Posters

Cyanobacteria are a group of phototrophic prokaryotes commonly known as blue-green algae. They grow embedded as biofilms in a thick matrix of extracellular polymeric substances (EPS) and can produce a highly diverse range of secondary metabolites, which are interesting in terms of their antimicrobial activity. Among these components, polyketide and polypeptide molecules are dominating. Antimicrobial polypeptide molecules are usually post-translational-modified or synthesised by non-ribosomal peptide synthetase (NRPS). Standard screening for antibiotics by inhibition tests is very time consuming and expression of antimicrobic activity highly depend on cultivation conditions. Therefore, they can vary between different cultivations. On a genomic level existing, but in this cultivation not synthesized, antibiotics are completely neglected. Due to the increasing amount of available genomic sequence data, screening for novel antibiotics can also be done in-silico. Highly homologous sequences to known antibiotic gen clusters can be determined in cyanobacterial genomes and eventually be detected in-vivo through PCR analysis. Compared to inhibition tests, a major advantage of PCR is the little amount of biomass needed. As the growth of cyanobacteria is slow, e.g. Trichocoleus sociatus (0.44 d^-1) compared to bacteria like Escherichia coli (2.08 h^-1), this leads to significant shorter cultivation and screening time. In addition, qPCR can be used to determine gene expression quantity of the considered genes. PCR with degenerated primers for specific gen cluster like NRPS, polyketide synthetases, lanthipeptides etc. can also be used to screen non-sequenced cyanobacteria for the possible origin of an unidentified antibiotic.

The following work is part of the iProcess project, whose overall scientific goal is to develop the process engineering fundamentals for using fungi and cyanobacteria as production organisms for pharmaceutically active substances. As part of the iProcess project, a semi-continuous process for the production of antibiotics from cyanobacteria biofilms in aerosol reactors shall be developed. Aim of the following work is the in-silico search for new polypeptide antibiotics, as well as the subsequent in-vivo detection to discover promising cyanobacteria as production strains. In the first instance, the screening is focusing on the intern cyanobacteria strain collection of the TU Kaiserslautern. Subsequently the new strains will be cultivated as biofilms in an aerosol reactor and the resulting extracellular polymeric substances can be analysed for their antimicrobial activity.

This project is financially supported by Ministry of Science, Further Education and Culture of Rhineland-Palatinate (mwwk.rlp) (iProcess intelligent process development – from modelling to product).

How to cite: Lenz, S., Walther, J., Strieth, D., and Ulber, R.: Genomic screening for novel peptide antibiotics in biofilm cyanobacteria by in-silico analysis and PCR , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-24, https://doi.org/10.5194/biofilms9-24, 2020.

Biofilm formation is a central process in the function of Microbial Electrochemical Technologies (METs). These technologies have emerged in recent years as a promising alternative green source of energy, in which microbes consume organic matter to produce energy or valuable by-products. It is the ability of performing extracellular electron transfer that allows these microbes, called electroactive organisms, to exchange electrons with an electrode in these systems. The low levels of current achieved have been the set-back for the large-scale application of METs. Shewanella oneidensis MR-1 is one of the most studied electroactive organisms, and it has been demonstrated that its increased biofilm formation can lead to higher current generation. The bolA gene has been identified as a central player in biofilm formation in different organisms, with its overexpression leading to increased biofilm production. In this work, we explored the effect of this gene in biofilm formation and current production by S. oneidensis MR-1. Our results demonstrate that this gene is involved in the biofilm formation by this organism, with its over expression leading to an increased biofilm formation. We could also show that this increase in biofilm formation lead to a consequent higher current generation. This information is a relevant step for the optimization of electroactive organisms towards their practical application in METs.

How to cite: V. Silva, A., Edel, M., Gescher, J., and M. Paquete, C.: Increased biofilm in Shewanella oneidensis MR-1 leads to higher current generation in METs, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-18, https://doi.org/10.5194/biofilms9-18, 2020.

Carbon-based and stainless steel-based materials are widely utilized as anode/cathode electrodes in bio electrochemical systems (BESs) due to its low capital cost, high conductivity and large specific surface area. Carbon-based materials such as carbon veil are mostly applied in lab-scale reactors because of its versatile shape and configuration. Moreover, stainless steel type materials show higher strength and are easier to incorporate within flow field. Optical coherence tomography (OCT) as an image technique is a suitable method to monitor biofilm growth and fluid-structure interactions at the meso-scale. In BESs, investigating bulk-biofilm interface (fluid-structure interactions) is of particular interest to optimize the mass transfer under suitable hydrodynamic condition and enhances the overall effectivity of BESs systems. To extend the knowledge about the influence of different anode electrodes as substratum on OCT monitoring and quantification, the biofilm structural properties analyzed by OCT image processing and bioelectrochemical systems performance were compared.  

