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Background: Biofilm formation on implant-abutment surfaces can cause inflammatory reactions. Ethical concerns often limit intraoral testing, necessitating preliminary in vitro or animal studies. Here, we propose an in vitro model using human saliva and hypothesize that this model has the potential to closely mimic the dynamics of biofilm formation on implant-abutment material surfaces in vivo. Methods: A saliva stock was mixed with modified Brain-Heart-Infusion medium to form biofilms on Titanium-Aluminum-Vanadium (Ti6Al4V) and Yttria-partially Stabilized Zirconia (Y-TZP) discs in 24-well plates. Biofilm analyses included crystal violet staining, intact cell quantification with BactoBox, 16S rRNA gene analysis, and short-chain fatty acids measurement. As a control, discs were worn in maxillary splints by four subjects for four days to induce in vivo biofilm formation. Results: After four days, biofilms fully covered Ti6Al4V and Y-TZP discs both in vivo and in vitro, with similar cell viability. There was a 60.31% overlap of genera between invitro and invivo biofilms in the early stages, and 41% in the late stages. Ten key oral bacteria, including Streptococcus, Haemophilus, Neisseria, Veillonella, and Porphyromonas, were still detectable in vitro, representing the common stages of oral biofilm formation. Conclusion: This invitro model effectively simulates oral conditions and provides valuable insights into biofilm dynamics. [ABSTRACT FROM AUTHOR]

Polyphosphate (polyP) is an intriguing molecule that is found in almost any organism, covering a multitude of cellular functions. In industry, polyP is used due to its unique physiochemical properties, including pH buffering, water binding, and bacteriostatic activities. Despite the importance of polyP, its analytics is still challenging, with the gold standard being 31P NMR. Here, we present a simple staining method using the fluorescent dye JC‐D7 for the semi‐quantitative polyP evaluation in yeast extracts. Notably, fluorescence response was affected by polyP concentration and polymer chain length in the 0.5–500 µg/mL polyP concentration range. Hence, for polyP samples of unknown chain compositions, JC‐D7 cannot be used for absolute quantification. Fluorescence of JC‐D7 was unaffected by inorganic phosphate up to 50 mM. Trace elements (FeSO4 > CuSO4 > CoCl2 > ZnSO4) and toxic mineral salts (PbNO3 and HgCl2) diminished polyP–induced JC‐D7 fluorescence, affecting its applicability to samples containing polyP–metal complexes. The fluorescence was only marginally affected by other parameters, such as pH and temperature. After validation, this simple assay was used to elucidate the degree of polyP production by yeast strains carrying gene deletions in (poly)phosphate homeostasis. The results suggest that staining with JC‐D7 provides a robust and sensitive method for detecting polyP in yeast extracts and likely in extracts of other microbes. The simplicity of the assay enables high‐throughput screening of microbes to fully elucidate and potentially enhance biotechnological polyP production, ultimately contributing to a sustainable phosphorus utilization. Take‐Away: The commercially available fluorescence sensor JC‐D7 was used to establish a fast, highly sensitive polyP detection assay tailored for yeast samples.First‐time evaluation of JC‐D7's robustness to various abiotic factors, including polyP counteracting metal cations and polyP extraction agents.Pre‐screening of yeast strains and rapid classification based on JC‐D7 polyP detection.Researchers and industry professionals can benefit from this powerful, easy‐to‐use method for detecting polyphosphates in yeasts and other organisms. [ABSTRACT FROM AUTHOR]

Chemically defined mineral media are widely used in bioprocesses, as these show less batch to batch variation compared with complex media. Nonetheless, the recommended media formulations often lead to the formation of precipitants at elevated pH values. These precipitates are insoluble and reduce the availability of macronutrients to the cells, which can result in limiting growth rates and lower productivity. They can also damage equipment by clogging pipes, hoses, and spargers in stirred tank fermenters. In this study, the observed precipitate was analyzed via X‐ray fluorescence spectroscopy and identified as the magnesium ammonium phosphate salt struvite (MgNH4PO4 × 6H2O). The solubility of struvite crystals is known to be extremely low, causing the macronutrients magnesium, phosphate, and ammonium to be bound in the struvite crystals. Here, it was shown that struvite precipitates can be redissolved under common fermentation conditions. Furthermore, it was found that the struvite particle size distribution has a significant effect on the dissolution kinetics, which directly affects macronutrient availability. At a certain particle size, struvite crystals rapidly dissolved and provided unlimiting growth conditions. Therefore, struvite formation should be considered during media and bioprocess development, to ensure that the dissolution kinetics of struvite are faster than the growth kinetics. [ABSTRACT FROM AUTHOR]

Screening Pichia pastoris (Komagataella phaffii) strain libraries utilizing the methanol inducible alcohol oxidase 1 (AOX1) promoter requires addition of methanol as an inducer during cultivation. As the AOX1 promoter is repressed in the presence of glucose or glycerol, common screening procedures include long incubation times before methanol induction by successive multiple additions. In this study, a P. pastoris MutS strain, secreting a green fluorescent protein (GFP), was used to characterize a standard screening protocol in complex medium. Based on these results, an auto-induction screening method for P. pastoris strains was developed using mineral SYN6 medium, where methanol was added from the beginning. To gain more insight into growth and production behavior, the cultures were online-monitored, using the Respiration Activity Monitoring System and the BioLector system. Monitoring the oxygen transfer rate indicated that prolonged cultivation phases before induction can be avoided and that high glucose concentrations hinder inducible protein production. The results show the effects of using 20 g/L glucose, and the resulting ethanol formation on the screening process. The newly developed auto-induction method was then used to screen and rank a strain library of high, medium, and low GFP producers. These results demonstrate that the auto-induction method facilitates the identification of high producers during primary screening. It is significantly less time consuming and labor intensive. Nonetheless, it leads to the same conclusion as the conventional screening with manual methanol additions. [ABSTRACT FROM AUTHOR]

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a widely used host for recombinant protein production. In this study, a clonal library of P. pastoris MutS strains (S indicates slow methanol utilization) was screened for high green fluorescent protein (GFP) production. The expression cassette was under the control of the methanol inducible AOX promoter. The growth behavior was online-monitored in 48-well and 96-well microtiter plates by measuring the oxygen transfer rate (OTR). By comparing the different GFP producing strains, a correlation was established between the slope of the cumulative oxygen transfer during the methanol metabolization phase and the strain's production performance. The correlation corresponds to metabolic burden during methanol induction. The findings were validated using a pre-selected strain library (7 strains) of high, medium, and low GFP producers. For those strains, the gene copy number was determined via Whole Genome Sequencing. The results were consistent with the described OTR correlation. Additionally, a larger clone library (45 strains) was tested to validate the applicability of the proposed method. The results from this study suggest that the cumulative oxygen transfer can be used as a screening criterion for protein production performance that allows for a simple primary screening process, facilitating the pre-selection of high producing strains. [ABSTRACT FROM AUTHOR]