fat at laboratory- and pilot-scale.

Bioconversion of waste animal fat (WAF) to polyhydroxyalkanoates (PHAs) is an approach to lower the production costs of these plastic alternatives. However, the solid nature of WAF requires a tailor-made process development. In this study, a double-jacket feeding system was built to thermally liquefy the WAF to employ a continuous feeding strategy. During laboratory-scale cultivations with Ralstonia eutropha Re2058/pCB113, 70% more PHA (45 gPHA L-1 ) and a 75% higher space-time yield (0.63 gPHA L-1  h-1 ) were achieved compared to previously reported fermentations with solid WAF. During the development process, growth and PHA formation were monitored in real-time by in-line photon density wave spectroscopy. The process robustness was further evaluated during scale-down fermentations employing an oscillating aeration, which did not alter the PHA yield although cells encountered periods of oxygen limitation. Flow cytometry with propidium iodide staining showed that more than two-thirds of the cells were viable at the end of the cultivation and viability was even little higher in the scale-down cultivations. Application of this feeding system at 150-L pilot-scale cultivation yielded in 31.5 gPHA L-1 , which is a promising result for the further scale-up to industrial scale.

Polyhydroxyalkanoate production from animal by-products: Development of a pneumatic feeding system for solid fat/protein-emulsions.

Fat-containing animal by-product streams are locally available in large quantities. Depending on their quality, they can be inexpensive substrates for biotechnological processes. To accelerate industrial polyhydroxyalkanoate (PHA) bioplastic production, the development of efficient bioprocesses that are based on animal by-product streams is a promising approach to reduce overall production costs. However, the solid nature of animal by-product streams requires a tailor-made process development. In this study, a fat/protein-emulsion (FPE), which is a by-product stream from industrial-scale pharmaceutical heparin production and of which several hundred tons are available annually, was evaluated for PHA production with Ralstonia eutropha. The FPE was used as the sole source of carbon and nitrogen in shake flask and bioreactor cultivations. A tailored pneumatic feeding system was built for laboratory bioreactors to facilitate fed-batch cultivations with the solid FPE. The process yielded up to 51 g L-1 cell dry weight containing 71 wt% PHA with a space-time yield of 0.6 gPHA L-1  h-1 without using any carbon or nitrogen sources other than FPE. The presented approach highlights the potential of animal by-product stream valorization into PHA and contributes to a transition towards a circular bioeconomy.

Native feedstock options for the polyhydroxyalkanoate industry in Europe: A review.

The United Nations defined 17 Sustainable Development Goals (SDGs) in 2016 and agreed on fighting to confront the climate change and protecting the oceans and forests. Subsequently, the sustainable production of bioplastics is gradually gaining reputation and significance. With the usage of bioplastics such as biodegradable polyhydroxyalkanoates (PHAs) various SDGs would be tackled, but costs remain a crucial factor for competing against fossil-based plastics. Appropriate local feedstock selection can help to reduce the production costs and minimize transportation routes. In this work, four feedstock generations are introduced and respective conversion strategies to PHA are presented. Whilst the focus is on mapping the abundances of feedstocks and potential PHA production capacities in Europe, utilization of animal by-product streams is also highlighted as a rather unconventional but highly abundant feedstock for PHA production.

Untargeted metabolomics analysis of Ralstonia eutropha during plant oil cultivations reveals the presence of a fucose salvage pathway.

Process engineering of biotechnological productions can benefit greatly from comprehensive analysis of microbial physiology and metabolism. Ralstonia eutropha (syn. Cupriavidus necator) is one of the best studied organisms for the synthesis of biodegradable polyhydroxyalkanoate (PHA). A comprehensive metabolomic study during bioreactor cultivations with the wild-type (H16) and an engineered (Re2058/pCB113) R. eutropha strain for short- and or medium-chain-length PHA synthesis has been carried out. PHA production from plant oil was triggered through nitrogen limitation. Sample quenching allowed to conserve the metabolic states of the cells for subsequent untargeted metabolomic analysis, which consisted of GC-MS and LC-MS analysis. Multivariate data analysis resulted in identification of significant changes in concentrations of oxidative stress-related metabolites and a subsequent accumulation of antioxidative compounds. Moreover, metabolites involved in the de novo synthesis of GDP-L-fucose as well as the fucose salvage pathway were identified. The related formation of fucose-containing exopolysaccharides potentially supports the emulsion-based growth of R. eutropha on plant oils.

High-cell-density fed-batch cultivations of Vibrio natriegens.

