7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Chair

Professor

Understanding microbially induced calcite formation by Bacilli for application in bio-based, green construction materials

Biotechnology & Synthetic Microbiology

The construction industry, particularly the use of cement-based materials, has become a concern amid growing unrest over climate issues. Cement production and use accounts for 8% of global anthropogenic CO2 emissions. One exciting approach to reduce the use of cement is to take advantage of Microbially Induced Calcite Precipitation (MICP). This describes the ability of some bacteria to form calcium carbonate minerals, specifically calcite, the main component of limestone. Application of spore-forming MICP-bacteria into concrete, for example, can be used to develop self-healing technologies that autonomously heal microcracks in structures and thus prevent further deterioration and the need for repairs. Other uses include consolidation of soils, or, more recently, the development of engineered living materials, where bacterial activity replaces the use of cement entirely, e.g. for the production of bricks.A major drawback of current technologies is the use of expensive complex nutrients, which leads to high costs of the biobased materials, reducing economic feasibility. Optimisation, however, is hampered by a limited understanding of the molecular and physiological mechanisms of MICP. Here, we report on detailed profiling of environmental Bacillus sp. and close relatives to identify optimally suited MICP-strains. Exploration of the relationship between MICP and the metabolism of organic acids showed that acetate metabolism is a key driver of precipitation in some species, leading to rapid and almost complete precipitation of soluble calcium nitrate to insoluble calcite. Our findings further indicate that calcium homeostasis in bacteria may play a role in MICP. Detailed insights into how bacteria contribute to or even control the formation of calcite minerals opens the way for targeted design of economically viable self-healing or de novo-built construction materials. Bio-based cement alternatives could thus pave the road towards a greener, more sustainable infrastructure of the future.

Oral Presentation

Prof. Dr.

Designing a genetic switch for controlled magnetosome formation in Magnetospirillum gryphiswaldense

The alphaproteobacterium Magnetospirillum gryphiswaldense biosynthesizes magnetosomes, which consist of membrane-enveloped magnetite crystals aligned in linear chains. As one of only few tractable magnetic bacteria it has emerged as a widely used model for the study of prokaryotic organelle formation and the bioproduction of magnetic nanoparticles. However, analysis and engineering of magnetosome biogenesis has been hampered by the limited toolset for controlled gene expression in this bacterium.Here, we investigated novel candidate promoters for strong and tunable gene expression. Transcription from a set of putative and known promoters from M. gryphiswaldense and other bacteria was explored in M. gryphiswaldense using the luxABCDE luciferase operon as reporter. The resulting bioluminescence was measured in the absence and presence of cognate repressors. Among all tested promoters, Pcym and cymR, a promoter-repressor system derived from Pseudomonas putida, caused the highest expression of lux genes, thereby exceeding the activity of all currently used constitutive and inducible foreign and endogenous promoters in M. gryphiswaldense such as PmamG, Plac and Ptet. Furthermore, Pcym was tightly repressed in the absence of its inducer cumate, but could be rapidly induced at different cumate concentrations.To explore cymR/Pcym for the induced expression of single genes, we placed this system in front of mamI, mamE, mamL, mamM, mamO, mamQ, mamB, respectively, all encoding essential magnetosome proteins. Wildtype-like magnetosome formation could be restored in non-magnetic single deletion mutants in the presence of the inducer. By varying the conditions, we are currently exploring the system for the tuned (over)expression of entire accessory magnetosome gene clusters, such as mms6, mamGFDC and mamXYZ. This might enable the tuning of size, shape, and number of magnetosome crystals. In conclusion, this novel genetic tool is expected to be used for functional analysis and engineering of prokaryotic organelle formation, specifically to identify the influential parameters for controlled magnetosome biogenesis in M. gryphiswaldense.

