Low-pressure airlift fermenter for single cell protein production: II. Continuous culture of pichia yeast

Experiments using Pichia yeast grown on n-paraffins have been conducted in laboratory 10-L airlift fermenters and in a 640-L module of commercial scale. Results confirmed the design concept of combining oxygen transfer and fermenter cooling with low-pressure air. However, in the absence of mass transport constraints, the build up of toxic factors in the fermenter appeared to be a major variable limiting cell productivity. Foaming in the large fermenter also presented a serious problem, which must be solved before low-pressure airlift fermenters become practical.     

Characterization of Kloeckera apiculata strains from the Friuli regionin Northern Italy

Forty-nine strains of Kloeckera apiculata, isolated from the Friuli region in Italy, were differentiated on the basis of fermentation behaviour and production of secondary compounds in two different grape musts at 18 °C. The isolates exhibited a controlled production of acetic acid, only in a few cases more that 1 g/l. In Moscato grape must the strains exhibited a more uniform behaviour for the production of higher alcohols, ethyl acetate and acetoin than in red grapes. In general, higher levels of ethanol, glycerol and acetic acid were produced in red grape must fermentation. Apiculate strains behaved differently in the two musts, with different metabolic phenotypes dominating the fermentation process. The existence of different metabolic phenotypes correlated with the must composition underlines the need to perform a selection of indigenous apiculate yeasts to obtain the desired consistent products.     

Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate

The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP.     

Improvement of Nitrogen Assimilation and Fermentation Kinetics under Enological Conditions by Derepression of Alternative Nitrogen-Assimilatory Pathways in an Industrial Saccharomyces cerevisiae Strain

Metabolism of nitrogen compounds by yeasts affects the efficiency of wine fermentation. Ammonium ions, normally present in grape musts, reduce catabolic enzyme levels and transport activities for nonpreferred nitrogen sources. This nitrogen catabolite repression severely impairs the utilization of proline and arginine, both common nitrogen sources in grape juice that require the proline utilization pathway for their assimilation. We attempted to improve fermentation performance by genetic alteration of the regulation of nitrogen-assimilatory pathways in Saccharomyces cerevisiae. One mutant carrying a recessive allele of ure2 was isolated from an industrial S. cerevisiae strain. This mutation strongly deregulated the proline utilization pathway. Fermentation kinetics of this mutant were studied under enological conditions on simulated standard grape juices with various nitrogen levels. Mutant strains produced more biomass and exhibited a higher maximum CO₂ production rate than the wild type. These differences were primarily due to the derepression of amino acid utilization pathways. When low amounts of dissolved oxygen were added, the mutants could assimilate proline. Biomass yield and fermentation rate were consequently increased, and the duration of the fermentation was substantially shortened. S. cerevisiae strains lacking URE2 function could improve alcoholic fermentation of natural media where proline and other poorly assimilated amino acids are the major potential nitrogen source, as is the case for most fruit juices and grape musts.     

A quick screening method to identify β-glucosidase activity in native wine yeast strains: application of Esculin Glycerol Agar (EGA) medium

One hundred and fifty-four yeast strains were isolated from grapes and musts of Uruguayan vineyards and wineries. Only thirty strains showed β-glucosidase activity in Esculin Glycerol Agar (EGA) solid medium. Twenty-one were non-Saccharomyces and nine were Saccharomyces cerevisiae strains. The objective of this study was to evaluate the suitability of Esculin Glycerol Agar (EGA) solid medium for screening β-glucosidase activity in native yeasts strains. Halo sizes measured in the EGA solid medium were correlated to the Glycosyl-Glucose (GG) indexes measured after fermentation of grape musts with each strain. The two S. cerevisiae strains with the best performance were selected for further fermentations on a Muscat Miel grape must, rich in bound monoterpenes. The levels of free linalool, hodiol I and geraniol increased significantly as compared to fermentation with a commercial wine yeast strain. These results show the suitability of this simple and economic medium to identify S. cerevisiae glucosidase producers with a potential impact on real winemaking conditions. On the other hand, great variability was found for the non-Saccharomyces strains, and this would demand further studies for each species. In conclusion, the use of EGA solid medium shows that the screening method is suitable for exploring the glucosidase activity of native strains of S. cerevisiae and shows good correlation with its real impact on free aroma compounds in the final wine.     

