Data on mixed trophies biofilm for continuous cyclohexane oxidation to cyclohexanol using Synechocystis 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 Synechocystis sp. PCC 6803 and the chemoheterotrophic P. taiwanensis VLB 120 enabled HCDs up to 51.8 gCDW L−1. Respective biofilms have been grown as a biofilm in capillary flow-reactors, and oxygen evolution, total biomass, as well as the ratio of the two strains, have been followed under various cultivation conditions. Furthermore, biofilm formation on a microscopic level was analyzed via confocal laser scanning microscopy using a custom made flow-cell setup. The concept of mixed trophies co-cultivation was coupled to biotransformation, namely the oxyfunctionalization of cyclohexane to cyclohexanol. For benchmarking, the performance of the phototrophic reaction was compared to the chemical process, and to a biotechnological approach using a heterotrophic organism only. The data presented refer to our research paper “Mixed-species biofilms for high-cell-density application of Synechocystis sp. PCC 6803 in capillary reactors for continuous cyclohexane oxidation to cyclohexanol” Hoschek et al., 2019.


Data
This dataset contains information on strain development, biofilm cultivation devices, and imaging techniques, as well as analysis tools for characterizing productive biofilms converting cyclohexane to cyclohexanol. Bacterial strains and plasmids used for biocatalyst development are listed in Table 1, and their genetic features are briefly described. The schematic representation of the cultivation system developed for biofilm imaging using a confocal laser scanning microscope (CLSM) is given in Fig. 1. The central cultivation device is a flow cell made of stainless steel with the dimensions 65 mm Â 4.5 mm fitting beneath the microscope. The respective volumina of the biological specimen recorded using CLSM were calculated after 3D reconstruction from the acquired images using Imaris 8.2.0 [1] and are presented in Table 2.
In Fig. 2, a schematic representation of the biofilm reactor system developed for the transformation of cyclohexane to cyclohexanol using mixed trophies biofilms comprising photoautotrophic and chemoheterotrophic organisms is shown. Biofilms are cultivated in capillaries with the dimensions 20 cm Â 0.3 cm. Performance parameters like oxygen concentration, citrate consumption, and biofilm dry weight are summarized in Table 3, while average cyclohexanol production rates in light and dark conditions are given in Fig. 3. Different process concepts for cyclohexanol production are compared in Table 4.   Specifications table   Subject area  Biotechnology  More specific subject  area   Phototrophic mixed trophies biofilm cultivation and biotransformation   Type of data  Table, text file, graph How data was acquired Gas chromatography, dissolved oxygen measurement via oxygen microsensor, surface calculation of 3 D reconstructed images acquired by confocal laser scanning microscopy Data format analyzed Experimental factors The biofilm was cultivated in a custom made flow-cell developed for in-vivo confocal microscopy. For the dissolved oxygen determination in the liquid phase of the capillary reactors, microsensors were used. Whereas, oxygen in the gas phase was quantified via gas chromatography. For biomass analysis, the total biomass was mechanically removed from the capillary, re-suspended, and taken directly for coulter counter measurements and cell dry weight determination. To follow cyclohexane conversion, the substrate and the product were extracted from the aqueous phase with diethyl ether and quantified directly by gas chromatography.

Experimental features
The biofilm was either cultivated in the custom made flow-cell or in the capillary flow-reactor Data source location Leipzig, Germany Data accessibility All data are available in this document and in the Mendeley data sets (https://doi.org/10.17632/ vgx2hgxsc4.1).

Value of the data
The biofilm growth and development in a mixed-trophies format was investigated to gain insight on the role of individual species in biofilm formation. The recorded data of oxygen produced per biomass and the ratio between different species are valuable to understand and optimize high cell density culture of phototrophic organisms. The applicability of the bioreactor concept was demonstrated by continuous biotransformation of volatile and toxic substrates. For benchmarking, the performance of the mixed-trophies biofilm was compared to the chemical process, and to a biotechnological approach using a heterotrophic organism only

Monitoring biofilm growth in a flow-cell by confocal laser scanning microscopy
The development of a mixed trophies biofilm consisting of P. taiwanensis VLB 120_egfp and Synechocystis sp. PCC 6803 (pAH050) was analyzed by confocal laser scanning microscopy (CLSM). The Fig. 3. The average cyclohexanol production rate in g CHXOL m ¡2 d ¡1 utilizing Synechocystis sp. PCC 6803 (pAH050) and P. taiwanensis VLB 120 (pAH050) as a dual-species mixed-trophies biofilm under light and dark conditions. Experiments were conducted at 50 mE m À2 s À1 providing organic carbon free YBG11 medium and air segments at a flow rate of 52 mL min À1 . Green and grey bars represent product formation under light and dark conditions, respectively. CHXOL ¼ cyclohexanol. schematic representation of the experimental set-up is given in Fig. 1. The respective volumina of the biological specimen were calculated after 3D reconstruction from the acquired images using Imaris 8.2.0 [1] and are presented in Table 2. The eGFP signal of Pseudomonas sp. as well as the autofluorescence of Synechocystis sp. was recorded individually so that the volume could be calculated for each channel individually. The total volume of the biofilm equals the sum of the volume occupied by each species. Ps ¼ P. taiwanensis VLB120_egfp, Syn ¼ Synechocystis sp. PCC 6803 (pAH050). For the volume ratio of Ps/Syn, the calculated volume of Pseudomonas sp. was divided by the volume of Synechocystis sp. PCC 6803.

Biofilm cultivation in capillary reactors
Biofilms were cultivated as mixed and single species biofilms of Synechocystis sp. PCC 6803 and P. taiwanensis VLB120 both containing the plasmid pAH032. The schematic representation of the experimental set-up is given in Fig. 2.
The amount of oxygen produced as well as the biofilm dry weight and composition regarding bacterial species was determined for each cultivation condition (Table 3). For further cultivation details, please refer to [1].

Biotransformation of cyclohexane to cyclohexanol in capillary reactors
After 36 days of cultivation, the biotransformation was initiated for a mixed species biofilm of Synechocystis sp. PCC 6803 (pAH050) and P. taiwanensis VLB 120 (pAH050) by the addition of cyclohexane. The biotransformation substrate cyclohexane was supplied via saturation of the medium and air phase by a silicon membrane, before the reactor inlet. The productivity of 3.76 g CHXOH m À2 day À1 was reached after 1 day of adaptation and was stable for 30 days. After 31 days, the setup was actively terminated [1]. The light was turned off during day 8 and 10 so that Synechocystis sp. PCC 6803 (pAH050) was no longer able to perform photosynthesis, and this resulted in to decrease of the productivity 1.0e1.3 g CHXOH m À2 day À1 [1]. The average volumetric productivities during the light and dark conditions are 3.71 g CHXOH m À2 day À1 and 1.35 g CHXOH m À2 day À1 , respectively (Fig. 3).

Benchmarking
The here presented biotransformation using a mixed-trophies biofilm consisting of a photoautotrophic and a chemoheterotrophic strain has been compared to the conventional chemical process and to a biotechnological approach using a heterotrophic organism only (Table 4). Thereby, the advantages and disadvantages of the different concepts become obvious, and new engineering targets may be identified to develop an economic and sustainable process.