Orals

biofilms 9.4

We define productive biofilms as microbial communities utilised in biotechnological processes as biocatalysts for the production of value-added chemicals. For successful implementation, it is essential to merge engineering and natural sciences to equally address biological aspects like biofilm growth, structure and physiology, as well as technical challenges like reactor configuration, mass transfer issues and scale up. Productive biofilms growing on active substrate like membranes (delivering gaseous substrates) or electrodes (acting as electron donor or acceptor) are perfect model systems to study the benefits, challenges and limitations of continuous productive biofilm systems. Hence, we welcome contributions that present new processes and/or biocatalysts thriving on active substrata, describe new solutions for reactor design or highlight the impact of the biofilm matrix and heterogeneity on productivity.

Chair: Johannes Gescher | Co-chair: Katja Bühler
Orals
| Wed, 30 Sep, 15:40–16:00, Thu, 01 Oct, 11:10–12:30
Posters
| Attendance Wed, 30 Sep, 16:40–18:10, Attendance Thu, 01 Oct, 16:30–18:00

Topic assets

Wednesday, 30 September 2020 | virtual conference room

Invited Talk by Alfred M. Spormann, Stanford University, Civil and Environmental Engineering | Stanford, USA
15:40–16:00 |
biofilms9-54
Guillaume Pillot, Soniya Sunny, Victoria Comes, and Sven Kerzenmacher

The electrosynthesis of valuable compounds by biofilms on electrodes is intensively studied since few years. However, the actual biofilms growing so far on cathode produce mainly small inexpensive compounds such as acetate or ethanol. A novel Knallgas bacteria, Kyrpidia spormannii have been recently described to grow on cathode in thermophilic and microaerophilic conditions, producing significant amount of PolyHydroxyAlkanoates (PHAs) (Reiner et al., 2018). These PHA are promising sustainable bioplastic polymers with the potential to replace petroleum-derived plastics in a variety of applications. However, the effect of culture conditions and electrode properties on the growth of K. spormannii biofilm and PHA production is still unclear.

We present in this study the successful development and operation of autotrophic biocathode whereby the electroactive biofilm was able to grow by utilizing CO2 and a cathode as the sole carbon and electron source, respectively. We report for the first time, the effect of operating conditions of the Bioelectrochemical system (BES), cathode materials and cathode surface modification on current consumption, biofilm formation, PHA productivity and overall coulombic efficiency of a K. spormannii culture growing on electrodes. In particular, the focus of this study lies on optimization of three main operating conditions, which are the applied cathode potential, pH buffer and the oxygen concentration in the feed gas. Increased biofilm formation and PHA production was observed at an applied potential of -844mV vs. SCE, pH 6.5, O2 saturation of 2.5%, and for a graphite cathode modified by CO2 activation. The PHA concentration in the biofilm reached a maximum of ≈40 μg·cm-2 after optimization. The resultant PHA yield reported after optimization is increased by 12.2 times in comparison to previous results. In conclusion, these findings take microbial electrosynthesis of PHA a step forward towards practical implementation.

How to cite: Pillot, G., Sunny, S., Comes, V., and Kerzenmacher, S.: Optimization of PolyHydroxyAlkanoate Bioelectrosynthesis by the thermophilic bacterium Kyrpidia spormannii, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-54, https://doi.org/10.5194/biofilms9-54, 2020.

Thursday, 1 October 2020 | virtual conference room

11:10–11:30 |
biofilms9-55
Max Hackbarth, Tobias Jung, Johannes Eberhard Reiner, Andrea Hille-Reichel, Michael Wagner, Johannes Gescher, and Harald Horn

In the last 40 years, bioelectrochemical systems (BESs) have been increasingly discussed within the scope of debates about sustainable energy sources and production of value added chemicals independent of fossil sources. Since the produced current in microbial fuel cells as well as the turnover rates in microbial electrosynthesis cells are dependent on the biocatalysts´ activity, control of the growing biofilm plays a major role in BESs. Moreover, the knowledge about the interplay between biofilm development and electrochemical parameters is crucial for optimizing these sytems.

