The formation mechanism by yeast of 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone in Miso

The mechanism of the formation of 4-hydroxy-2(or 5)-ethyl-5-(or 2)-methyl-3(2H)-furanone (HEMF) with yeast under caltivation in a medium containing amino-carbonyl reactants of ribose and glycine was investigated using stable isotopes of the corresponding compounds. It was confirmed that the skeleton of the five-membered ring and the methyl group of the side chain of HEMF was formed from ribose, and that the ethyl group was derived from the glucose metabolite by yeast. The formation of HEMF was confirmed when acetaldehyde as the glucose metabolite and a cell-free extract from yeast were added to the medium containing amino-carbonyl reactants. These results suggest that the role of yeast in HEMF formation is not only to provide the glucose metabolite, but also in combining the amino-carbonyl reactants with the glucose metabolite.     

Screening of High-Level 4-Hydroxy-2 (or 5)-Ethyl-5 (or 2)-Methyl-3(2H)-Furanone-Producing Strains from a Collection of Gene Deletion Mutants of Saccharomyces cerevisiae

4-Hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3(2H)-furanone (HEMF) is an important flavor compound that contributes to the sensory properties of many natural products, particularly soy sauce and soybean paste. The compound exhibits a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. HEMF is produced by yeast species in soy sauce manufacturing; however, the enzymes involved in HEMF production remain unknown, hindering efforts to breed yeasts with high-level HEMF production. In this study, we identified high-level HEMF-producing mutants among a Saccharomyces cerevisiae gene deletion mutant collection. Fourteen deletion mutants were screened as high-level HEMF-producing mutants, and the ADH1 gene deletion mutant (adh1Δ) exhibited the maximum HEMF production capacity. Further investigations of the adh1Δ mutant implied that acetaldehyde accumulation contributes to HEMF production, agreeing with previous findings. Therefore, acetaldehyde might be a precursor for HEMF. The ADH1 gene deletion mutant of Zygosaccharomyces rouxii, which is the dominant strain of yeast found during soy sauce fermentation, also produces HEMF effectively, suggesting that acetaldehyde accumulation might be a benchmark for breeding industrial yeasts with excellent HEMF production abilities.     

Effects of the amino-carbonyl reaction of ribose and glycine on the formation of the 2(or 5)-ethyl-5(or 2)-methyl-4-hydroxy-3(2H)-furanone aroma component specific to miso by halo-tolerant yeast

The formation of HEMF[2(or 5)-ethyl-5(or 2)-methyl-4-hydroxy-3(2H)-furanone], the aroma component specific to miso and soy sauce, was promoted by cultivating the halo-tolerant yeast, Zygosaccharomyces rouxii, in a medium including the amino-carbonyl reaction products based on ribose and glycine. The glucose concentration in the medium influenced the HEMF formation by Z. rouxii.     

Effect of media constituents on the formation by halophilic yeast of the 2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone aroma component specific to miso.

The formation of HEMF [2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone] by yeast was examined in an attempt to investigate its mechanism and involved factors. HEMF formation was promoted by yeast cultivation in a heat-sterilized medium which included glucose, ribose, and a nitrogenous compound such as an extract of shoyu koji, poly-peptone, casamino acid, or an amino acid (glutamic acid, threonine, serine, or alanine).     

Formation by Yeast of the HEMF (4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone) Aroma Component in Miso with Aging

Two kinds of miso, one with added precultured yeast and the other without, were compared with respect to the changes in the concentration of HEMF formed and the number of yeast cells in the process of aging. In miso without added yeast, the HEMF concentration increased with the increasing number of existing yeast cells. In miso without yeast aged for 21 days after the miso mash, 0.06 ppm HEMF was detected when the cell number was 2.2 × 10³ cell/g. In yeast-added miso aged for 7 days after the miso mash, no HEMF was detected, although the number of yeast cells was 1.6 × 10⁶ cell/g. In yeast-added miso aged for 14 days after the miso mash, HEMF was first detected. The pH levels of miso without yeast and with added yeast when HEMF was first detected were 5.59 and 5.57, respectively. It is suggested that the formation of HEMF in miso containing a high concentration of reducing sugar and salt was related to the growth of yeast and started when the pH level fell to less than 5.6.     