A custom-designed dual-chamber setup was constructed by two transparent optical flow cells and fixed in the automated monitoring platform (Evobot). Herein, we applied OCT to in-situ characterize and quantify the biofilm structure properties on two different anode electrodes (carbon veil-CV and porous stainless steel-SS) as substratum in microbial fuel cell (MFC) mode.  3D OCT dataset analysis presented 3 structural parameters for biofilm-carbon veil interface and 5 structural parameters for biofilm-stainless steel interface, separately. Biofilm volume (BioV) was calculated to compare CV and SS anode electrodes.

In this study, a time-series of biofilm development was performed on both CV and SS materials. At the fourth day, the biofilm almost covered the entire anode surface and achieved 97% substratum coverage. Afterwards the biofilm grew mostly in vertical direction. With the further biofilm growth along height the electric resistance increased and power production gradually reached the equilibrium. Nevertheless, both materials did not show predominant advantage on power production. Furthermore, a relatively small error appeared on quantitative analysis of Biofilm volume using stainless steel. Whereas, the predictability of biofilm volume on the carbon veil anodes was hindered by the appearance of shadowing effects. Thus, it can be concluded that stainless steel flat plate electrode is preferable as anode material to investigate the interaction between biofilm structure, hydrodynamic conditions and mass transfer in BESs via OCT.

How to cite: Liu, J., Horn, H., and Wagner, M.: In situ probing and evaluation of two different electrode materials in bio electrochemical systems by means of Optical Coherence Tomography on automated robotic platform, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-110, https://doi.org/10.5194/biofilms9-110, 2020.

How to cite: Walther, J., Erdmann, N., Wastian, K., Strieth, D., and Ulber, R.: Novel photobioreactor for moving bed biofilm cultivation of terrestrial cyanobacteria, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-28, https://doi.org/10.5194/biofilms9-28, 2020.

Electroactive biofilms are routinely characterized in-operando by dynamic electrochemical measurement techniques such as cyclic voltammetry or electrochemical impedance spectroscopy. Since electrical signals can be recorded and processed very quickly, these techniques allow to investigate slow and fast electron transfer processes.

In contrast, the dynamics of species production rates are usually not addressed because standard measurement techniques for the quantification of reaction products such as gas chromatography are slow. Instead it is often assumed that species production rates are either directly proportional to the current - under so called turnover conditions - or equal zero - under so called non-turnover conditions.

To challenge this assumption, we measured species production rates of a biofilm electrode with a high time resolution by differential electrochemical mass spectrometry (DEMS). An acetate oxidizing biofilm electrode was placed just micrometers away from the mass spectrometer inlet in which enabled us to observe CO₂ production directly at the electrode during cyclic voltammetry (CV) and potential steps.

The measurement results showed that the CO₂ production deviates significantly from the expected value calculated from the current by Faraday’s law under certain operating conditions. We analyze this effect in detail and show that it can be explained with biofilm storage capacities for charge and substrate. These capacities are quantified by deconvoluting the faradaic and non-faradaic currents. [1]

Also, the onset of the complete oxidation of acetate to CO₂ during CVs was determined to be just 22 mV above the standard potential for acetate oxidation. Determining this value by directly measuring CO₂ instead of current is advantageous because capacitive effects can be excluded. [1]

In conclusion, we demonstrate that electrical current and CO₂ production can be partly decoupled in biofilm electrodes and that DEMS is a valuable technique for analyzing processes in such electrodes.

[1] Kubannek, F., Schröder, U., Krewer, U. (2018). Revealing metabolic storage processes in electrode respiring bacteria by differential electrochemical mass spectrometry. Bioelectrochemistry, 121, 160–168, doi: 10.1016/j.bioelechem.2018.01.014

How to cite: Kubannek, F., Schröder, U., and Krewer, U.: Online Analysis of CO2 Production in Electroactive Biofilms by Differential Electrochemical Mass Spectrometry, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-102, https://doi.org/10.5194/biofilms9-102, 2020.

