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Cell Retention as a Viable Strategy for PHA Production from Diluted VFAs with Bacillus megaterium.

ABSTRACT: The production of biodegradable and biocompatible materials such as polyhydroxyalkanoates (PHAs) from waste-derived volatile fatty acids (VFAs) is a promising approach towards implementing a circular bioeconomy. However, VFA solutions obtained via acidification of organic wastes are usually too diluted for direct use in standard batch or fed-batch processes. To overcome these constraints, this study introduces a cell recycle fed-batch system using Bacillus megaterium uyuni S29 for poly(3-hydroxybutyrate) (P3HB) production from acetic acid. The concentrations of dry cell weight (DCW), P3HB, acetate, as well as nitrogen as the limiting substrate component, were monitored during the process. The produced polymer was characterized in terms of molecular weight and thermal properties after extraction with hypochlorite. The results show that an indirect pH-stat feeding regime successfully kept the strain fed without prompting inhibition, resulting in a dry cell weight concentration of up to 19.05 g/L containing 70.21% PHA. After appropriate adaptations the presented process could contribute to an efficient and sustainable production of biopolymers.

SUBMITTER: Kacanski M

PROVIDER: S-EPMC8945770 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Cell Retention as a Viable Strategy for PHA Production from Diluted VFAs with <i>Bacillus megaterium</i>.

Kacanski Milos M Pucher Lukas L Peral Carlota C Dietrich Thomas T Neureiter Markus M

Bioengineering (Basel, Switzerland) 20220316 3

The production of biodegradable and biocompatible materials such as polyhydroxyalkanoates (PHAs) from waste-derived volatile fatty acids (VFAs) is a promising approach towards implementing a circular bioeconomy. However, VFA solutions obtained via acidification of organic wastes are usually too diluted for direct use in standard batch or fed-batch processes. To overcome these constraints, this study introduces a cell recycle fed-batch system using <i>Bacillus megaterium</i> uyuni S29 for poly(3- ...[more]

PMID: 35324811

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Project description:BackgroundSilk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry.ResultsIn this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures.ConclusionIt is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.

Project description:BACKGROUND: Vitamin B12 is a fascinating molecule which acts as a co-factor in the metabolism of many organisms, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production. The synthesis of vitamin B12 is limited to a few of bacteria and archaea. Therefore, industrial microbial fermentation is used to meet annual demands worldwide of vitamin B12 and as an alternative method to the chemical synthesis which requires at least 60 steps that is uneconomical. Bacillus megaterium is one of vitamin B12 producers and an ideal host for many biotechnology applications and being one of the best tools for the industrial production of several enzymes. Therefore, a two-step optimization strategy was established to produce high yield of vitamin B12 by B. megaterium through the provision of the production requirements and the suitable conditions for the biosynthesis of vitamin B12. RESULTS: We achieved the optimum conditions for the fermentation process of B. megaterium to produce high yield of vitamin B12 in a practical way based on statistical design and analysis which allowed vitamin B12 production to increase up to 759-fold (204.46 ?g/l) as compared with control without parameters (0.26 ?g/L). High performance liquid chromatography coupled to variable wavelength detector and mass spectrometry has been used to identify vitamin B12 forms and confirm the results. CONCLUSIONS: We developed the fermentation process of B. megaterium to enhance the production of vitamin B12 by providing the required supplements for the synthesis of vitamin B12 (CoCl2, ?-aminolevulinic acid (ALA) and 5,6-dimethylbenzimidazole (DMB)) and dividing the fermentation process into three stages. In addition, the optimum incubation times of the three fermentation stages were investigated and performed with reducing number of experimental and evaluated multiple parameters and their interactions by using statistical experimental design and analysis. All of these strategies has proven successful in enhancing the production of vitamin B12 up to 204.46 ?g/l and demonstrated that B. megaterium could be a good candidate for the industrial production of vitamin B12.

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Dataset Information

Cell Retention as a Viable Strategy for PHA Production from Diluted VFAs with Bacillus megaterium.

Publications

Cell Retention as a Viable Strategy for PHA Production from Diluted VFAs with <i>Bacillus megaterium</i>.

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