In‐situ monitoring of Saccharomyces cerevisiae ITV01 bioethanol process using near‐infrared spectroscopy NIRS and chemometrics

The application feasibility of in‐situ or in‐line monitoring of S. cerevisiae ITV01 alcoholic fermentation process, employing Near‐Infrared Spectroscopy (NIRS) and Chemometrics, was investigated. During the process in a bioreactor, in the complex analytical matrix, biomass, glucose, ethanol and glycerol determinations were performed by a transflection fiber optic probe immersed in the culture broth and connected to a Near‐Infrared (NIR) process analyzer. The NIR spectra recorded between 800 and 2,200 nm were pretreated using Savitzky‐Golay smoothing and second derivative in order to perform a partial least squares regression (PLSR) and generate the calibration models. These calibration models were tested by external validation and then used to predict concentrations in batch alcoholic fermentations. The standard errors of calibration (SEC) for biomass, ethanol, glucose and glycerol were 0.212, 0.287, 0.532, and 0.296 g/L and standard errors of prediction (SEP) were 0.323, 0.369, 0.794, and 0.507 g/L, respectively. Calibration and validation criteria were defined and evaluated in order to generate robust and reliable models for an alcoholic fermentation process matrix. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:510–517, 2016     

On-line determination of glucose and lactate concentrations in animal cell culture based on fibre optic detection of oxygen in flow-injection analysis.

A flow-injection analysis (FIA) system based on fibre optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the on-line monitoring of glucose and lactate concentrations in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by molecular oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fibre optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an r.s.d. of 5% at 30 mM (five measurements) and for 0-30 mM lactate, with an r.s.d. of 5% at 30 mM (five measurements). The enzyme reactors used were stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per hour. The system has been successfully applied to the on-line monitoring of glucose and lactate concentrations of an animal cell culture designed for the production of recombinant human antithrombine III (AT-III). Results of the on-line measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyser from Yellow Springs Instrument Company (YSI).     

Effect of mixed organic substrate on α-tocopherol production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in photoheterotrophic culture

Effects of organic carbon sources on cell growth and alpha-tocopherol productivity in wild and chloroplast-deficient W14ZUL strains of Euglena gracilis under photoheterotrophic culture were investigated. In both strains, the increase in cell growth was particularly high when glucose was added as the sole organic carbon source. On the other hand, alpha-tocopherol production per dry cell weight was enhanced by adding ethanol. Ethanol addition also increased the chlorophyll concentration in wild strain and mitochondria activity in W14ZUL strain. For effective alpha-tocopherol production, the effects of mixture of glucose and ethanol were investigated. The results showed that, when a mixture of glucose (6 g/l) and ethanol (4 g/l) was used, alpha-tocopherol productivity per culture broth was 3.89 x 10(-2) mg l(-1) h(-1), which was higher than the value obtained without addition of organic carbon source (0.92 x 10(-2) mg l(-1) h(-1)). In addition, under fed-batch cultivation using an internally illuminated photobioreactor, the alpha-tocopherol production per culture broth was 23.43 mg/l, giving a productivity of 16.27 x 10(-2) mg l(-1) h(-1).     

Process development with nitrogenase-producing Escherichia coli C-M74 (pUS1) CellS

A process for the continuous fermentation of the genetically modified, nitrogenase-producing Escherichia coli C-M74 (pUS1)-strain has been developed. This strain, which is able to fix molecular nitrogen, has the nifgenes of the bacterium Klebsiella pneumoniae. Cell growth and nitrogenase activity of the enzyme have been optimized both in batch and continuous fermentations. For the fermentations, trial runs were performed by cultivating the E. coli cells in 50-ml culture bottles. The medium composition was varied in order to provide high biomass production and nitrogenase activity. For an effective fermentation control, an on-line analysis was built up for the substrates ammonium and glucose. Other medium components such as ampicillin, citric acid, acetic acid, nitrogenase activity, and protein were measured by using different off-line methods. Modern optical methods like in-line microfluorometry for monitoring the culture fluorescence and laser flow cytometry for the estimation of DNA and protein content were also employed. Plasmid stability was also determined.     

Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control

Alcaligenes eutrophus NCIMB 11599 was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from glucose by the automatic fed-batch culture technique. The glucose concentration of the culture broth was controlled at 10 to 20 g/L by two methods: using exit gas data obtained from a mass spectrometer and using an on-line glucose analyzer. The effect of ammonium limitation on PHB synthesis at different culture phases was studied. The final cell concentration, PHB concentration, and PHB productivity increased as ammonia feeding was stopped at a higher cell concentration. High concentrations of PHB (121 g/L) and total cells (164 g/L) were obtained in 50 h when ammonia feeding was stopped at the cell concentration of 70 g/L. The maximum PHB content reached 76% of dry cell weight and the productivity was 2.42 g/L h with the yield of 0.3 g PHB/g glucose.     

