Abstract Phosphate is mined from phosphate rock, which is a limited resource on a human time scale. For a sustainable phosphate supply, strategies for efficient use and recycling of phosphate must be developed. A German chemical company produces annually wash water containing phosphate and other inorganic substances (e.g., sodium, potassium, sulfate, and chloride) at a ton scale. Chemical precipitation is mostly used for phosphate removal. In this study, a biotechnological process utilizing Saccharomyces cerevisiae to upcycle phosphate‐containing wastewater into pure sodium polyphosphate in powder form was developed. The process comprises fermentation and downstream processing (polyphosphate yields: 25% and 36%, respectively). The polyphosphate quality was independent of the wash water composition. Polyphosphate with a purity of 23% molar ratio Na to Na, K, and Mg of > 90%, and with an average chain length of 12.5 phosphate subunits was produced. The upcycled polyphosphate can be reused compared to phosphate fertilizer in many different applications. Overall, the here developed process can contribute to truly slowing down phosphate mining and finally enable a sustainable utilization of phosphate. Thereby, the benefit of the process is the cascade use of phosphate, reducing the need for phosphate rock before the phosphate ends up in the soil and ultimately in the sea.
Abstract 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 Mut S 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.
High gene expression of enzymes partaking in recombinant production pathways is a desirable trait among cell factories belonging to all different kingdoms of life. High enzyme abundance is generally aimed for by utilizing strong promoters, which ramp up gene transcription and mRNA levels. Increased protein abundance can alternatively be achieved by optimizing the expression on the post-transcriptional level. Here, we evaluated protein synthesis with a previously proposed optimized gene expression architecture, in which mRNA stability and translation initiation are modulated by genetic parts such as self-cleaving ribozymes and a bicistronic design, which have initially been described to support the standardization of gene expression. The optimized gene expression architecture was tested in Pseudomonas taiwanensis VLB120, a promising, novel microbial cell factory. The expression cassette was employed on a plasmid basis and after single genomic integration. We used three constitutive and two inducible promoters to drive the expression of two fluorescent reporter proteins and a short acetoin biosynthesis pathway. The performance was confronted with that of a traditional expression cassette harboring the same promoter and gene of interest but lacking the genetic parts for increased expression efficiency. The optimized expression cassette granted higher protein abundance independently of the expression basis or promoter used proving its value for applications requiring high protein abundance.
A multi-step reductive approach for the selection ofin situextraction solvents for bioprocesses was developed enabling a foam-free biotechnological production of rhamnolipids in stirred-tank reactors.
Volatile organic compounds play an essential role in every domain of life, with diverse functions. In this study, we use novel secondary electrospray ionisation high-resolution Orbitrap mass spectrometry (SESI-Orbitrap MS) to monitor the complete yeast volatilome every 2.3 s. Over 200 metabolites were identified during growth in shake flasks and bioreactor cultivations, all with their unique intensity profile. Special attention was paid to ethanol as biotech largest product and to acetaldehyde as an example of a low-abundance but highly-volatile metabolite. While HPLC and Orbitrap measurements show a high agreement for ethanol, acetaldehyde could be measured five hours earlier in the SESI-Orbitrap MS. Volatilome shifts are visible, e.g. after glucose depletion, fatty acids are converted to ethyl esters in a detoxification mechanism after stopped fatty acid biosynthesis. This work showcases the SESI-Orbitrap MS system for tracking microbial physiology without the need for sampling and for time-resolved discoveries during metabolic transitions.
Novel applications of bioelectrochemical systems (BES) are emerging constantly, but the majority still lacks economic viability. Especially the use of electrochemical system components without adaptation to BES requirements causes poor exploitation of the potential system performance. The electrode material is one central component that determines BES performance. While commercial carbon fiber (CF) fabrics are commonly used, their customizability as two- or three-dimensional electrode material for BES is rarely investigated. Using pure cultures of S. oneidensis MR-1, we identified CF properties impacting bacterial current generation: 1. The removal of the sizing (protective coating) is of great importance for all the fibers studied, as it acts as an electrical insulator. By desizing, the maximum current density (jmax) is increased by up to 40-fold. 2. Alteration of the filament surface chemistry results in an accelerated initial development of current generation, but the maximum current density (jmax) is hardly affected. 3. A specific yarn structure, the stretch-broken yarn, supports exceptionally high current densities. The good electrode performance is correlated to the presence of free filament ends (responsible for 41 % current increase), which are characteristic for this yarn. 4. Moreover, a combination of these free filament ends with a high degree of graphitization enhances electrode performance of a commercial fabric by 100 %. The results demonstrate that the CF selection can greatly influence the achievable electrode performance of CF fabrics, and thereby contributes to rational engineering of CF based electrodes that can be tailored for the many BES applications envisaged.
Understanding the composability of genetic elements is central to synthetic biology. Even for seemingly long-time known elements such as a sigma 70 promoter the genetic context-dependent variability of promoter activity remains poorly understood. The lack of understanding of sequence to function results in highly limited de novo design of novel genetic elements combinations. To address this issue, we characterized in detail concatenated synthetic promoters ("stacked") including varying spacer sequence lengths and compared the transcription strength to the output of the individual promoters. The proxy for promoter activity, the msfGFP synthesis was from stacked promoters consistently lower than expected from the sum of the activities of the single promoters. While the spacer sequence itself had no activity, it drastically affected promoter activities when placed up- or downstream of a promoter. Single promoter-spacer combinations revealed a bivalent effect on msfGFP synthesis. By systematic analysis of promoter and spacer combinations, a semi-empirical correlation was developed to determine the combined activity of stacked promoters.
Abstract Itaconic acid is a platform chemical with a range of applications in polymer synthesis and is also discussed for biofuel production. While produced in industry from glucose or sucrose, co‐feeding of glucose and acetate was recently discussed to increase itaconic acid production by the smut fungus Ustilago maydis . In this study, we investigate the optimal co‐feeding conditions by interlocking experimental and computational methods. Flux balance analysis indicates that acetate improves the itaconic acid yield up to a share of 40% acetate on a carbon molar basis. A design of experiment results in the maximum yield of 0.14 itaconic acid per carbon source from 100 glucose and 12 acetate. The yield is improved by around 22% when compared to feeding of glucose as sole carbon source. To further improve the yield, gene deletion targets are discussed that were identified using the metabolic optimization tool OptKnock. The study contributes ideas to reduce land use for biotechnology by incorporating acetate as co‐substrate, a C2‐carbon source that is potentially derived from carbon dioxide.
The bioeconomy is a paramount pillar in the mitigation of greenhouse gas emissions and climate change. Still, the industrialization of bioprocesses is limited by economical and technical obstacles. The synthesis of biosurfactants as advanced substitutes for crude-oil-based surfactants is often restrained by excessive foaming. We present the synergistic combination of simulations and experiments towards a reactor design of a submerged membrane module for the efficient bubble-free aeration of bioreactors. A digital twin of the combined bioreactor and membrane aeration module was created and the membrane arrangement was optimized in computational fluid dynamics studies with respect to fluid mixing. The optimized design was prototyped and tested in whole-cell biocatalysis to produce rhamnolipid biosurfactants from sugars. Without any foam formation, the new design enables a considerable higher space-time yield compared to previous studies with membrane modules. The design approach of this study is of generic nature beyond rhamnolipid production.
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