Biofilms最新文献

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).

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 Scien