Measurement of ethanol concentration using solvent extraction and dichromate oxidation and its application to bioethanol production process

A method for measuring the ethanol concentration in a yeast culture broth was developed using both microtubes and a 96-deepwell microplate. The strategy involved first the solvent extraction of ethanol from the yeast culture broth and measurements of the ethanol concentration using the dichromate oxidation method. Particular focus was made on selecting the extraction solvent as well as determining the measurable range of ethanol concentrations using this solvent extraction-dichromate oxidation method. This method was developed as an assay format in 2.0-ml microtubes and 1.2-ml 96-deepwell microplates, and the ethanol concentration in the batch cultures and fed-batch fermentations was measured. Tri-n-butyl phosphate [non-alcoholic solvent, density = 0.9727, solubility in water = 0.028% (w/v)] was used for solvent extraction when measuring the ethanol concentration from the yeast culture broth. The maximum detectable ethanol concentration was 8% (v/v) when 10 g potassium dichromate in 100 ml of 5 M sulfuric acid was used. The concentrations determined from the solvent extraction-dichromate oxidation methods were remarkably similar to those of gas chromatography in which samples were prepared from seven experiments, such as four batch cultures and three fed-batch fermentations.     

A New Evaluation Method for 2‐D Fluorescence Spectra Based on Theoretical Modeling

This article presents a new evaluation procedure of 2‐D fluorescence spectra obtained during a yeast cultivation without performing a calibration measurement. The 2‐D fluorescence spectra are used to predict the process variables biomass, glucose and ethanol. The new calibration procedure uses a theoretical model of these process variables, i.e., differential equations, to replace any calibration measurement. The theoretical model parameters are identified simultaneously during the calculation of the chemometric models. The root mean square error of prediction of the chemometric models with respect to off‐line measurements are 1.5 g/L, 0.40 g/L and 0.56 g/L for glucose, biomass and ethanol, respectively.     

Simple fed-batch cultivation strategy for the enhanced production of a single-sugar glucuronic acid-based oligosaccharides by a cellulose-producing <Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis> strain

The production of water-soluble single-sugar glucuronic acid-based oligosaccharides (WSOS) by a cellulose producing strain Gluconacetobacter hansenii PJK was studied in a periodically recycled and fed-batch cultivations using glucose/ethanol or glucose only. Fermentations were carried out in a 2 L jar fermenter equipped with a turbine impeller with 6 flat blades. WSOS were produced constantly but the bacterial cellulose (BC) production stopped at 48 h of cultivation in a periodically recycled culture using the exhausted medium supplemented with glucose and ethanol. Tremendous quantities of WSOS were obtained in fed-batch cultivations using glucose/ethanol (35.6 g/L at 132 h of cultivation) or glucose only (86 g/L after 240 h of cultivation) as the nutritional source. However, the BC production yield under these nutritional conditions decreased significantly in comparison to previous studies about the BC production by the same strain. The overall results revealed that G. hansenii is capable of producing enormous quantities of WSOS compared to those reported previously for compounds of a related chemical nature. Moreover, the WSOS production was found to be dependent on the pH of the culture broth.     

Production of acetone butanol ethanol (ABE) by a hyper-producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation.

A silicone membrane was used to study butanol separation from model butanol solutions and fermentation broth. Depending upon the butanol feed concentration in the model solution and pervaporation conditions, butanol selectivities of 20.88-68.32 and flux values of 158.7-215.4 g m(-)(2) h(-)(1) were achieved. Higher flux values (400 g m(-)(2) h(-)(1)) were obtained at higher butanol concentrations using air as sweep gas. In an integrated process of butanol fermentation-recovery, solvent productivities were improved to 200% of the control batch fermentation productivities. In a batch reactor the hyper-butanol-producing mutant strain C. beijerinckii BA101 utilized 57.3 g/L glucose and produced 24.2 g/L total solvents, while in the integrated process it produced 51.5 g/L (culture volume) total solvents. Concentrated glucose medium was also fermented. The C. beijerinckii BA101 mutant strain was not negatively affected by the pervaporative conditions. In the integrated experiment, acids were not produced. With the active fermentation broth, butanol selectivity was reduced by a factor of 2-3. However, the membrane flux was not affected by the active fermentation broth. The butanol permeate concentration ranged from 26.4 to 95.4 g/L, depending upon butanol concentration in the fermentation broth. Since the permeate of most membranes contains acetone, butanol, and ethanol (and small concentrations of acids), it is suggested that distillation be used for further purification.     

Use of fluorometry for monitoring and control of a bioreactor

A new fluorescent bioreactor monitoring probe-multiple excitation fluorometric system (MEFS)-has been developed. This probe was compared to the commercially available BioChem Technology FluroMeasure system (NADH probe). In this task the fluorescence behavior of three model fermentation systems, ethanol fermentation by Candida utilis, phenol fermentation by Pseudomonas putida, and glucose fermentation by Saccharomyces cerevisiae, were examined. The results indicated that the fluorescence intensity and behavior of various cellular fluorophors vary significantly between the different fermentation systems. Monitoring a fermentation process using only NAD(P)H fluorescence provided limited information. The NAD(P)H fluorescence was found not to be the best fluorescence signal for monitoring cell concentrations. The best way of monitoring a bioreactor by fluorometry may be to monitor several fluorophors in the whole culture broth simultaneously and to relate these fluorescence signals to various biological parameters.     