A microbial fuel cell (MFC) is a renewable energy converter, which transforms organic biomass directly into electricity, using biofilm-electrode metabolic interaction within a bioelectrochemical cell. Efficiency of this transformation can be enhanced through miniaturisation. Miniaturisation of MFCs offers higher surface-area-to-volume ratio and improved mass transfer.

The development of mL-scale; power dense and low cost MFCs, are of great interest in diverse areas of research, ranging from modern bio-robotics, internet-of-things devices, electrical energy generation, remote sensing to wastewater treatment and mineral recovery. The biofilms increased ability in converting organic pollutants into electric power more efficiently, makes mL-sized MFCs attractive for the development of multi-modular stacks and usable off-grid power sources with an ability of enhanced wastewater treatment. This work focuses on small scale MFCs; i) minimising the distance between feeding stream and the biofilm, ii) construction and analysis of a  millilitre scale prototype, using a low cost ceramic separator for higher energy recovery efficiency and sensitivity enhancement to substrates and pollutants. The study aims to test efficient cathode modifications, using graphene ink and magnetite (Fe₃O₄); in order to improve the oxygen reduction reaction (ORR). This in turn is envisioned in an increase of the output, reaching comparable power levels to the larger MFC prototypes tested so far. The additives are chosen such that,  both graphene and iron–based oxides are known from the literature to be catalysts for electrochemical processes, this work focusses on their incorporation into the open-to air cathode in novel, low cost MFC bioreactors.

The miniaturised MFC construction constituted of an in-house fabricated small scale ceramic cylinder of internal volume of 3.88 mL. An anode, made of carbon veil fibre with a coating of activated carbon powder, was placed inside the ceramic cylinder, while the cathode was attached to the outer surface of the structure. Three types of cathodes were tested: i) activated carbon as the control (AC), ii) AC with a graphene ink coating (AC+G) and iii) AC with graphene ink and magnetite powder blend (AC+G+M). Experiments were conducted in triplicate using activated sludge and urine inoculum and thereafter continuously supplemented with 100% real human urine. The results show that the control produced up to 0.85 mW (219 W/m³), while AC+G produced 1.22 mW (312 W/m³), and AC+G+M 1.12 (288 W/m³) which is a 44 % and a 32 % increase respectively in comparison to the control. Comparison of linear sweep voltammetry (LSV) showed superior performance of both modified electrodes against the unmodified AC cathode; further resulting in an enhancement of ORR reaction rate. Power outputs from this work show over 14 times improvement in power density levels in comparison to larger reactors of 20 times the volume, as well as comparable raw (actual) power levels. This makes these novel small-scale bioreactors particularly attractive for use in numerous practical applications such as energy autonomous robots (e.g. EcoBots) and multi-modular stacks for off-grid energy sources.

How to cite: Gajda, I., Mendis, B. A., Greenman, J., and Ieropoulos, I.: Power output enhancement in ceramic, mL-scale Microbial Fuel Cell , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-159, https://doi.org/10.5194/biofilms9-159, 2020.

Biofilms are typically characterized as a consortium of microorganisms, which adhere to each other and often to surfaces. This adhesion is realized by extracellular polymeric substances (EPS), which are secreted by the microorganisms and mainly consist of water, polysaccharides, proteins and lipids as well as nucleic acids and lysis products [1]. Although cultured plant cells are not typically considered biofilms, parallels can be found in the properties of plant calli. These callus cells tend to form cohesive aggregates, owing to their extracellular matrix, and often strongly adhere to the agar plates they are kept on. The extracellular matrix of plant cells is mainly composed of structural polysaccharides, such as xyloglucans, arabinogalactans [2], homogalacturonan and extensins [3] among others. Cultured plant cells were found to adhere to surfaces before [4]. Surface-associated plant cell culture may have potential in a (semi‑)continuous cultivation including product secretion, as was shown in principle for alginate-embedded plant cells [5]. For cyanobacterial biofilms, an efficient strategy for EPS extraction was recently developed [6]. The transferability of these protocols to biofilm-like growing plant calli of Ocimum basilicum is currently being investigated. Subsequently, the composition of the extracellular matrix extracted from cultured O. basilicum cells is of interest. Furthermore, the adhesive properties of O. basilicum suspension cultures to microstructured surfaces and the potential role of the extracellular matrix are under investigation. An investigation of culture properties in an aerosol photobioreactor [7] is planned as well.

This project is financially supported by the German research foundation (DFG, project number SFB 926-C03).

