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.

Bioprocess development to produce a hyperthermostable S-methyl-5'-thioadenosine phosphorylase in Escherichia coli.

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5'-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1-90.1 g L-1 were observed together with an overproduction of ApMTAP in a 1.9%-3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg-1 (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.

Workflow for shake flask and plate cultivations with fats for polyhydroxyalkanoate bioproduction.

Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L-1 of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L-1 CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production. KEY POINTS: • Evaluation of shake flask designs for cultivating with hydrophobic raw materials • Development of a workflow for microwell plate cultivations with hydrophobic raw materials • Production of polyhydroxyalkanoate in small scale experiments from waste animal fat.

Implementation of a high cell density fed-batch for heterologous production of active [NiFe]-hydrogenase in Escherichia coli bioreactor cultivations.

BACKGROUND: O2-tolerant [NiFe]-hydrogenases offer tremendous potential for applications in H2-based technology. As these metalloenzymes undergo a complicated maturation process that requires a dedicated set of multiple accessory proteins, their heterologous production is challenging, thus hindering their fundamental understanding and the development of related applications. Taking these challenges into account, we selected the comparably simple regulatory [NiFe]-hydrogenase (RH) from Cupriavidus necator as a model for the development of bioprocesses for heterologous [NiFe]-hydrogenase production. We already reported recently on the high-yield production of catalytically active RH in Escherichia coli by optimizing the culture conditions in shake flasks. RESULTS: In this study, we further increase the RH yield and ensure consistent product quality by a rationally designed high cell density fed-batch cultivation process. Overall, the bioreactor cultivations resulted in ˃130 mg L-1 of catalytically active RH which is a more than 100-fold increase compared to other RH laboratory bioreactor scale processes with C. necator. Furthermore, the process shows high reproducibility of the previously selected optimized conditions and high productivity. CONCLUSIONS: This work provides a good opportunity to readily supply such difficult-to-express complex metalloproteins economically and at high concentrations to meet the demand in basic and applied studies.

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.

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.

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products.

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (ß-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from ß-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.

High-cell-density fed-batch strategy to manufacture tailor-made P(HB-co-HHx) by engineered Ralstonia eutropha at laboratory scale and pilot scale.

The transition towards a sustainable bioeconomy requires the development of highly efficient bioprocesses that enable the production of bulk materials at a competitive price. This is particularly crucial for driving the commercialization of polyhydroxyalkanoates (PHAs) as biobased and biodegradable plastic substitutes. Among these, the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)) shows excellent material properties that can be tuned by regulating its monomer composition. In this study, we developed a high-cell-density fed-batch strategy using mixtures of fructose and canola oil to modulate the molar composition of P(HB-co-HHx) produced by Ralstonia eutropha Re2058/pCB113 at 1-L laboratory scale up to 150-L pilot scale. With cell densities >100 g L-1 containing 70-80 wt% of PHA with tunable HHx contents in the range of 9.0-14.6 mol% and productivities of up to 1.5 g L-1 h-1, we demonstrate the tailor-made production of P(HB-co-HHx) at an industrially relevant scale. Ultimately, this strategy enables the production of PHA bioplastics with defined material properties on the kilogram scale, which is often required for testing and adapting manufacturing processes to target diverse applications.

Microbially synthesized poly(hydroxybutyrate-co-hydroxyhexanoate) with low to moderate hydroxyhexanoate content: Properties and applications.

Plastic pollution is the biggest environmental concern of our time. Breakdown products like micro- and nano-plastics inevitably enter the food chain and pose unprecedented health risks. In this scenario, bio-based and biodegradable plastic alternatives have been given a momentum aiming to bridge a transition towards a more sustainable future. Polyhydroxyalkanoates (PHAs) are one of the few thermoplastic polymers synthesized 100 % via biotechnological routes which fully biodegrade in common natural environments. Poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] is a PHA copolymer with great potential for the commodity polymers industry, as its mechanical properties can be tailored through fine-tuning of its molar HHx content. We have recently developed a strategy that enables for reliable tailoring of the monomer content of P(HB-co-HHx). Nevertheless, there is often a lack of comprehensive investigation of the material properties of PHAs to evaluate whether they actually mimic the functionalities of conventional plastics. We present a detailed study of P(HB-co-HHx) copolymers with low to moderate hydroxyhexanoate content to understand how the HHx monomer content influences the thermal and mechanical properties and to link those to their abiotic degradation. By increasing the HHx fractions in the range of 2 - 14 mol%, we impart an extension of the processing window and application range as the melting temperature (Tm) and glass temperature (Tg) of the copolymers decrease from Tm 165 °C to 126 °C, Tg 4 °C to -5.9 °C, accompanied by reduced crystallinity from 54 % to 20 %. Elongation at break was increased from 5.7 % up to 703 % at 14 mol% HHx content, confirming that the range examined was sufficiently large to obtain ductile and brittle copolymers, while tensile strength was maintained throughout the studied range. Finally, accelerated abiotic degradation was shown to be slowed down with an increasing HHx fraction decreasing from 70 % to 55 % in 12 h.

