Two-tier vessel for photoautotrophic high-density cultures

Bähr, Lars; Wüstenberg, Arne; Ehwald, R.

doi:10.1007/s10811-015-0614-5

Cited by 33 publications

(32 citation statements)

References 35 publications

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“…For conventional cultivation, cells were grown in 100-ml Erlenmeyer flasks with a final volume of 25 ml under white light at an intensity of 30 mol photons/m 2 s without shaking. For high-density cultivation, a two-tier vessel system was used (9). Essentially, cells in a liquid culture chamber were connected through a highly gas-permeable polypropylene membrane with a second chamber containing a bicarbonate buffer that maintains a high CO 2 concentration in the gas atmosphere within the membrane pores.…”

Section: Methodsmentioning

confidence: 99%

“…Two major problems complicate high-density (HD) cultivation of cyanobacteria: light attenuation in dense cultures that strongly limits the available light for net photosynthesis and an insufficient supply of inorganic carbon (9). Although the first problem was successfully addressed in flat-plate bioreactors with rapid turbulent mixing, media buffered with saturating concentrations of HCO 3 pose a considerable risk of developing a high pH as a result of photosynthesis that, in turn, can ultimately lead to chronic photoinhibition.…”

Section: Importancementioning

confidence: 99%

“…Although the first problem was successfully addressed in flat-plate bioreactors with rapid turbulent mixing, media buffered with saturating concentrations of HCO 3 pose a considerable risk of developing a high pH as a result of photosynthesis that, in turn, can ultimately lead to chronic photoinhibition. Ehwald and coworkers recently developed a two-tier vessel for photoautotrophic growth in which a cyanobacterial culture and a high-CO 2 buffer are shaken in two distinct compartments that are separated by a gas-permeable polypropylene membrane (9). The device enabled rapid growth of the unicellular model cyanobacteria Synechocystis sp.…”

Section: Importancementioning

confidence: 99%

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High-Density Cultivation of Terrestrial Nostoc Strains Leads to Reprogramming of Secondary Metabolome

Guljamow

Kreische

Ishida

et al. 2017

Appl Environ Microbiol

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Terrestrial symbiotic cyanobacteria of the genus Nostoc exhibit a large potential for the production of bioactive natural products of the nonribosomal peptide, polyketide, and ribosomal peptide classes, and yet most of the biosynthetic gene clusters are silent under conventional cultivation conditions. In the present study, we utilized a high-density cultivation approach recently developed for phototrophic bacteria to rapidly generate biomass of the filamentous bacteria up to a density of 400 g (wet weight)/liter. Unexpectedly, integrated transcriptional and metabolomics studies uncovered a major reprogramming of the secondary metabolome of two Nostoc strains at high culture density and a governing effect of extracellular signals in this process. The holistic approach enabled capturing and structural elucidation of novel variants of anabaenopeptin, including one congener with potent allelopathic activity against a strain isolated from the same habitat. The study provides a snapshot on the role of cell-type-specific expression for the formation of natural products in cyanobacteria.IMPORTANCE Terrestrial filamentous cyanobacteria are a largely untapped source of small-molecule natural products. Exploitation of the phototrophic organisms is hampered by their slow growth and the requirement of photobioreactors. The present study not only demonstrates the suitability of a recently developed two-tier vessel cultivation approach for the rapid generation of biomass of Nostoc strains but also demonstrates a pronounced upregulation of high value natural products at ultrahigh culture densities. The study provides new guidelines for high-throughput screening and exploitation of small-molecule natural products and can facilitate the discovery new bioactive products from terrestrial cyanobacteria.

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Section: Methodsmentioning

confidence: 99%

Section: Importancementioning

confidence: 99%

Section: Importancementioning

confidence: 99%

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High-Density Cultivation of Terrestrial Nostoc Strains Leads to Reprogramming of Secondary Metabolome

Guljamow

Kreische

Ishida

et al. 2017

Appl Environ Microbiol

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“…High density cultivation (HDC) is a recently developed cultivation system that tackles these problems by implementing a membrane-mediated CO 2 supply technology, combined with optimised nutrient supply and high light intensities 27 . This system facilitates rapid, sustainable biomass accumulation and was demonstrated to further raise the potential for the recovery of natural products from cyanobacteria [28][29][30] .…”