References:

[1]      H. C. Flemming, T. R. Neu, and D. J. Wozniak, “The EPS matrix: The ‘House of Biofilm Cells,’” J. Bacteriol., vol. 189, no. 22, pp. 7945–7947, 2007.

[2]      I. M. Sims, K. Middleton, A. G. Lane, A. J. Cairns, and A. Bacic, “Characterisation of extracellular polysaccharides from suspension cultures of members of the Poaceae,” Planta, vol. 210, no. 2, pp. 261–268, Jan. 2000.

[3]      M. Popielarska-Konieczna, K. Sala, M. Abdullah, M. Tuleja, and E. Kurczyńska, “Extracellular matrix and wall composition are diverse in the organogenic and non-organogenic calli of Actinidia arguta,” Plant Cell Rep., no. 0123456789, 2020.

[4]      R. J. Robins, D. O. Hall, D. ‐J Shi, R. J. Turner, and M. J. C. Rhodes, “Mucilage acts to adhere cyanobacteria and cultured plant cells to biological and inert surfaces,” FEMS Microbiol. Lett., vol. 34, no. 2, pp. 155–160, 1986.

[5]      Y. Kobayashi, H. Fukui, and M. Tabata, “Berberine production by batch and semi-continuous cultures of immobilized Thalictrum cells in an improved bioreactor,” Plant Cell Rep., vol. 7, no. 4, pp. 249–252, 1988.

[6]      D. Strieth, J. Stiefelmaier, B. Wrabl et al., “A new strategy for a combined isolation of EPS and pigments from cyanobacteria,” J. Appl. Phycol., no. Fromme 2008, Feb. 2020.

[7]        S. Kuhne, D. Strieth, M. Lakatos, K. Muffler, and R. Ulber, “A new photobioreactor concept enabling the production of desiccation induced biotechnological products using terrestrial cyanobacteria,” J. Biotechnol., vol. 192, no. Part A, pp. 28–33, 2014.

How to cite: Mehring, A., Stiefelmaier, J., and Ulber, R.: Surface-associated plant cell culture, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-79, https://doi.org/10.5194/biofilms9-79, 2020.

Cyanobacteria belong to the oldest known microorganisms and are capable of oxygenic photosynthesis. Depending on their habitat aquatic and terrestrial cyanobacteria are distinguished. Terrestrial cyanobacteria grow embedded in a matrix of extracellular polymeric substances (EPS) as phototrophic biofilms. Those EPS serve as nutrient storage, protection from desiccation and play an important role in surface adhesion. For cultivation of phototrophic biofilms different biofilm reactors have been developed in the last years. One interesting parameter when cultivating biofilms is the surface material and structure, since it can influence the surface adhesion and thus biofilm formation. Therefore, different materials as cultivation surfaces were investigated as well as the strain specific behavior of different cyanobacteria and the impact on EPS formation. In this work the adhesion of the terrestrial cyanobacteria Coleofasciculus chthonoplastes and Trichocoleus sociatus to different materials was investigated. For characterization of materials measurements concerning surface roughness were conducted using atomic force microscopy. Biofilms were cultivated in an aerosol and the development of surface adhesion in connection with biofilm age was analyzed using two different methods. In the first set-up biofilms were placed in a specially designed flow-through chamber and overflown with medium at increasing flow speed. The detachment of the biofilm was documented with optical coherence tomography (OCT). Additionally, the experiments were supplemented with CFD-simulation for quantification of shear forces. The second method analyzed adhesion forces using rotational rheometry. Hereby, differences between cyanobacteria strains and surface materials could be observed as well as an increasing adhesion with increasing cultivation time. The developed flow-through chamber, which could as well be utilized with a camera instead of OCT, offers a simple low-priced possibility for investigation of surface adhesion.

This project is financially supported by the German Research Foundation (DFG; Project number: UL 170/16-1; MU 2985/3-1 and SFB 926) and the Landesförderung Rheinland-Pfalz (Project: iProcess).

How to cite: Stiefelmaier, J., Strieth, D., Schaefer, S., Kronenberger, D., Wrabl, B., Bröckel, U., and Ulber, R.: Surface adhesion of phototrophic biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-37, https://doi.org/10.5194/biofilms9-37, 2020.

