Evaluating scaling of capillary photo-biofilm reactors for high cell density cultivation of mixed trophies artificial microbial consortia.

Capillary biofilm reactors (CBRs) are attractive for growing photoautotrophic bacteria as they allow high cell-density cultivation. Here, we evaluated the CBR system's suitability to grow an artificial consortium composed of sp. PCC 6803 and sp.

Photosynthesis driven continuous hydrogen production by diazotrophic cyanobacteria in high cell density capillary photobiofilm reactors.

Hydrogen (H) is a promising fuel in the context of climate neutral energy carriers and photosynthesis-driven H-production is an interesting option relying mainly on sunlight and water as resources. However, this approach depends on suitable biocatalysts and innovative photobioreactor designs to maximize cell performance and H titers. Cyanobacteria were used as biocatalysts in capillary biofilm photobioreactors (CBRs).

Cyanobacteria as whole-cell factories: current status and future prospectives.

Cyanobacteria as phototrophic microorganisms bear great potential to produce chemicals from sustainable resources such as light and CO. Most studies focus on either strain engineering or tackling metabolic constraints. Recently gained knowledge on internal electron and carbon fluxes and their regulation provides new opportunities to efficiently channel cellular resources toward product formation.

Rational orthologous pathway and biochemical process engineering for adipic acid production using Pseudomonas taiwanensis VLB120.

Microbial bioprocessing based on orthologous pathways constitutes a promising approach to replace traditional greenhouse gas- and energy-intensive production processes, e.g., for adipic acid (AA).

Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation.

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h.

biofilms for continuous conversion of cyclohexanone in drip flow and rotating bed reactors.

In this study, the biocatalytic performance of a Baeyer-Villiger monooxygenase (BVMO) catalyzing the reaction of cyclohexanone to ε-caprolactone was investigated in biofilms. Biofilm growth and development of two VLB120 variants, Ps_BVMO and Ps_BVMO_DGC, were evaluated in drip flow reactors (DFRs) and rotating bed reactors (RBRs). Engineering a hyperactive diguanylate cyclase (DGC) from into Ps_BVMO resulted in faster biofilm growth compared to the control Ps_BVMO strain in the DFRs.

One-pot synthesis of 6-aminohexanoic acid from cyclohexane using mixed-species cultures.

6-Aminohexanoic acid (6AHA) is a vital polymer building block for Nylon 6 production and an FDA-approved orphan drug. However, its production from cyclohexane is associated with several challenges, including low conversion and yield, and severe environmental issues. We aimed at overcoming these challenges by developing a bioprocess for 6AHA synthesis.

Whole-cell biocatalysis using the Acidovorax sp. CHX100 Δ6HX for the production of ω-hydroxycarboxylic acids from cycloalkanes.

Omega hydroxycarboxylic acids (ω-HAs) possess two functional groups, a hydroxyl group and a carboxyl group, and are essential precursors for the production of biodegradable polyester polymers. In this work, an Acidovorax mutant was investigated as a whole-cell biocatalyst for the conversion of cycloalkanes to their respective ω-hydroxycarboxylic acids. This Acidovorax sp.

The Impact of Glass Material on Growth and Biocatalytic Performance of Mixed-Species Biofilms in Capillary Reactors for Continuous Cyclohexanol Production.

In this study, the growth and catalytic performance of mixed-species biofilms consisting of photoautotrophic sp. PCC 6803 and chemoheterotrophic sp. VLB120 was investigated.

Rational Engineering of a Multi-Step Biocatalytic Cascade for the Conversion of Cyclohexane to Polycaprolactone Monomers in Pseudomonas taiwanensis.

The current industrial production of polymer building blocks such as ε-caprolactone (ε-CL) and 6-hydroxyhexanoic acid (6HA) is a multi-step process associated with critical environmental issues such as the generation of toxic waste and high energy consumption. Consequently, there is a demand for more eco-efficient and sustainable production routes. This study deals with the generation of a platform organism that converts cyclohexane to such polymer building blocks without the formation of byproducts and under environmentally benign conditions.

Maximizing Biocatalytic Cyclohexane Hydroxylation by Modulating Cytochrome P450 Monooxygenase Expression in VLB120.

Cytochrome P450 monooxygenases (Cyps) effectively catalyze the regiospecific oxyfunctionalization of inert C-H bonds under mild conditions. Due to their cofactor dependency and instability in isolated form, oxygenases are preferably applied in living microbial cells with strains constituting potent host organisms for Cyps. This study presents a holistic genetic engineering approach, considering gene dosage, transcriptional, and translational levels, to engineer an effective Cyp-based whole-cell biocatalyst, building on recombinant VLB120 for cyclohexane hydroxylation.

Cultivation of Productive Biofilms in Flow Reactors and Their Characterization by CLSM.

Biofilms, a natural form of immobilized whole cells, are currently being investigated as a robust biocatalyst for the production of chemicals. Fluidic conditions and reactor geometry severely influence biofilm growth, development, and reaction performance. However, there is a missing link between the in situ characterization of biofilms on microscale setups and macroscale reactors because of the difference in reactor geometry and fluidic conditions.

Mixed-trophies biofilm cultivation in capillary reactors.

