Handheld methanol detector for beverage analysis: interlaboratory validation.

Methanol is a toxic alcohol contained in alcoholic beverages as a natural byproduct of fermentation or added intentionally to counterfeits to increase profit. To ensure consumer safety, many countries and the EU have established strict legislation limits for methanol content. Methanol concentration is mostly detected by laboratory instrumentation since mobile devices for routine on-site testing of beverages in distilleries, at border stations or even at home are not available. Here, we validated a handheld methanol detector for beverage analysis in an ISO 5725 interlaboratory trial: a total of 119 measurements were performed by 17 independent participants (distilleries, universities, authorities, and competence centers) from six countries on samples with relevant methanol concentrations (0.1, 1.5 vol%). The detector was based on a microporous separation filter and a nanostructured gas sensor allowing on-site measurement of methanol down to 0.01 vol% (in the liquid) within only 2 min by laymen. The detector showed excellent repeatability (<5.4%), reproducibility (<9.5%) and small bias (<0.012 vol%). Additional measurements on various methanol-spiked alcoholic beverages (whisky, rum, gin, vodka, tequila, port, sherry, liqueur) indicated that the detector is not interfered by environmental temperature and spirit composition, featuring excellent linearity (R2 > 0.99) down to methanol concentrations of 0.01 vol%. This device has been recently commercialized (Alivion Spark M-20) with comparable accuracy to the gold-standard gas chromatography and can be readily applied for final product inspection, intake control of raw materials or to identify toxic counterfeit products.

Nitrogen recovery from low-value biogenic feedstocks via steam gasification to methylotrophic yeast biomass.

Carbon and nitrogen are crucial elements for life and must be efficiently regenerated in a circular economy. Biomass streams at the end of their useful life, such as sewage sludge, are difficult to recycle even though they contain organic carbon and nitrogen components. Gasification is an emerging technology to utilize such challenging waste streams and produce syngas that can be further processed into, e.g., Fischer-Tropsch fuels, methane, or methanol. Here, the objective is to investigate if nitrogen can be recovered from product gas cleaning in a dual fluidized bed (DFB) after gasification of softwood pellets to form yeast biomass. Yeast biomass is a protein-rich product, which can be used for food and feed applications. An aqueous solution containing ammonium at a concentration of 66 mM was obtained and by adding other nutrients it enables the growth of the methylotrophic yeast Komagataella phaffii to form 6.2 g.L-1 dry yeast biomass in 3 days. To further integrate the process, it is discussed how methanol can be obtained from syngas by chemical catalysis, which is used as a carbon source for the yeast culture. Furthermore, different gas compositions derived from the gasification of biogenic feedstocks including sewage sludge, bark, and chicken manure are evaluated for their ability to yield methanol and yeast biomass. The different feedstocks are compared based on their potential to yield methanol and ammonia, which are required for the generation of yeast biomass. It was found that the gasification of bark and chicken manure yields a balanced carbon and nitrogen source for the formation of yeast biomass. Overall, a novel integrated process concept based on renewable, biogenic feedstocks is proposed connecting gasification with methanol synthesis to enable the formation of protein-rich yeast biomass.

An investigation into gene copy number determination in transgenic yeast; The importance of selecting a reliable real-time PCR standard.

Nowadays, Pichia pastoris is a well-known yeast for the production of recombinant proteins. The yield of protein production tightly depends on the copy number of the gene of interest into the host chromosome. Real-time PCR has been used as a high throughput method for molecular detection of gene copy number. In light of determining an absolute gene copy number, the reliability of the qPCR quantification standard is a major issue and it can be a potential source of errors in the final results. Since the literature on this issue is inconclusive, we set out to find a reliable quantification method that allows comparing results in different laboratories. We generated standard curves for two genomic loci (5'UTR AOX1 and ARG4) and for plasmid DNA carrying hGM-CSF coding sequence. These data was used to calculate the integrated hGM-CSFcDNA copy number in a recombinant P. pastoris clone. In our expriments the 5'UTR AOX1 gene showed a more accurate quantification standard, based on more efficient amplification and better reproducibility. The results obtained in this study showed that the differences in terms of structure and length between circular plasmid and linear gDNA could be the source of significant differences in the pattern of DNA amplification.

A Novel Methanol-Free Platform for Extracellular Expression of rhGM-CSF in Pichia pastoris.

The production of the recombinant proteins under the control of AOX1 promoter is a one of the most common expression systems in the methylotrophic yeast Pichia pastoris which is induced by methanol. The application of this expression platform is restricted by the toxicity and inflammatory nature of methanol, especially in food and pharmaceutical products. Human granulocyte macrophage-colony stimulating factor (hGM-CSF) is an important pharmaceutical protein, playing a crucial role in the proliferation and differentiation of innate immune cells. In this study, a methanol-free expression platform for extracellular expression of hGM-CSF was developed. To attain this goal, a novel constructed expression vector pEP(α)101, carrying the FMD promoter regulating recombinant expression by glycerol derepression was designed. The optimized hGM-CSF gene was subcloned into pEP(α)101 and transformed into P. pastoris. The expression of rhGM-CSF in three different culture media were investigated. Based on the observed heterogeneous glycosylation pattern on SDS-PAGE and western blot, the glycoproteins were deglycosylated to remove carbohydrate units. According to the results, the novel methanol independent PFMD expression platform would be a suitable candidate for driving heterologous gene expression especially for the production of food-grade and therapeutically important recombinant proteins.