mentioning

confidence: 99%

High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803

Dienst

Wichmann

Mantovani

et al. 2020

Sci Rep

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cyanobacteria and microalgae are attractive photoautotrophic host systems for climate-friendly production of fuels and other value-added biochemicals. However, for economic applications further development and implementation of efficient and sustainable cultivation strategies are essential. Here, we present a comparative study on cyanobacterial sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803 using a commercial lab-scale High Density Cultivation (HDC) platform in the presence of dodecane as in-situ extractant. operating in a two-step semi-batch mode over a period of eight days, volumetric yields of (E)-α-bisabolene were more than two orders of magnitude higher than previously reported for cyanobacteria, with final titers of 179.4 ± 20.7 mg * L −1. Likewise, yields of the sesquiterpene alcohols (−)-patchoulol and (−)-α-bisabolol were many times higher than under reference conditions, with final titers of 17.3 ± 1.85 mg * L −1 and 96.3 ± 2.2 mg * L −1 , respectively. While specific productivity was compromised particularly for (E)-α-bisabolene in the HDc system during phases of high biomass accumulation rates, volumetric productivity enhancements during linear growth at high densities were more pronounced for (E)-α-bisabolene than for the hydroxylated terpenoids. together, this study provides additional insights into cell density-related process characteristics, introducing HDC as highly efficient strategy for phototrophic terpenoid production in cyanobacteria. Cyanobacteria are oxygenic phototrophs with a versatile metabolism and therefore constitute an economically appealing platform for the sustainable production of a diversity of industrially relevant chemicals. In particular, the natural occurrence of the methylerythritol-4-phosphate (MEP) pathway 1 allows the engineered biosynthesis of a diversity of high-value terpenoid compounds such as pharmaceuticals, cosmetics, and biofuels (reviewed in 2,3). While numerous pioneering proof-of-concept studies already demonstrated the heterologous biosynthesis of isoprene (C 5), mono-(C 10), sesqui-(C 15), di-(C 20) and tri-(C 30) terpenes in cyanobacterial host strains 4-9 , the reported titers of these compounds are typically two to three orders of magnitudes lower than those of e.g. alcoholic fuels (cf. overviews in 3,10). The major limitation of these processes arise from the inherent carbon partitioning characteristics, which favour primary metabolism and biomass accumulation, resulting in weak carbon fluxes through the native MEP pathway 11. In accordance, a strategy to bypass these inherent flux limitations by heterologous expression of the complete bacterial mevalonate (MVA) pathway in Synechocystis sp. PCC 6803 (from here: Synechocystis) resulted in 2.5-fold higher isoprene yields 12. Likewise, numerous rational design studies targeting selected bottleneck steps within the MEP pathway as well as selected steps of upstream carbon fluxes lent support to this concept 8,9,13-19. Further strategies sought to challenge the problem of inherently weak terpene s...

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“…High density cultivation (HDC) is a recently developed cultivation system that tackles these problems by implementing a membrane-mediated CO2 supply technology, combined with optimized nutrient supply and high light intensities [27]. This system facilitates rapid, sustainable biomass accumulation and was demonstrated to further raise the potential for the recovery of natural products from cyanobacteria [28][29][30].…”

Section: Introductionmentioning

confidence: 99%

High density cultivation for efficient sesquiterpenoid biosynthesis inSynechocystissp. PCC 6803

Dienst

Wichmann

Mantovani

et al. 2019

Preprint

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Cyanobacteria and microalgae are attractive phototrophic host systems for climate-friendly production of fuels and other high-value chemicals. The biosynthesis of an increasing diversity of industrially relevant compounds such as terpenoids has been demonstrated in recent years. To develop economically feasible and sustainable process designs, major challenges still remain regarding intracellular carbon partitioning, specific metabolic pathway activities and efficient cultivation strategies. Here, we present a technical study on comparative characteristics of sesquiterpene and sesquiterpene alcohol accumulation in engineered strains of Synechocystis sp. PCC 6803 (substrain GT-U) under different growth conditions and cell densities. This study particularly focuses on the basic applicability of a commercial High Density Cultivation platform in the presence of a dodecane overlay, which serves as a standard in-situ extractant and sink for various hydrophobic biochemicals. Significantly, the presented data demonstrate high volumetric productivities of (E)-α-bisabolene under high-density conditions that are more than two orders of magnitude higher than previously reported for cyanobacteria. Operating in a two-step semi-batch mode over a period of eight days, average final volumetric titers of 179.4 ± 20.7 mg * L -1 were detected. Likewise, the sesquiterpene alcohols (-)patchoulol and (-)-α-bisabolol accumulated to many times higher levels in high density cultivation than under standard batch conditions, with final titers of 17.3 ± 1.85 mg * L -1 and 96.3 ± 2.2 mg * L -1 , respectively. In contrast, specific product accumulation (mg * L -1 * OD750 -1 ) was compromised particularly for bisabolene in the high density system during phases of high biomass accumulation rates. Volumetric productivities were high during linear growth at high densities, distinctly outperforming standard batch systems. While the presented data highlight the benefits of high-density strategies for highly efficient phototrophic terpenoid production, they further point at the presence of major metabolic bottlenecks for engineered terpenoid biosynthesis and the requirement for systematic and/or targeted strategies to sustainably redirect inherent carbon fluxes in cyanobacteria. Together, our data provide additional insights into growth-and density-related effects on the efficiency of product accumulation, introducing low-scale High Density Cultivation as a rapid and efficient platform for screening of heterologous terpenoid production in cyanobacteria.

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Two-tier vessel for photoautotrophic high-density cultures

Cited by 33 publications

References 35 publications

High-Density Cultivation of Terrestrial Nostoc Strains Leads to Reprogramming of Secondary Metabolome

High-Density Cultivation of Terrestrial Nostoc Strains Leads to Reprogramming of Secondary Metabolome

High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803

High density cultivation for efficient sesquiterpenoid biosynthesis inSynechocystissp. PCC 6803

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