Butanol and Isopropanol are naturally produced by the bacteria C. beijerinckii. Those products are used in large field of applications such as fuel and bulk chemicals. Since butanol is toxic at small concentration for cells, bacterial growth and metabolism are inhibited during classical batch fermentation (1). These phenomena lead to the production of low solvent concentration (around 7 g.L^-1) and a low volumetric productivity (0,13 g.L^-1.h^-1) (2). Continuous fermentation can be performed in order to avoid product inhibition by  a continuous removal of fermentation broth. However, the solvent productive biomass is easily washout at high dilution rate because of the low maximum growth rate of the strain in this metabolism phase  (0,05 h^-1) (3). To overcome this issue, cell immobilization of  C. beijerinckii by biofilm formation on solid support is the best solution. As a result, the biomass residence time can be uncorrelated from the hydraulic residence time leading to a higher viable biomass concentration in the bioreactor and consequently a higher volumetric productivity (up to 5 g.L^-1.h-1 ) (4). Our study aimed  at evaluating biofilm viability which is an important parameter that is linked to process productivity and has been little studied in the case of the IBE fermentation (5).

In this study we developed two techniques to monitor biofilm viability during immobilized cell fermentation: Flow cytometry (FC) and PMA qPCR. After FC analysis, a high background noise due to the biofilm extra polymeric substance is obtained. Consequently, an enzymatic  sequential enzymatic biofilm deconstruction using Dnase I and Proteinase K was developed . This pre-treatment successfully lowered the background noise of this analysis. The suspensions obtained were stained with carboxyfluoresceine diacetate (cFDA) and propidium iodide (PI) which are indicators of cellular activity and alteration of membrane integrity, respectively,  and analyzed by flow cytometry. The percentage of viable cells obtained after pre-treatment compared to the control sample is increased from 2.6 ± 0.9 % to 22.8 ± 8.6% because of the background noise decrease. PMA-qPCR confirmed the results obtained by flow cytometry without using enzymatic pre-treatment. Although FC is less accurate than PMA-qPCR, this technique is less time-consuming, cheaper and reliable to study biofilm viability.

References

How to cite: Carrié, M., Velly, H., Gabelle, J.-C., and Ben-Chaabane, F.: Viability of mono-specie biofilm formed by the solvent producer Clostridium beijerinckii during continuous fermentation in packed bed bioreactor., biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-47, https://doi.org/10.5194/biofilms9-47, 2020.

Biofilm-based algal processes are increasingly used for wastewater treatment, carbon capture, and production of biofuels and other valuable products. They provide high cell densities, are more robust, and are easier to harvest and concentrate than suspended algae. However, algal biofilms are more likely to experience carbon limitation, O₂ inhibition, and pH limitations, especially when thick and exposed to high light intensities. To address these limitations, we studied a novel photobioreactor based on CO₂-supplying hollow-fiber membranes, where the algal biofilms grow directly on the membranes. We used modelling and experiments to study our membrane biofilm photobioreactor (MB-PBR) system and to compare it to a control with atmospheric CO₂ and bicarbonate supplied in the bulk liquid.

Mathematical models of the MB-PBR and the control were developed using COMSOL Multiphysics®. The models included phototrophic growth, diffusion of gases (CO₂, O₂, N₂) across the membrane, nutrient diffusion from the bulk liquid, pH-dependent carbonate speciation, and light attenuation. Experimentally, we compared the MB-PBR and control using bench-scale photobioreactors with hollow-fiber membranes attached to them, 10% BG-11 media and white light from an LED lamp. The MB-PBR membranes were supplied with 5% CO₂ and 95% N₂.  The control system had sealed membranes, to prevent gas exchange.  We measured the biomass dry weight gravimetrically and the biofilm growth rates by daily measurement of the thicknesses using optical coherence tomography (OCT).

Both modeling and experiments suggested that MB-PBR biofilms grow significantly faster than the control. Using our model, we studied the effect of light intensity, pH, buffer concentration and light and oxygen inhibition on MB-PBR behavior. Growth was inhibited by excessively high levels of light and O₂. By providing CO₂ through the membrane, the carbon limitation was minimized, O₂ was stripped from the biofilm, and pH shifts were attenuated. These results suggest the MB-PBR may provide a more efficient platform for algal biofilm processes.

How to cite: Garg, M., Perez-Calleja, P., Garcia-Perez, J. S., Ontiveros-Valencia, A., Picioreanu, C., Parra-Saldivar, R., and Nerenberg, R.: A membrane-based biofilm photobioreactor for enhanced algal growth rates, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-97, https://doi.org/10.5194/biofilms9-97, 2020.