OBJECTIVES: With generation times of less than 10 min under optimal conditions, the halophilic Vibrio natriegens is the fastest growing non-pathogenic bacterium isolated so far. The availability of the full genome and genetic engineering tools and its ability to utilize a wide range of carbon sources make V. natriegens an attractive host for biotechnological production processes. However, high-cell-density cultivations, which are desired at industrial-scale have not been described so far. RESULTS: In this study we report fed-batch cultivations of V. natriegens in deep-well plates and lab-scale bioreactor cultivations at different temperatures in mineral salt medium (MSM). Upon switching from exponential glucose to constant glucose-feeding cell death was induced. Initial NaCl concentrations of 15-18 g L-1 and a temperature reduction from 37 to 30 °C had a positive effect on cell growth. The maximal growth rate in MSM with glucose was 1.36 h-1 with a specific oxygen uptake rate of 22 mmol gCDW-1 h-1. High biomass yields of up to 55 g L-1 after only 12 h were reached. CONCLUSIONS: The shown fed-batch strategies demonstrate the potential of V. natriegens as a strong producer in industrial biotechnology.

Low-quality animal by-product streams for the production of PHA-biopolymers: fats, fat/protein-emulsions and materials with high ash content as low-cost feedstocks.

OBJECTIVE: The rapid accumulation of crude-oil based plastics in the environment is posing a fundamental threat to the future of mankind. The biodegradable and bio-based polyhydroxyalkanoates (PHAs) can replace conventional plastics, however, their current production costs are not competitive and therefore prohibiting PHAs from fulfilling their potential. RESULTS: Different low-quality animal by-products, which were separated by thermal hydrolysis into a fat-, fat/protein-emulsion- and mineral-fat-mixture- (material with high ash content) phase, were successfully screened as carbon sources for the production of PHA. Thereby, Ralstonia eutropha Re2058/pCB113 accumulated the short- and medium-chain-length copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)]. Up to 90 wt% PHA per cell dry weight with HHx-contents of 12-26 mol% were produced in shake flask cultivations. CONCLUSION: In future, the PHA production cost could be lowered by using the described animal by-product streams as feedstock.

Recovery of the PHA Copolymer P(HB-co-HHx) With Non-halogenated Solvents: Influences on Molecular Weight and HHx-Content.

Biodegradable and biocompatible polyhydroxyalkanoates (PHAs) are promising alternatives to conventional plastics. Based on the chain length of their monomers they are classified as short chain length (scl-) or medium chain length (mcl-) PHA polymers. The type of monomers, the composition and the molecular weight (MW) define the polymer properties. To accelerate the use of PHA as a bulk material, the downstream associated costs need to be minimized. This study focuses on the evaluation of non-halogenated solvents, especially acetone as a scl-PHA non-solvent, for the recovery of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) - P(HB-co-HHx) - with an mcl-HHx content >15 mol% and a MW average (M w) < 2 × 105 Da. Solvents and precipitants were chosen regarding zeotrope formation, boiling point differences, and toxicity. Non-halogenated solvent-precipitant pairs were evaluated regarding the MW characteristics (MWCs) of the extracted polymer. Acetone and 2-propanol as a low toxic and zeotropic solvent-precipitant pair was evaluated at different extraction temperatures and multiple extraction times. The extraction process was further evaluated by using impure acetone for the extraction and implementing a multi-stage extraction process. Additionally, P(HB-co-HHx) extracted with three different solvents was characterized by 1H and 13C-APT NMR. The screening of precipitants resulted in a negative influence on the MWCs by ethanol precipitation for extractions with acetone and ethyl acetate, respectively. It was observed, that extractions with acetone at 70°C extracted a higher fraction of PHA from the cells compared to extractions at RT, but the M w was decreased by 9% in average. Acetone with a 2-propanol fraction of up to 30% was still able to extract the polymer 95% as efficient as pure acetone. Additionally, when acetone and ethyl acetate were used in a multi-stage extraction process, a two-stage process was sufficient to extract 98-99% of the polymer from the cells. 1H and 13C-APT NMR analysis confirmed the monomer fraction and structure of the extracted polymers and revealed a random copolymer structure. The presented strategy can be further developed to an ecological and economically feasible PHA downstream process and thus contributes to the commercialization of low-cost PHAs.

In-Line Monitoring of Polyhydroxyalkanoate (PHA) Production during High-Cell-Density Plant Oil Cultivations Using Photon Density Wave Spectroscopy.

Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. Plant oils are excellent carbon sources for a cost-effective PHA production, due to their high carbon content, large availability, and comparatively low prices. Additionally, efficient process development and control is required for competitive PHA production, which can be facilitated by on-line or in-line monitoring devices. To this end, we have evaluated photon density wave (PDW) spectroscopy as a new process analytical technology for Ralstonia eutropha (Cupriavidus necator) H16 plant oil cultivations producing polyhydroxybutyrate (PHB) as an intracellular polymer. PDW spectroscopy was used for in-line recording of the reduced scattering coefficient µs' and the absorption coefficient µa at 638 nm. A correlation of µs' with the cell dry weight (CDW) and µa with the residual cell dry weight (RCDW) was observed during growth, PHB accumulation, and PHB degradation phases in batch and pulse feed cultivations. The correlation was used to predict CDW, RCDW, and PHB formation in a high-cell-density fed-batch cultivation with a productivity of 1.65 gPHB·L-1·h-1 and a final biomass of 106 g·L-1 containing 73 wt% PHB. The new method applied in this study allows in-line monitoring of CDW, RCDW, and PHA formation.