Investigation into struvite precipitation: A commonly encountered problem during fermentations on chemically defined media.

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 (MgNH4 PO4 × 6H2 O). 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.

Methods for the Analysis of Polyphosphate in the Life Sciences.

Inorganic polyphosphate (polyP) is the polymer of orthophosphate and can be found in all living organisms. For polyP characterization, one or more of six parameters are of interest: the molecular structure (linear, cyclic, or branched), the concentration, the average chain length, the chain length distribution, the cellular localization, and the cation composition. Here, the merits, limitations, and critical parameters of the state-of-the-art methods for the analysis of the six parameters from the life sciences are discussed. With this contribution, we aim to lower the entry barrier into the analytics of polyP, a molecule with prominent, yet often incompletely understood, contributions to cellular function.

Biotechnological synthesis of water-soluble food-grade polyphosphate with Saccharomyces cerevisiae.

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2-40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO3 , in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, 31 P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP.

Polyphosphate Chain Length Determination in the Range of Two to Several Hundred P-Subunits with a New Enzyme Assay and 31P NMR.

Currently, 31P NMR is the only analytical method that quantitatively determines the average chain length of long inorganic polyphosphate (>80 P-subunits). In this study, an enzyme assay is presented that determines the average chain length of polyphosphate in the range of two to several hundred P-subunits. In the enzyme assay, the average polyP chain length is calculated by dividing the total polyphosphate concentration by the concentration of the polyphosphate chains. The total polyphosphate is determined by enzymatic polyphosphate hydrolysis with Saccharomyces cerevisiae exopolyphosphatase 1 and S. cerevisiae inorganic pyrophosphatase 1, followed by colorimetric orthophosphate detection. Because the exopolyphosphatase leaves one pyrophosphate per polyphosphate chain, the polyphosphate chain concentration is assayed by coupling the enzymes exopolyphosphatase (polyP into pyrophosphate), ATP sulfurylase (pyrophosphate into ATP), hexokinase (ATP into glucose 6-phosphate), and glucose 6-phosphate dehydrogenase (glucose 6-phosphate into NADPH), followed by fluorometric NADPH detection. The ability of 31P NMR and the enzyme assay to size polyP was demonstrated with polyP lengths in the range from 2 to ca. 280 P-subunits (no polyP with a longer chain length was available). The small deviation between methods (-4 ± 4%) indicated that the new enzyme assay performed accurately. The limitations of 31P NMR (i.e., low throughput, high sample concentration, expensive instrument) are overcome by the enzyme assay that is presented here, which allows for high sample throughput and requires only a commonly available plate reader and micromole per liter concentrations of polyphosphate.

Saccharomyces cerevisiae containing 28% polyphosphate and production of a polyphosphate-rich yeast extract thereof.

Currently, inorganic polyphosphate is chemically synthesized from phosphate rock and added directly to food products. Yeast extract is a concentrate of soluble fractions of Saccharomyces cerevisiae and is, as a food additive, generally regarded as safe. The aim of this study was to biotechnologically produce a naturally polyphosphate-rich yeast extract. Polyphosphate-rich cells were produced with a wild type (non-genetically modified) S. cerevisiae by orthophosphate-starvation and subsequent orthophosphate-feeding, and contained 28% (w/w) polyphosphate (as KPO3) in cell dry weight, which is the highest content reported so far. Four yeast extract production protocols (autolysis, plasmolysis, enzymatic hydrolysis without and with prior heat inactivation) were tested, whereas the latter was the most promising. From the polyphosphate-rich cells, yeast extract paste and powder were produced containing 20% and 14% (w/w, as KPO3) polyphosphate with an average chain length of 31 and 3 P-subunits, 7% and 14% (w/w, as K1.5H1.5PO4) orthophosphate, 22% and 0% (w/w) water, respectively. For the first time, naturally polyphosphate-rich yeast extracts were produced, which possibly can be used as a clean-label food additive and biological alternative to chemically synthesized polyphosphate in food products.

Enzymatic quantification and length determination of polyphosphate down to a chain length of two.

Polyacrylamide gel electrophoresis, being the current method of choice for length determination of inorganic polyphosphate (polyP), requires a sequencing apparatus, relies on commercially not available polyP length standards and yields only a chain length distribution. State of the art polyP quantification involves enzymatic hydrolysis of polyP to orthophosphate with the Saccharomyces cerevisiae exopolyphosphatase 1 (scPpx1p) and subsequent colorimetric orthophosphate detection. Because scPpx1p leaves one pyrophosphate per polyP, short chain polyPs are only partially detected. To overcome this analytical limitation, a method involving both the scPpx1p and the S. cerevisiae inorganic pyrophosphatase (scIpp1p) is proposed. Differential enzymatic hydrolysis of polyP with scPpx1p, and a combination of scIpp1p and scPpx1p allows not only for comprehensive quantification of polyP (excluding cyclic polyP) down to a chain length of two, but also absolute average chain length determination in the range of two to approximately 80. An optimized one-reagent method for rapid (2 min) orthophosphate quantification is part of the assay. Biological phosphorous containing molecules at equimolar phosphorous concentrations regarding polyP do not interfere. The method requires 1.5 µg polyP and calls only for a plate reader. This is the first enzymatic method for simultaneous average polyP chain length determination as well as comprehensive quantification.

Analytical polyphosphate extraction from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, inorganic polyphosphate is analyzed by polyphosphate extraction and subsequent quantification. Recently, we developed a method for polyphosphate quantification, and length determination of short chain polyphosphate. However, the lack of a simple, optimized and validated method for analytical polyphosphate extraction has both hindered the advance in this research field, and prevented comparability of results between laboratories. Hence, the goal of this study was to develop an analytical method for polyphosphate extraction from S. cerevisiae. Several literature methods were compared with special attention to omission of polyphosphate precipitation steps, because these work neither at low polyphosphate concentrations nor quantitatively. The best literature protocol, which takes 5.5 h and requires five reaction tubes per sample, was optimized here in regards to the amount of extracted polyphosphate and simplification of the work flow. The final protocol extracts 40 % more polyphosphate than the best literature method, takes only 30 min, requires just one reaction tube per sample, and is, therefore, proposed as the new gold standard for analytical polyphosphate extraction from S. cerevisiae. In combination with our recently published polyphosphate quantification method, total polyphosphate in S. cerevisiae can now be analyzed within 2 h.