In the last 3 years, various electroactive biofilms (anodic and cathodic) were cultivated and characterized in a versatile and house made lab-scale flow cell system as well as in a rotating disc biofilm contactor (RDBC). Both systems allow for control of substrate (liquid and gaseous), and nutritional conditions as well as hydrodynamics and other physical parameters. The monitoring of biofilm development was conducted non-invasively by means of optical coherence tomography (OCT). For cathodic biofilms, quantitative analysis of generated 3D OCT data sets revealed a correlation between substratum coverage and measured current density. The increase of substratum coverage led to a decrease of measured current density due to less abiotic redox processes on the cathode surface. A stable current density was achieved when a substratum coverage of 99 % was reached. Furthermore, calculated biofilm accumulation rates could also be correlated with the substratum coverage. The overall biofilm accumulation rate decreased when the substratum was fully covered. Both correlations support the hypothesis that the availability of electrons from the cathode surface is a limiting factor in microbial electrosynthesis.

A 10-liter RDBC was designed to continuously harvest biomass from the electrode to extract intracellularly stored products. In future, this approach could be applied for biotechnological processes. Additionally, the RDBC can be used to obtain reliable mass balances and turnover rates because of its larger scale.

How to cite: Hackbarth, M., Jung, T., Reiner, J. E., Hille-Reichel, A., Wagner, M., Gescher, J., and Horn, H.: Monitoring and quantification of bioelectrochemical biofilms by means of OCT in novel and customized reactor-setups, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-55, https://doi.org/10.5194/biofilms9-55, 2020.

11:30–11:50 |
biofilms9-65
Luciana C. Gomes, Gabriel A. Monteiro, and Filipe J. Mergulhão

Escherichia coli biofilms have a great biotechnological potential since this organism has been one of the preferred hosts for recombinant protein production for the past decades and it has been successfully used in metabolic engineering for the production of high-value compounds.

In a previous study, we have demonstrated that the non-induced enhanced green fluorescent protein (eGFP) expression from E. coli biofilm cells was 30-fold higher than in the planktonic state without any optimization of cultivation parameters [1]. The aim of the present work was to evaluate the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of eGFP by planktonic and biofilm cells of E. coli JM109(DE3) transformed with a plasmid containing a T7 promoter.

It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Recombinant protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the recombinant protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of recombinant protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth [2].

It is expected that this work will be of great value to elucidate the mechanisms of induction on recombinant protein production, especially in biofilm cells which have shown potential to be used as protein factories.

 

 

References:

[1] Gomes, L.C., & Mergulhão, F.J. (2017) Heterologous protein production in Escherichia coli biofilms: A non-conventional form of high cell density cultivation. Process Biochemistry, 57, 1-8. https://doi.org/10.1016/j.procbio.2017.03.018

[2] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms. International Journal of Molecular Sciences, 21(2), 576. https://doi.org/10.3390/ijms21020576

How to cite: Gomes, L. C., Monteiro, G. A., and Mergulhão, F. J.: Recombinant Protein Production and Plasmid Stability in Escherichia coli Biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-65, https://doi.org/10.5194/biofilms9-65, 2020.

11:50–12:10 |
biofilms9-80
Katja Bühler, Anna Hoschek, Andreas Schmid, Ingeborg Heuschkel, and Rohan Karande

 

Despite photo-biocatalysis developing remarkably and the huge potential of photoautotrophic microorganisms for eco-efficient production scenarios, photo-biotechnology is still in its infancy. The lack of scalable photo-bioreactors that provide efficient light transmission, CO2 supply, and O2 degassing and thus enable high cell densities (HCD), constitutes a key bottleneck, especially if cost-sensitive bulk chemicals are the product of choice. Commercialized tubular photo-bioreactors with 100 to 600 mm inner diameter offer a surface area to volume ratio (SA/V) of over 100 m2 m-3 enabling the efficient capturing of incident solar radiation.1 Here we introduce a new generation of photo-bioreactors based on capillary biofilm reactors. The biofilm is composed of two strains, namely the photoautotrophic strain Synechocystis sp. PCC 6803 and the chemoheterotrophic strain Pseudomonas taiwanensis VLB120, which serves as a biofilm supporter strain. Pseudomonas sp. is lowering the pO2 in the system, which otherwise would toxify the Cyanobacteria. Furthermore, it produces extrapolymeric substances (EPS) and produces a kind of seeding layer promoting the attachment of Synechocystis sp.. Synechocystis sp. on the other hand produces organic compounds and oxygen consumed by Pseudomonas sp. The system is run completely without any organic carbon source.