Nitrogen catabolite repression modulates the production of aromatic thiols characteristic of Sauvignon Blanc at the level of precursor transport

The free thiols 3-mercapto-hexanol (3MH) and its acetate, practically absent from musts, are liberated by yeast during fermentation from a cysteinylated precursor [S-3-(hexan-1-ol)-l-cysteine (Cys-3MH)] present in the grape must and contribute favorably to the flavor of Sauvignon white wines. Production of 3MH is increased when urea is substituted for diammonium phosphate (DAP) as the sole nitrogen source on a synthetic medium. On grape must, complementation with DAP induces a decrease of 3MH production. This observation is reminiscent of nitrogen catabolite repression (NCR). The production of 3MH is significantly lower for a gap1Delta mutant compared with the wild type, during fermentation of a synthetic medium containing Cys-3MH as the precursor and urea as the sole nitrogen source. Mutants isolated from an enological strain with a relief of NCR on GAP1 produce significantly higher amounts of 3MH on synthetic medium than the parental strain. These phenotypes were not confirmed on grape must. It is concluded that on synthetic medium, Cys-3MH enters the cell through at least one identified transporter, GAP1p, whose activity is limiting the release of volatile thiols. On grape must, the uptake of the precursor through GAP1p is not confirmed, but the effect of addition of DAP, eventually prolonging NCR, is shown to decrease thiol production.     

Stimulation of malo-lactic fermentation in eastern grape musts

Induced malo-lactic fermentation was stimulated in Eastern grape musts by the addition of a new fermentation enhancer product.     

Contribution of different natural yeasts to the aroma of two alcoholic beverages

The aroma formation in the fermentation of two types of natural musts by 12 different yeasts has been analysed. In grape must fermentation Pichia fermentans Colección Española de Cultivos Tipo (CECT) 11773, Clavispora lusitaniae OJ6 and Pichia anomala OJ5 produced the best balance between concentrations of ethyl acetate and high alcohols. When orange juice was fermented with the 12 yeasts, Pichia fermentans CECT 11773, Rhodotorula mucilaginosa OJ2 and Hanseniaspora uvarum CECT 10885 produced a good beverage with low alcoholic grade. For both types of natural musts Pichia fermentans CECT 11773 increased the presence of higher alcohols and ethyl acetate. After using this strain both alcoholic beverages obtained the highest evaluation in the sensory analysis.     

The optimization of L-lysine fermentation and a mass transfer model

The efficiency of L-lysine biosynthesis is essentially determined by the power input and aeration ratio in the stirred fermenter. A mass transfer model was developed by means of the results of lysine fermentations in four geometrically similar fermenters with working volumes of 10 1, 50 1, 100 1 and 2500 1 which allows the optimization of lysine fermentation from the energetical point of view. The usefullness of this k_La-model is demonstrated with an example where the power input for an unknown fermenter is calculated.     

Modelling of the process yields of a whey fermentation

Summary The biomass yields (y) and COD reduction efficiencies (η) of a whey fermentation by Kluyveromyces fragilis were studied in a 100-1 fermenter at various stirrer speeds and lactose concentrations, and compared to those obtained in 10-1 and 15-1 fermenters at constant values of the oxygen transfer coefficient (k_La) and air velocity. The empirical models previously constructed by using the 15-1 fermenter data could be used to predict the yields on the other scales by calculating for each run the 15-1 fermenter which would provide the same oxygen transfer coefficient measured by the sulphite method on each fermenter under study. To make this model independent of stirrer speeds used in each generic fermenter, the effect of aeration and mixing was incorporated into an overall parameter (k_La) and the values of y and η were correlated only with temperature, lactose level and k_L a, since these variables were approximately orthogonal. The validity of this model was finally checked against the yields reported by Wasserman et al. (1961) in a 6-m³ fermenter, thus confirming the capability of the model to provide a reliable basis for further scale-up on the production scale.     