Hydrogen sulfide (H₂S) is a toxic pollutant and harmful to human health. Industries such as pulp and paper manufacturing, rayon production, natural gas extraction and refining, and crude petroleum refineries generate waste gas streams with high H₂S concentrations. Both physico-chemical and biological methods are used for H₂S removal from the gas stream. Biological methods offer several advantages such as environmental friendly, less expensive and require simple operation and maintenance compared to physico-chemical methods. In this study, a hydrophilic hollow fibre membrane (HFM) based bioreactor configuration has been tested for biological H₂S removal. Three reactors were fabricated and operated for ~ 3 months where two reactors were used for biological conversion process and the third reactor was used for abiotic process. The effective membrane area of a HFM module used in each reactor was 0.0138 m². The bioreactors demonstrated efficient gas-liquid mass transfer through the HFM module and achieved ~ 99% removal efficiency with an elimination capacity of ~ 17.0 g m^-3 h^-1. The H₂S flux of the bioreactor was ~ 0.20 g m^-2 day^-1 which was ~ 9 times higher than the abiotic reactor for an inlet H₂S concentration of ~ 0.90 g m^-3. The overall mass transfer coefficient value for the biotic process was 17.2 µm s^-1 which was ~ 25 times higher than the abiotic process. The bioreactors demonstrated both microbial attached growth on the membrane surface and suspended growth in the liquid phase. Microbial community analysis confirmed the presence of diverse sulfur-oxidizing bacteria at genus level including Acinetobacter, Dechloromonas, Hydrogenophaga, Rhodopseudomonas and Sulfurospirillum. Moreover, the enrichment of other bacterial genera such as ammonia-oxidizing (e.g. Nitrosospira), organic matter degrading (e.g. Trichococcus) and methanogenic (e.g. Methanosaeta) microorganisms demonstrate the diverse microbial ecology of the sludge growing in the bioreactor.

How to cite: Das, J., Ravishankar, H., and Lens, P.: Application of hollow fibre membrane reactor for biological removal of H2S, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-45, https://doi.org/10.5194/biofilms9-45, 2020.

Microbial nanowires are nanofilaments that could offer an extracellular electron transfer (EET) pathway linking the bacterial respiratory chain to external surfaces, such as oxidized metals in the environment and engineered electrodes in renewable energy devices. Filaments proposed to function as nanowires have been reported in multiple bacteria, yet it remains largely unclear about the composition and electron transfer mechanism of bacterial nanowires. Pseudomonas aeruginosa is an environmental and electrochemically active bacterium. In this study, we found nanotube-like extracellular filaments in P. aeruginosa biofilms, which were bacterial membrane extensions similar to the nanowires reported in Shewanella oneidensis. Remarkably, conductive probe atomic force microscope showed measurable conductivity of these extracellular filaments, suggesting that they may function as nanowires in P. aeruginosa. Our results also indicated that the electron shuttle pyocyanin significantly affected the conductivity of P. aeruginosa nanowires, suggesting that the electron transfer mechanism of P. aeruginosa nanowires was different from S. oneidensis. Furthermore, factors that impact biofilm formation, such as flagella, type IV pili, and exopolysaccharides, were not essential for nanowires formation, while affect the formation and length of nanowires of P. aeruginosa. Taken together, this is the first report that investigated the role of electron shuttle on the conductivity of nanowires and factors that affected nanowires formation.

How to cite: ma, L.: Bacterial nanotubes and their role as bacterial nanowires in Pseudomonas aeruginosa biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-156, https://doi.org/10.5194/biofilms9-156, 2020.

Biofilm Engineering Approaches for Improving the Performance of Bioelectrochemical Systems

ABSTRACT:

Bio-electrochemical devices are realized as promising technologies for a wide range of applications such bioenergy, and in biocommodity engineering.  Bioelectrochemical systems make use of electroactive biofilms as electrocatalysts for converting chemical energy to electrical energy and vice versa. In this presentation, surface engineering of electrodes (using biopolymers such as chitosan/alginate, nanomaterials such as reduced graphene oxide), extremophilic bioprocessing, and biofilm engineering strategies for enhancing the biofilm formation and performance of bio-electrochemical systems will be discussed. This talk will also cover the applications of biofilm for energy and environment.

Keywords: Electrochemical devices, Electrode materials, Bioelectricity, Biofilm Engineering, Biopolymers

How to cite: Sani, R.: Biofilm Engineering Approaches for Improving the Performance of Bioelectrochemical Systems, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-126, https://doi.org/10.5194/biofilms9-126, 2020.

