Sebastian L. Riedel

Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

ABSTRACT 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 worlds 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.

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 19mol% 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). Biotechnol. Bioeng. 2012;109: 74–83.

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.

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. Biotechnol. Bioeng. 2013; 110: 461–470.

Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here. An emulsification strategy, without any mechanical or chemical pre-treatment, was developed to increase the bioavailability of solid, poorly-consumable fats. Wild type R. eutropha strain H16 produced 79-82% (w/w) polyhydroxybutyrate (PHB) per cell dry weight (CDW) when cultivated on various fats. A productivity of 0.3g PHB/(L × h) with a total PHB production of 24 g/L was achieved using tallow as carbon source. Using a recombinant strain of R. eutropha that produces poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], 49-72% (w/w) of PHA per CDW with a HHx content of 16-27 mol% were produced in shaking flask experiments. The recombinant strain was grown on waste animal fat of the lowest quality available at lab fermenter scale, resulting in 45 g/L CDW with 60% (w/w) PHA per CDW and a productivity of 0.4 g PHA/(L × h). The final HHx content of the polymer was 19 mol%. The use of low quality waste animal fats as an inexpensive carbon feedstock exhibits a high potential to accelerate the commercialization of PHAs.

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 (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-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.

An integrative study on biologically recovered polyhydroxyalkanoates (PHAs) and simultaneous assessment of gut microbiome in yellow mealworm

Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that - purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome.