Online capillary electrophoresis for monitoring carboxylic acid production by yeast during bioreactor cultivations.

Bioprocess monitoring can improve the understanding and control of biotechnological processes. When analyses are carried out as automated online measurements, manual steps of the analysis procedures are avoided, thus decreasing both the time required for analyses and systematic errors. In this study, an online capillary electrophoresis (CE) system with flow-through sample vial made in-house and action control programming was assembled to monitor carboxylic acid production by Kluyveromyces lactis and Saccharomyces cerevisiae during two different bioreactor cultivations. The relative standard deviations were less than 0.6% for intraday migration times and the total analysis time was less than 20 min. The system operated continuously and automatically up to 6 days and produced data concerning carboxylic acid production during the cultivations. The successful test runs demonstrated that this system has potential for the monitoring of biotechnological processes.

Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis.

BACKGROUND: Glycolic acid is a C2 hydroxy acid that is a widely used chemical compound. It can be polymerised to produce biodegradable polymers with excellent gas barrier properties. Currently, glycolic acid is produced in a chemical process using fossil resources and toxic chemicals. Biotechnological production of glycolic acid using renewable resources is a desirable alternative. RESULTS: The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are suitable organisms for glycolic acid production since they are acid tolerant and can grow in the presence of up to 50 g l(-1) glycolic acid. We engineered S. cerevisiae and K. lactis for glycolic acid production using the reactions of the glyoxylate cycle to produce glyoxylic acid and then reducing it to glycolic acid. The expression of a high affinity glyoxylate reductase alone already led to glycolic acid production. The production was further improved by deleting genes encoding malate synthase and the cytosolic form of isocitrate dehydrogenase. The engineered S. cerevisiae strain produced up to about 1 g l(-1) of glycolic acid in a medium containing d-xylose and ethanol. Similar modifications in K. lactis resulted in a much higher glycolic acid titer. In a bioreactor cultivation with D-xylose and ethanol up to 15 g l(-1) of glycolic acid was obtained. CONCLUSIONS: This is the first demonstration of engineering yeast to produce glycolic acid. Prior to this work glycolic acid production through the glyoxylate cycle has only been reported in bacteria. The benefit of a yeast host is the possibility for glycolic acid production also at low pH, which was demonstrated in flask cultivations. Production of glycolic acid was first shown in S. cerevisiae. To test whether a Crabtree negative yeast would be better suited for glycolic acid production we engineered K. lactis in the same way and demonstrated it to be a better host for glycolic acid production.

Reporting Sysmex XN Absolute Neutrophil Count in Samples with Leukocyte Analyzer Flagging.

OBJECTIVE: To provide faster laboratory data reporting, we evaluated the accuracy of Sysmex XN (Sysmex Inc, Kobe, Japan) absolute neutrophil count (ANC) in the presence of analyzer flagging. METHODS: Sysmex XN and manual microscopy ANC were compared with 80 autovalidated control specimens and with 280 study specimens with analyzer flagging regarding immature granulocytes (IG) >3% or other leukocyte abnormalities. Specimens with ambiguous neutrophil clusters were excluded. RESULTS: A slight positive overall method bias was seen for Sysmex XN compared to manual microscopy (n = 280), 0.025 (95% confidence interval [CI], -0.023 to 0.069) × 109/L. With IG > 10% (n = 123) the bias was larger, but not clinically significant, 0.17 (95% CI, 0.060-0.25) × 109/L. No clinically significant difference was seen in neutropenic (ANC < 1.5 × 109/L) specimens (n = 91), 0.070 (95% CI, -0.013 to 0.14) × 109/L. CONCLUSION: These data indicate that Sysmex XN ANC can be reported in the presence of certain analyzer flagging to improve patient care.

Predictive value of plasma proenkephalin and neutrophil gelatinase-associated lipocalin in acute kidney injury and mortality in cardiogenic shock.