The biocatalytic application of photoautotrophic organisms is a promising alternative for the production of biofuels and value-added compounds as they do not rely on carbohydrates as a source of carbon, electrons, and energy. Although the photoautotrophic organisms hold potential for the development of sustainable processes, suitable reactor concepts that allow high cell density (HCD) cultivation of photoautotrophic microorganisms are limited. Such reactors need a high surface to volume ratio to enhance light availability.

Data on mixed trophies biofilm for continuous cyclohexane oxidation to cyclohexanol using sp. PCC 6803.

Photosynthetic microorganisms offer promising perspectives for the sustainable production of value-added compounds. Nevertheless, the cultivation of phototrophic organisms to high cell densities (HCDs) is hampered by limited reactor concepts. Co-cultivation of the photoautotrophic sp.

Light-Dependent and Aeration-Independent Gram-Scale Hydroxylation of Cyclohexane to Cyclohexanol by CYP450 Harboring Synechocystis sp. PCC 6803.

Oxygenase-containing cyanobacteria constitute promising whole-cell biocatalysts for oxyfunctionalization reactions. Photosynthetic water oxidation thereby delivers the required cosubstrates, that is activated reduction equivalents and O , sustainably. A recombinant Synechocystis sp.

Mixed-species biofilms for high-cell-density application of Synechocystis sp. PCC 6803 in capillary reactors for continuous cyclohexane oxidation to cyclohexanol.

Photosynthetic microorganisms have enormous potential to produce fuels and value-added compounds sustainably. Efficient cultivation concepts that enable optimal light and CO supply are necessary for the realization of high cell densities (HCDs), and subsequently for process implementation. We introduce capillary biofilm reactors with a high surface to volume ratio, and thus enhanced light availability, enabling HCDs of photo-autotrophic microorganisms.

Biocatalytic conversion of cycloalkanes to lactones using an in-vivo cascade in Pseudomonas taiwanensis VLB120.

Chemical synthesis of lactones from cycloalkanes is a multi-step process challenged by limitations in reaction efficiency (conversion and yield), atom economy (by-products) and environmental performance. A heterologous pathway comprising novel enzymes with compatible kinetics was designed in Pseudomonas taiwanensis VLB120 enabling in-vivo cascade for synthesizing lactones from cycloalkanes. The respective pathway included cytochrome P450 monooxygenase (CHX), cyclohexanol dehydrogenase (CDH), and cyclohexanone monooxygenase (CHXON) from Acidovorax sp.

Continuous cyclohexane oxidation to cyclohexanol using a novel cytochrome P450 monooxygenase from Acidovorax sp. CHX100 in recombinant P. taiwanensis VLB120 biofilms.

The applications of biocatalysts in chemical industries are characterized by activity, selectivity, and stability. One key strategy to achieve high biocatalytic activity is the identification of novel enzymes with kinetics optimized for organic synthesis by Nature. The isolation of novel cytochrome P450 monooxygenase genes from Acidovorax sp.

Novel cyclohexane monooxygenase from Acidovorax sp. CHX100.

Acidovorax sp. CHX100 has a remarkable ability for growth on short cycloalkanes (C5-C8) as a sole source of carbon and energy under aerobic conditions via an uncharacterized mechanism. Transposon mutagenesis of Acidovorax sp.

Guiding efficient microbial synthesis of non-natural chemicals by physicochemical properties of reactants.

The recent progress in sustainable chemistry and in synthetic biology increased the interest of chemical and pharmaceutical industries to implement microbial processes for chemical synthesis. However, most organisms used in biotechnological applications are not evolved by Nature for the production of hydrophobic, non-charged, volatile, or toxic compounds. In order to overcome this discrepancy, bioprocess design should consist of an integrated approach addressing pathway, cellular, reaction, and process engineering.

Segmented flow is controlling growth of catalytic biofilms in continuous multiphase microreactors.

Biofilm reactors are often mass transfer limited due to excessive biofilm growth, impeding reactor performance. Fluidic conditions play a key role for biofilm structural development and subsequently for overall reactor performance. Continuous interfacial forces generated by aqueous-air segmented flow are controlling biofilm structure and diminish mass transfer limitations in biofilm microreactors.

Integrated one-pot enrichment and immobilization of styrene monooxygenase (StyA) using SEPABEAD EC-EA and EC-Q1A anion-exchange carriers.

A straightforward one-pot procedure combining enrichment and immobilization of recombinantely expressed FADH₂ dependent styrene monooxygenase (StyA) directly from Escherichia coli cell extracts was investigated. Sepabeads EC-EA and EC-Q1A anion-exchange carriers were employed to non-covalently adsorb StyA from the cell extracts depending on basic parameters such as varying initial protein concentrations and pH. The protein fraction of the cell extract contained around 25% StyA.

Enzyme catalysis in an aqueous/organic segment flow microreactor: ways to stabilize enzyme activity.

Multiphase flow microreactors benefit from rapid mixing and high mass transfer rates, yet their application in enzymatic catalysis is limited due to the fast inactivation of enzymes used as biocatalysts. Enzyme inactivation during segment flow is due to the large interfacial area between aqueous and organic phases. The Peclet number of the system points to strong convective forces within the segments, and this results in rapid deactivation of the enzyme depending on segment length and flow rate.