Recovery of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Ralstonia eutropha cultures with non-halogenated solvents.

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA-based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non-halogenated solvents. Several non-halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17-30 mol%) remained completely in solution, while polymer with a lower HHx content (11-16 mol%) formed a gel-like phase. All PHA in solution could be precipitated by addition of threefold volumes of n-hexane or n-heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non-halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA.

Tailoring the HHx monomer content of P(HB-co-HHx) by flexible substrate compositions: scale-up from deep-well-plates to laboratory bioreactor cultivations.

The enhanced material properties exhibited by the microbially synthetized polyhydroxyalkanoate (PHA) copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] evidence that this naturally biodegrading biopolymer could replace various functionalities of established petrochemical plastics. In fact, the thermal processability, toughness and degradation rate of P(HB-co-HHx) can be tuned by modulating its HHx molar content enabling to manufacture polymers à-la-carte. We have developed a simple batch strategy to precisely control the HHx content of P(HB-co-HHx) to obtain tailor-made PHAs with defined properties. By adjusting the ratio of fructose to canola oil as substrates for the cultivation of recombinant Ralstonia eutropha Re2058/pCB113, the molar fraction of HHx in P(HB-co-HHx) could be adjusted within a range of 2-17 mol% without compromising polymer yields. The chosen strategy proved to be robust from the mL-scale in deep-well-plates to 1-L batch bioreactor cultivations.

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.

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by Ralstonia eutropha in high cell density palm oil fermentations.

Improved production costs will accelerate commercialization of polyhydroxyalkanoate (PHA) polymer and PHA-based products. Plant oils are considered favorable feedstocks, due to their high carbon content and relatively low price compared to sugars and other refined carbon feedstocks. Different PHA production strategies were compared using a recombinant strain of Ralstonia eutropha that produces high amounts of P(HB-co-HHx) when grown on plant oils. This R. eutropha strain was grown to high cell densities using batch, extended batch, and fed batch fermentation strategies, in which PHA accumulation was triggered by nitrogen limitation. While extended batch culture produced more biomass and PHA than batch culture, fed batch cultivation was shown to produce the highest levels of biomass and PHA. The highest titer achieved was over 139 g/L cell dry weight (CDW) of biomass with 74% of CDW as PHA containing 19 mol% HHx. Our data suggest that the fermentation process is scalable with a space time yield (STY) better than 1 g PHA/L/h. The achieved biomass concentration and PHA yield are among the highest reported for the fermentation of recombinant R. eutropha strains producing P(HB-co-HHx).

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.

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains.

The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.

Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium.

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.

Substrate-Flexible Two-Stage Fed-Batch Cultivations for the Production of the PHA Copolymer P(HB-co-HHx) With Cupriavidus necator Re2058/pCB113.

Recent studies of the impact and dimension of plastic pollution have drawn the attention to finding more sustainable alternatives to fossil-based plastics. Microbially produced polyhydroxyalkanoates (PHAs) biopolymers are strong candidates to replace conventional plastic materials, due to their true biodegradability and versatile properties. However, widespread use of these polymers is still hindered by their high cost of production. In the present study, we target high yields of the PHA copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] using a substrate-flexible two-stage fed-batch approach for the cultivation of the recombinant Cupriavidus necator strain Re2058/pCB113. A more substrate-flexible process allows to cope with constant price fluctuations and discontinuous supply of feedstocks on the market. Utilizing fructose for biomass accumulation and rapeseed oil for polymer production resulted in a final biomass concentration of 124 g L-1 with a polymer content of 86 wt% holding 17 mol% of HHx. Productivities were further optimized by operating the biomass accumulation stage in a "drain and fill" modus where 10% of the culture broth was recycled for semi-continuous biomass accumulation, after transferring 90% to a second bioreactor for PHA production. This strategy succeeded in shortening process times rising productivity yields to ∼1.45 g L-1 h-1.

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.

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.