BACKGROUND: Acute kidney injury (AKI) is a frequent form of organ injury in cardiogenic shock. However, data on AKI markers such as plasma proenkephalin (P-PENK) and neutrophil gelatinase-associated lipocalin (P-NGAL) in cardiogenic shock populations are lacking. The objective of this study was to assess the ability of P-PENK and P-NGAL to predict acute kidney injury and mortality in cardiogenic shock. RESULTS: P-PENK and P-NGAL were measured at different time points between baseline and 48 h in 154 patients from the prospective CardShock study. The outcomes assessed were AKI defined by an increase in creatinine within 48 h and all-cause 90-day mortality. Mean age was 66 years and 26% were women. Baseline levels of P-PENK and P-NGAL (median [interquartile range]) were 99 (71-150) pmol/mL and 138 (84-214) ng/mL. P-PENK > 84.8 pmol/mL and P-NGAL > 104 ng/mL at baseline were identified as optimal cut-offs for AKI prediction and independently associated with AKI (adjusted HRs 2.2 [95% CI 1.1-4.4, p = 0.03] and 2.8 [95% CI 1.2-6.5, p = 0.01], respectively). P-PENK and P-NGAL levels at baseline were also associated with 90-day mortality. For patients with oliguria < 0.5 mL/kg/h for > 6 h before study enrollment, 90-day mortality differed significantly between patients with low and high P-PENK/P-NGAL at baseline (5% vs. 68%, p < 0.001). However, the biomarkers provided best discrimination for mortality when measured at 24 h. Identified cut-offs of P-PENK24h > 105.7 pmol/L and P-NGAL24h > 151 ng/mL had unadjusted hazard ratios of 5.6 (95% CI 3.1-10.7, p < 0.001) and 5.2 (95% CI 2.8-9.8, p < 0.001) for 90-day mortality. The association remained significant despite adjustments with AKI and two risk scores for mortality in cardiogenic shock. CONCLUSIONS: High levels of P-PENK and P-NGAL at baseline were independently associated with AKI in cardiogenic shock patients. Furthermore, oliguria before study inclusion was associated with worse outcomes only if combined with high baseline levels of P-PENK or P-NGAL. High levels of both P-PENK and P-NGAL at 24 h were found to be strong and independent predictors of 90-day mortality. TRIAL REGISTRATION: NCT01374867 at www.clinicaltrials.gov , registered 16 Jun 2011-retrospectively registered.

Capillary electrophoresis with laser-induced fluorescence detection for studying amino acid uptake by yeast during beer fermentation.

The amino acid composition of cultivation broth is known to affect the biomass accumulation, productivity, and vitality of yeast during cultivation. A separation method based on capillary electrophoresis with laser-induced fluorescence (LIF) detection was developed for the determination of amino acid consumption by Saccharomyces cerevisiae during beer fermentation. Intraday relative standard deviations were less than 2.1% for migration times and between 2.9% and 9.9% for peak areas. Interday relative standard deviations were less than 2.5% for migration times and between 4.4% and 18.9% for peak areas. The quantification limit was even as low as 62.5 pM which equals to below attomole level detection. The method was applied to study the rate of amino acid utilization during beer fermentation.

Capillary electrophoresis for the monitoring of phenolic compounds in bioprocesses.

Hydrolysates of lignocellulosic biomass, used as substrates for the sustainable production of fuels and chemicals often contain high amounts of phenolic compounds inhibiting the production microbiota. Quantification of these inhibitor compounds may help to understand possible difficulties in bioprocessing and further the development of more efficient, robust and tolerable processes. A separation method based on capillary electrophoresis with UV detection was developed for the simultaneous quantification of 10 phenolic compounds that may have inhibitor properties. Intraday relative standard deviations were less than 0.7% for migration times and between 2.6% and 6.4% for peak areas. Interday relative standard deviations were less than 3.0% for migration times and between 5.0% and 7.2% for peak areas. The method was applied to demonstrate that Saccharomyces cerevisiae was able to decrease the concentrations of vanillin, coniferyl aldehyde, syringaldehyde, acetoguaiacone and cinnamic acid during the cultivation, whereas the concentrations of phenols increased.

Capillary electrophoresis for the monitoring of carboxylic acid production by Gluconobacter oxydans.

Determination of carboxylic acids in Gluconobacter oxydans fermentations of wheat straw hydrolyzate was carried out. This matrix is of complex composition containing carbohydrates, organic compounds (e.g., amino acids, toxins), and inorganic salts making the analysis challenging even with separation techniques. A method based on capillary electrophoresis with indirect UV detection was developed for the simultaneous quantification of 18 carboxylic acids. The background electrolyte solution of ammonia, 2,3-pyridinedicarboxylic acid, and Ca2+ and Mg2+ salts, containing myristyltrimethylammonium hydroxide as a dynamic capillary coating reagent, was validated for the robust and repeatable separation of the carboxylic acids. Intraday relative standard deviations in the optimized method were less than 1.6% for migration times and between 1.0% and 5.9% for peak area. Interday relative standard deviations were less than 5.0% for migration times and between 5.7% and 9.3% for peak area. With 11 nl injected, detection limits for the analytes were between 10 and 43 micromol/l. Detection limits ranged from 0.1 to 0.5 pmol at signal-to-noise ratio of 3. The results demonstrated that wheat straw hydrolyzate was a suitable substrate for G. oxydans with a product yield of 45% for the formation of xylonic acid from xylose and 96% for the formation of gluconic acid from glucose.