Assessment of in-line near-infrared spectroscopy for continuous monitoring of fermentation processes

The application of NIR in-line to monitor and control fermentation processes was investigated. Determination of biomass, glucose, and lactic and acetic acids during fermentations of Staphylococcus xylosus ES13 was performed by an interactance fiber optic probe immersed into the culture broth and connected to a NIR instrument. Partial least squares regression (PLSR) calibration models of second derivative NIR spectra in the 700-1800 nm region gave satisfactory predictive models for all parameters of interest: biomass, glucose, and lactic and acetic acids. Batch, repeated batch, and continuous fermentations were monitored and automatically controlled by interfacing the NIR to the bioreactor control unit. The high frequency of data collection permitted an accurate study of the kinetics, supplying lots of data that describe the cultural broth composition and strengthen statistical analysis. Comparison of spectra collected throughout fermentation runs of S. xylosus ES13, Lactobacillus fermentum ES15, and Streptococcus thermophylus ES17 demonstrated the successful extension of a unique calibration model, developed for S. xylosus ES13, to other strains that were differently shaped but growing in the same medium and fermentation conditions. NIR in-line was so versatile as to measure several biochemical parameters of different bacteria by means of slightly adapted models, avoiding a separate calibration for each strain.     

A study of sulfite-tolerant yeasts from comminuted lamb products

The sulfite tolerance of meat yeasts was shown to be determined by pH, sulfite concentration, substrate availability, and the composition of the preincubation medium. Acetaldehyde production by Candida norvegica was sulfite-induced and occurred during the exponential growth phase in sulfited (500 micrograms SO2 ml-1) lab lemco glucose broth cultures buffered at pH 5, 6, or 7. Growth at pH 4, however, was inhibited by sulfite. Acetaldehyde production occurred in sulfited medium containing fructose or ethanol but not lactate nor a range of other assimilable substrates. A non-acetaldehyde-producing yeast, Candida vini, grew in sulfited (500 micrograms SO2 ml-1) lab lemco broth containing glucose or lactate buffered at pH 6 or 7 but not at pH 4 or 5.     

On-line characterization of a hybridoma cell culture process

The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.     

Gas chromatography and gateway sensors for on-line state estimation of complex fermentations (butanol-acetone fermentation)

A fermentation system has been designed to demonstrate the use of gas chromatography (GC) for on-line monitoring of the butanol-acetone and other complex saccharolytic fermentations. Tangential flow ultrafiltration was used to sterilely and continuously obtain a cell-free filtrate from the fermentation broth for on-line GC analysis of butanol, butyrate, acetate, acetone, ethanol, and acetoin. The liquid injection system consists of a phosphoric acid contactor, a slider-type injection valve, and a heater to address the difficulties (ghosting) encountered in the analysis of carboxylic acids. The fermentation headspace gas was also analyzed by on-line GC for nitrogen and carbon dioxide, while hydrogen was measured by difference. Raw chromatographic data were analyzed by a chromatography data system. Both raw and processed data were transmitted to a VAX 11/750 computer for further processing (using the fermentation equation) and archiving. The fermentation equation, which has recently been derived and tested on completed fermentation data, was also found to be valid during transient fermentations and thus useful as a gateway sensor for calculating various fermentation parameters on-line. Such parameters include glucose concentration and gas composition, as well as a number of unobservable parameters (such as Y(ATP), excess ATP, and NAD reduced by FdH(2)), which characterize the state of the fermentation.     

The effect of CO2 ventilation on kinetics and yields of cell-mass and ethanol in batch cultures ofZymomonas mobilis

Summary The effect of CO₂ removal by continuous sparging of N₂ in batch cultures ofZymomonas mobilis (ATCC10988) was examined. N₂ sparging considerably reduces lag times in batch cultures, possibly because of continuous removal of CO₂ from the culture media. Ventilation of CO₂ from culture media results in an increase of about 15% in the average specific growth rate and about 12% in the cell-mass yield with no noticeable trend in the average specific glucose uptake and ethanol production rates. The overall ethanol yield on glucose, however, decreases slightly by 5%. Analysis of ventilated experiments show that the CO₂ production is directly coupled with the ethanol formation but not necessarily with the cell-mass production, indicating a decoupling of growth from ethanol production. Further, comparison of ventilated and non-ventilated experiments rules out the possibility of CO₂ accumulation in the culture media as a factor responsible for increasing growth inhibition and decoupling of growth from ethanol fermentation at increasing initial glucose concentrations in batch cultures.