Depending on the functionalities engineered into the biofilm forming organisms, these systems can be used for biotechnological applications. Here, we will present data on the physiology of the mixed trophies biofilm, and the challenging conversion of cyclohexane to caprolactone, and further on to 6-hydroxyadipic acid, both being important monomers for Nylon production.

 

References

  • [1] Posten, C. Eng. In Life Science. (2009) 9:165-177
  • [2] Hoschek, A. et al Bioresource Technology. (2019) 282: 171-178

 

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How to cite: Bühler, K., Hoschek, A., Schmid, A., Heuschkel, I., and Karande, R.: Mixed-trophies two species biofilms driven by Cyanobacteria for biotechnnological applications, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-80, https://doi.org/10.5194/biofilms9-80, 2020.

12:10–12:30 |
biofilms9-158
Miriam Edel and Johannes Gescher

A shift from petrochemical processes to a bio-based economy is inevitable to establish a sustainable industry. Bioelectrochemical systems (BESs) are a future technology for the environment-friendly production of platform chemicals. In BESs exoelectrogenic bacteria such as Shewanella oneidensis can directly transfer respiratory electrons to the anode, which serves as a non-depletable electron acceptor. So far, the main limiting factor in BESs is the achievable current density which correlates to some extend with the density, thickness and metabolic activity of anode biofilms composed of exoelectrogenic microorganisms. This is especially true for S. oneidensis as the organism forms rather thin biofilms under anoxic conditions on anode surfaces.

In order to enhance the organisms’ biofilm formation capabilities Bursac et al. deleted the λ-prophage from the genome. The deletion of the λ-prophage led to a 2.3-fold increased cell number on the anode ongoing with a 1.34-fold increased mean current density (Bursac et al., 2017). Furthermore, we just recently discovered that exogenous riboflavin enhances biofilm formation by the upregulation of the Ornithine-decarboxylase speC. This is probably based on a quorum sensing effect of riboflavin. Taken together the upregulation of speC ongoing with the deletion of the λ-prophage leads to a 4-fold increase in current density ongoing with a 6.1-fold increased biofilm formation on the anode.

However, to ensure an optimal performance of the biofilm in BESs, biofilm thickness itself is not sufficient. The biofilm also needs to be conductive. Our aim is to establish the Spytag-/Spycatcher-tool to synthetically steer biofilm conductivity. Spytag and Spycatcher are two protein residues from the fibronectin binding protein of Streptococcus pyogenes (Spy). These two protein residues form a spontaneous isopeptide bond under a variety of temperatures, pH values and buffers (Zakeri et al., 2012). By coupling Spytag and Spyctacher to different outer membrane c-type cytochromes of S. oneidensis the cells are covalently bound to each other while the biofilm remains conductive. In a first application the production of acetoin as one of the top 30 platform chemicals world-wide is desired (US Department of Energy, 2004).

In order to render S. oneidensis producing acetoin instead of the native end product acetate, Bursac et al. deleted the key genes for acetate production and introduced the acteoin production pathway (Bursac et al., 2017). To broaden the substrate spectrum of S. oneidensis further genes for glucose metabolism were introduced. Through a long term adaption, the glucose degradation, the biofilm formation abilities and the bioelectrochemical performance were significantly enhanced.

Merging all genetic optimizations into one production strain will enable us to produce acetoin from glucose as a platform chemical with high space-time yields. This will give rise to a production process that is competitive with existing oxic process routines without being dependent on expensive aeration.

References:

Bursac, T., Gralnick, J.A.,Gescher, J. (2017) Acetoin production via unbalanced fermentation in Shewanella oneidensis. Biotechnol Bioeng 114: 1283–1289.

Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., Howarth, M. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109: E690.

How to cite: Edel, M. and Gescher, J.: Biotechnological production of platform chemicals through anode assisted fermentation by using an artificial biofilm of S. oneidensis, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-158, https://doi.org/10.5194/biofilms9-158, 2020.