Genetic engineering of Agrobacterium increases production of the industrially useful polymer curdlan from apple waste

Curdlan is a water-insoluble polymer that has structure and gelling properties that are useful in a wide variety of applications such as in medicine, cosmetics, packaging and the food and building industries. The capacity to produce curdlan has been detected in certain soil-dwelling bacteria of various phyla, although the role of curdlan in their survival remains unclear. One of the major limitations of wider use of curdlan in industry is the high cost of production during fermentation, partly because production involves specific nutritional requirements such as nitrogen limitation. For broader application in industry, the production of curdlan needs to be significantly cheaper and avoid competing with human nutritional resources such as glucose, sucrose or starch. Engineering of the industrially relevant curdlan-producing strain Agrobacterium sp. ATTC31749 is a promising approach that could decrease the cost of production so that agricultural waste flows could be used as fermentation substrates. Here, during investigations on curdlan production, it was found that curdlan was deposited as a capsule. Curiously, only a part of the bacterial population produced a curdlan capsule. This heterogeneous distribution appeared to be due to the activity of Pcrd, the native promoter responsible for the expression of the crdASC biosynthetic gene cluster. To improve curdlan production, Pcrd was replaced by a promoter (PphaP) from another Alphaproteobacterium, Rhodobacter sphaeroides. Compared to Pcrd, PphaP was stronger and only mildly affected by nitrogen levels. Consequently, PphaP dramatically boosted crdASC gene expression and curdlan production. Importantly, the genetic modification overrode the strict nitrogen depletion regulation that presents a hindrance for maximal curdlan production from complex media. With this biotechnological innovation, apple pomace was used as an example of agricultural waste for curdlan production and demonstrated excellent potential for achieving high yields using cheap substrates under relaxed fermentation conditions.

Development of THERMOphilic microorganisms for SYNgas CONversion to biobased fuels and chemicals

The decreasing availability of fossil fuels and the consequences of climate change increase the demand for biofuels and bioproducts from renewable resources and waste within a circular economy. Syngas, which contains various ratios of CO2, CO, and H2, is generated as waste gas in industrial processes. Acetogenic bacteria convert syngas into value-added carbon-based chemicals. Given the high temperatures of these industrial gas streams, syngas conversions (fermentations) at temperatures between 50–80°C are attractive. Within the BMBF-funded consortium ThermoSynCon, four partners from different German universities bundled their expertise in a research consortium to establish a syngas fermentation production platform using new thermophilic isolates or engineered strains.Within the project, we enriched and isolated new thermophilic acetogenic bacteria and created a strain collection of thermophilic acetogens. Small-scale growth studies and fermentation studies of the new isolates on H2+CO2, CO, and syngas were performed, and the isolates were characterized and compared in their performance to the few known thermophilic acetogens, including the promising species T. kivui (TOPT 66°C) and M. thermoacetica. We improved the bioenergetics models for T. kivui and M. thermoacetica based on detailed omics, biochemical, physiological, and genetic studies. We developed bioreactor technologies for (syn)gas fermentation at elevated temperatures (50–80°C), optimized the process control, and evaluated the performance of T. kivui, and M. thermoacetica in bioreactors. Genetic tools for T. kivui were improved, with the goal of gaining a fundamental understanding of its physiology and constructing superior strains to produce alcohols from syngas. We subsequently genetically engineered T. kivui for ethanol production and tested the strains in the bioreactor, achieving a 50-mM titer in a continuous bioreactor.Overall, the project provided deep insights into the metabolism of thermophilic acetogens, and proof-of-concept results of syngas conversion to ethanol in a continuous bioreactor. The road is paved towards a thermophilic syngas fermentation platform!