Static incubation of liquid microcosms results in a physically heterogeneous environment, where depletion of O₂ in the lower region creates a relatively high-O₂ niche directly below the air-liquid (A-L) interface. This has been investigated using the model bacterium Pseudomonas fluorescens SBW25 and the biofilm-forming adaptive mutant known as the Wrinkly Spreader. In this system, colonisation of the A-L interface by the Wrinkly Spreader provides a fitness advantage over non-biofilm-forming competitors, including the ancestral SBW25, due to better access to O₂ in an otherwise O₂-growth limiting environment. Our current research seeks to understand how the ecological interactions of this simple system applies in more complex communities, where biofilms can be produced by multiple competing or co-operative strains and the low-O₂ region colonised by a range of strains capable of micro-aerobic growth. Here we report the effect of selection on the productivity of A-L interface biofilm-forming communities initiated by soil-wash (SW) inocula, which were serially transferred across ten microcosms and sixty days with mixed-community or biofilm-only samples. Initial analysis of the serial transfer experiments shows a decrease in community productivity which is explained by the accumulation of toxic metabolites, though small increases in community biofilm strength and attachment were also observed. Isolate-level analysis revealed a decrease in community diversity and a biofilm-associated phenotypic shift between the SW inocula and final-transfer communities, and these changes provide evidence of selection within our system.

Cell-localisation experiments confirm enrichment at the top of the liquid column in the high-O₂ region, but also show high cell densities in the low-O₂ region, even within the biofilm-only final-transfer communities. Samples taken from the biofilm and lower region of these communities were able to re-colonise both in fresh microcosms, indicating that community members were capable of migration within the liquid column. Despite the over-all decrease seen in community productivity in the serial transfer experiments, we suggest that communities maximised productivity by colonising both regions of the liquid column, with a resource trade-off between fast growth in the highly competitive high-O₂ region and slower growth in the less-competitive low-O₂ region. Many isolates from the final-transfer communities could occupy both regions and were capable of migration, with almost all isolates capable of flagella-mediated motility, and we interpret this ability to move between regions as a fitness advantage in A-L interface biofilm-forming communities. Although we have not been able to test this directly using the final-transfer communities or isolates, we have been able to demonstrate a fitness advantage in the less complex P. fluorescens SBW25 system, where biofilm-forming mutants capable of colonising both regions had a greater competitive fitness advantage over those with a poor ability to colonise the liquid column.

How to cite: Jerdan, R., Donaldson, E., Cameron, S., and Spiers, A.: Biofilm and productivity-associated community changes in serial-transfer experiments in heterogeneous liquid microcosms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-17, https://doi.org/10.5194/biofilms9-17, 2020.

Renewable raw materials from agriculture help to reduce greenhouse gases, as they expand the CO₂ cycle and release less greenhouse gases than fossil raw materials when used for energy purposes. However, since high levels of nitrous oxide are released, especially by organic fertilization, no emission-free production of renewable raw materials is possible so far. Accordingly, there is an urgent need to replace organic and mineral fertilizers with nitrogen-binding systems. Here, diazotrophic (atmospheric nitrogen fixing), terrestrial cyanobacteria provide a way to fix atmospheric nitrogen and pass it on to plants for the production of renewable raw materials. Especially terrestrial cyanobacteria grow embedded in a thick matrix of extracellular polymeric substances which can contribute to a desirable soil stabilization and thus protection against soil erosion and to promoting water retention in the soil.

The main goal of this research is the sustainable establishment of nitrogen-fixing terrestrial cyanobacteria, which are immobilized on biodegradable carriers, in the ground in agriculture. Based on the aerosol-based photobioreactors constructed and established at the chair of bioprocess engineering, a new reactor system for the cultivation of cyanobacteria on carriers was developed and characterized. In addition, a screening for potential diazotrophic terrestrial cyanobacteria including the formation from vegetative cells to heterocysts (nitrogen fixing cells), nitrogen uptake and release rates for the use in the German agricultural economy was performed. A proof-of-principle to prove the use of terrestrial cyanobacteria as fertilizer for a climate-friendly production of renewable resources was tested by sowing seeds of Arabidopsis thaliana on agar plates with and without nitrogen as well as with and without a diazotrophic phototrophic biofilm.

This project is financially supported by the def DFG (UL 170/16-1) and the “Landesforschungsschwerpunkt NanoKat”.

