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
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.
Antibiotic treatment regularly fails to cure patients suffering from infections caused by adaptively resistant microbial communities, referred to as biofilms. Even though at least two thirds of all clinical infections are associated with biofilms, there are no biofilm-specific therapies on the market or in clinical trials. Pseudomonas aeruginosa is a remarkably antibiotic resistant, nosocomial pathogen and biofilm-former that causes morbidity and mortality especially in cystic fibrosis and immunocompromised patients. This project aims to identify regulatory genes associated with drug resistance in P. aeruginosa biofilms to provide novel biofilm-specific targets for the design of potent drugs. A genome-wide screen of P. aeruginosa burn wound isolate UCBPP-PA14 identified 362 genes involved in biofilm formation, including dozens of regulatory and hypothetical genes. I will discuss regulatory as well as metabolic genes corresponding to the known resistome of antimicrobials.
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
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Terrestrial cyanobacteria grow quite poorly as suspension culture. This is one of the reasons why they have not yet been considered as producers of interesting metabolites such as antibacterial substances. Previous work in our group have shown that surface-associated growth can significantly increase productivity [1]. Moving bed bioreactor technology, which is already established in wastewater treatment, offers a possibility to carry out such growth on a larger scale. In these reactors, the bacteria grow on the surface of solid structured carrier particles in areas protected from mechanical abrasion (protected surface). These particles are usually about 1-5 cm in size and are made of high-density polyethylene (HDPE). Moving bed processes for microalgae have only been described for fabric as a solid substrate [2] whereby only 30% of the biomass was actually immobilized on the carrier particles. For this reason, different HDPE carrier particles and different cyanobacteria were investigated. Three different cyanobacteria could be successfully cultivated on two different particles in a 1.5-liter photobioreactor in a moving bed. As an up-scale step, a larger reactor was developed, which provided a larger cultivation surface in combination with a sufficient illumination.
Photobioreactor
The design of the reactor is similar to Zhuang et al. [2]. Based on an 80x35x40 cm tank, the reactor has a working volume of 65 liters. At a particle filling degree of 27 %, the reactor has a protected cultivation surface area of 11.26 m² within the particles. This corresponds to 173 m² per m³ reactor volume. Their circulation is generated by a gassing unit on the ground. An inclined plate is installed beside the gassing unit, to avoid a flow dead zone at the bottom of the reactor. The reactor is illuminated by LEDs located outside the reactor. The growth is monitored offline by the determination of the dry biomass (bdm) and the measurement of the biofilm thickness by optical coherence tomography (OCT).
Results
Cultivations with the cyanobacterium Trichocoleus sociatus were carried out. The inoculum was added to the reactor as suspended biomass with a concentration of 0.035 gbdm/L. After two weeks, the complete biomass was immobilized as a thin biofilm on the carrier particles. Between day 18 and day 45, an increase in the median biofilm thickness from 36 µm to 65 µm could be measured with an increase of the dry biomass from 0.44 to 1.56 g/L. This volume-specific yield is similar to cultivations in the 1.5-liter photobioreactors with carrier particles.
Funding
The project is financially supported by the DFG (Project number UL 170/16-1) and the Ministry of Education of Rhineland-Palatinate (bm.rlp) (iProcess intelligent process development – from modelling to product)
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