Novel multi-kingdom consortium for the production of antimicrobial caproic acid

Microbial fermentation systems can be designed to produce specific short chain carboxylic acids. The strong antimicrobial caproic acid can be produced by strains of the strictly anaerobe Clostridium kluyveri by chain elongation using acetate and ethanol as electron donor. We hypothesized that multi-kingdom microbial consortium composed of C. kluyveri, the heterofermentative Limosilactobacillus reuteri and the yeast Saccharomyces cerevisiae leads to the production of caproic acid in presence of glucose and fructose.Fermentations were performed in 400 ml bioreactors at 37°C and pH 6.8. We carried out first fed batch fermentation consisting of a bacterial consortium (BC) of L. reuteri and C. kluyveri with external addition of ethanol, and afterwards batch fermentations with a multi-kingdom consortium (MKC) of L. reuteri, C. kluyveri and S. cerevisiae. Substrate consumption and metabolite production were quantified by HPLC-RI, UHPLC-DAD and confirmed by 1H NMR, and the cells growth was monitored by qPCR. The minimum inhibitory dilution fold 50 % (MIDF50) value of the fermentates was tested against Salmonella enterica subsp. enterica CCM 4420. Fermentations were started with L. reuteri or L. reuteri with S. cerevisiae to initiate acetate and ethanol production. C. kluyveri was inoculated 24 h later in both BC and MKC.During 12 days of fermentation, only L. reuteri increased in cell counts by 1.5±0.7 and 0.7±0.1 log cells/ml in BC and MKC, respectively. Acetic acid levels were highest (about 12-18 mM) between days 2-5. In the same period, the concentration of butyric acid increased, and after 5-6 days of fermentation caproic acid was detected. After 12 days, 25.3± 0.1 and 31.2±11.0 mM of caproic acid were produced by BC and MKC, respectively. In addition to caproic acid, the fermentates contained lactic acid (116-108 mM), butyric acid (4-6.8 mM), propionic acid (4.1-5.1 mM) and ethanol (9.7-10.2 mM). Fermentates produced by BC and MKC showed high inhibitory effects against S. enterica.We designed a self-contained process to produce fermentates containing antimicrobial caproic acid by a novel multi-kingdom microbe consortium with potential in food preservation.

Engineering of a sticky therapeutic microbe

Innovative strategies are needed in the permanent fight against microbial pathogenic infections. Utilization of antibiotics is limited by the spread of resistant strains, the slow rate of new antibiotics discoveries and the deleterious off-target effect of these antibiotics against our microbiome. Probiotic strains represent an appealing alternative to antibiotics but suffer from weak efficacy and a poorly understood mode of action. In an effort to combine the best of the two strategies, the concept of therapeutic microbes has been developed in the last years. Therapeutic microbes are commensal organisms genetically engineered to have an enhanced therapeutic effect and they can be designed to specifically combat a microbial pathogenic strain. Thanks to synthetic biology tools, these natural hosts of our microbiome can be tuned to produce bioactive molecules against a wide variety of pathogens in situ. For that purpose, they can be upgraded by the integration of genetic elements called modules allowing detection of a target or ensuring biocontrol of the modified organism. In our lab, we work on implementing a cell-cell adhesion module, i.e. allow the specific attachment of our therapeutic microbe to a microbial target. With this module, higher bioactive compound concentrations can be achieved near the target and the therapeutic potential of our microbe will be enhanced. In a first proof of principle experiment, we describe the engineering of an adhesion module for the specific binding of the probiotic Escherichia coli Nissle to the fungal pathogen Candida albicans. Through membrane display of diverse carbohydrate binding proteins, we can show tight adhesion of the bacteria to the fungi hyphae. Adhesion between two members of the microbiome would not only be interesting to treat microbial infections but could also help to better understand mutualistic or antagonistic relationships inside the microbiome. Moreover, alternative adhesion modules could be engineered to target abiotic surfaces or different parts of the human body, therefore opening the way for innovative medical applications.

Oral Session
Biotechnology & Synthetic Microbiology 1

Appointment

Chair

Topic

Programme

Oral SessionBiotechnology & Synthetic Microbiology 1

Appointment

Chair

Topic

Programme

Oral Session
Biotechnology & Synthetic Microbiology 1