How to cite: Strieth, D., Stiefelmaier, J., Di Nonno, S., Kronenberger, D., and Ulber, R.: Climate-friendly production of renewable resources by the innovative use of diazotrophic, terrestrial cyanobacteria, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-25, https://doi.org/10.5194/biofilms9-25, 2020.

The aim of this study was to establish cathodic biofilms of the photosynthetic non sulfur purple bacterium Rhodobacter sphaeroides as biocatalyst for the production of platform chemicals from carbon dioxide as carbon source and an electrical current as energy and electron source.  Therefore, R. sphaeroides was cultivated in a bioelectrical system (BES) in which light, CO₂ and a stable current were provided. Chronopotentiometric measurements revealed the cathode potential necessary to maintain the applied current of I = 22,2 µA/cm². Interestingly, exposure of R. sphaeroides to the antibiotic kanamycin lead to increased biofilm production on the cathode although the organism expressed the necessary resistance marker. This enhanced biofilm production raised the potential by 170 mV to E = -1 V compared to the wildtype (E = -1,17 V) and hence increased the efficiency of the process. To date, the molecular basis of this effect remains unclear and is under investigation using a proteomic approach. To elucidate, if the productivity of R. sphaeroides as a production strain is also enhanced, the production of acetoin was established as proof of principle. After the confirmation of the acetoin production under autotrophic conditions, various approaches to increase the space-time yields of the process were conducted and their effect will be presented.  

How to cite: Schmid, F. and Gescher, J.: Electrode assisted production of platform chemicals in Rhodobacter sphaeroides., biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-135, https://doi.org/10.5194/biofilms9-135, 2020.

The use of phototrophic cyanobacteria in biotechnology is highly interesting as they represent a carbon neutral production platform, relying mainly on carbon dioxide, light and water for growth. However, one key bottleneck for utilizing cyanobacteria as production hosts is that in the currently established cultivation systems like tube or flatpanel reactors only cell densities of 2 to 4 g_CDW/L are possible, which is at least 20 times too low for most applications. One promising concept to solve this shortcoming is the cultivation of such microbes as dual trophies biofilms in microtubular systems in a segmented flow fashion with air bubbles, as recently reported in [1]. According to the aspects mentioned in Posten et. al [2], it becomes clear that the concept fulfils most requirements for photo-bioreactors. Firstly, the surface area to volume ratio is increasing with decreasing tube diameter. Hence, the path of the light through the reactor is reduced, leading to an optimal light supply. Secondly, using air segments increases the mixing within the reactor leading to a better supply of the cells with a carbon source as well as a better extraction of oxygen. Apart from that, the attached biofilm provides continuous cell regeneration and thus a continuous production system. All these aspects lead to a biomass concentration in this reactor system of up to 60 g_CDW/L [1].

The microtubular system was successfully applied in the challenging conversion of cyclohexane to cyclohexanol [1]. The reaction was conducted in a small lab scale system utilizing capillaries of 20 cm length, with a total volume of 1.4 mL. Here, we are evaluating the impact of larger scale on biofilm performance. Experiments were conducted in 1 m capillaries with 3 mm inner diameter. First, the impact of different flow rates was investigated. Results show, that a total minimal flow rate of 104 µL/min (52 µL air and 52 µL medium /min) leads to a significant biofilm detachment in various positions in the tube after one week of cultivation. A total flow rate of 520 µL/min (260 µL air and 260 µL medium /min) prevents detachment, however, it seems to hinder full surface coverage of the tube. An optimal condition turned out to be a cultivation of the biofilm with a starting flowrate of 520 µL/min for the initial attachment of the cells and a consecutive decrease of the flow to 104 µL/min after one week of cultivation. Thereby biofilm detachment was prevented and full surface coverage was achieved, while scaling the system by 5 fold. Respective data will be presented and discussed.

[1] Hoschek, Heuschkel, Mixed-species biofilms for high-cell-density application of Synechocystis sp. PCC 6803 in capillary reactors for continuous cyclohexane oxidation to cyclohexanol, Bioresource Technology, 2019

[2] Posten, Design principles of photo-bioreactors for cultivation of microalgae, Engineering in Life Sciences, 2009

How to cite: Kenkel, A., Schmid, A., Karande, R., and Bühler, K.: First steps for the scale up of a dual trophies microtubular biofilm reactor - preventing biofilm detachment, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-13, https://doi.org/10.5194/biofilms9-13, 2020.