The scale up of mycelial shake flask fermentations: A case study of gamma linolenic acid production byMucor hiemalis IRL 51

Summary Production of gamma linolenic acid (GLA) by the filamentous fungusMucor hiemalis IRL 51 was studied in both shake flask culture and in a 10-L stirred tank fermenter. This study was conducted to assess how the results from shake flask media screening trials compared to those obtained in a 10-L stirred tank fermenter, which is assumed to be more representative of an industrial system. The results show that the biological performance in 10-L fermenters is usually the same as that in shake flask culture. There were some inconsistencies which could possibly be attributed to scale, but no large differences were systematically seen. These results show that for this filamentous fungus, shake flask culture provides a quick and inexpensive way of optimizing medium composition.     

Managing high-density commercial scale wine fermentations

AIMS: To investigate the effects of grape juice dilution and different temperature/nitrogen addition regimes on commercial-scale, high-density Shiraz and Chardonnay fermentations. METHODS AND RESULTS: Duplicated fermentations (30 hl) were conducted at two temperatures for Shiraz and for Chardonnay. Two additional tanks of Chardonnay and Shiraz were diluted. Nitrogen was added once at inoculation or in aliquots over several days. Yeast concentration and viability was determined by flow cytometry. Fermentation chemistry was monitored by Fourier transform infrared spectroscopy. Fermentations arrested in both of the undiluted, higher temperature duplicate tanks of Shiraz. Different fermentation temperature resulted in sensorially different Shiraz, but not Chardonnay, wines made from undiluted musts. The converse was observed for wines made from diluted musts. CONCLUSIONS: High-density musts can be fermented completely using reduced fermentation temperature coupled with incremental nitrogen addition. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study in duplicated, commercial-scale, high-density grape juice fermentations to address temperature, nitrogen addition, and juice dilution effects on stuck fermentation potential and wine sensory properties.     

Dynamics of the yeast strain population during spontaneous alcoholic fermentation determined by CHEF gel electrophoresis

Wine yeasts were isolated from spontaneous alcoholic fermentations performed with white and red grape musts from vintages 1991 and 1992. Yeast cells were analysed by physiological tests and gel electrophoretic karyotyping. It was shown that there is a succession of different strains in the yeast population during the time course of the fermentation process. Furthermore, the composition of the yeast strain population differs from grape must to grape must and from year to year, and may therefore be considered vineyard (terrain)- and vintage-dependent.     

Solid substrate fermentation of wheat straw to fungal protein

Steam-treated wheat straw at a 70% (w/w) moisture level was subjected to solid substrate fermentation (SSF) with Trichoderma reesei (Riga, USSR) or a mixed culture of T. reesei and Endomycopsis fibuliger (R-574) in fermentation equipment of various design: some with mixing, some with stationary layers, including a mixedlayer 1.5-m(3) pilot plant scale fermenter. The best protein productivity was obtained in stationary layer fermenters with a product containing 13% protein. The main limitations of lignocellulose SSF, such as hindrance of fungal growth, limiting accessibility and availability of substrate, and difficulty in moisture and heat control, were analyzed. The technological parameters of SSF, submerged fermentation, and alternate lignocellulose conversion processes were compared. The SSF had lower overall efficiency but higher product concentration per reaction volume than other conversion schemes.     

The vinification of partially dried grapes: a comparative fermentation study of Saccharomyces cerevisiae strains under high sugar stress

AIMS: The study of the fermentation performance of Saccharomyces cerevisiae strains under high sugar stress during the vinification of partially dried grapes. METHODS AND RESULTS: Microvinification of partially dried grape must with sugar concentration of 35 degrees Brix was performed using four commercial strains to carry out alcoholic fermentation. A traditional red vinification without nutrients addition was applied. Yeasts displayed different efficiency to convert sugar in ethanol and varied in glycerol yield. Sugar consumption and ethanol level were attested at 80-87% and 143.5-158.0 g l(-1) respectively. High correlation between sugar and assimilable nitrogen consumption rate was observed. Statistical treatment of data by principal component analysis highlighted the different behaviours that strains exhibited in regard to the production of higher alcohols and other compounds important to wine quality. CONCLUSIONS: Saccharomyces cerevisiae strains displayed appreciable capability to overcome osmotic stress and to yield ethanol fermenting high sugar concentration grape must in winemaking condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provided insights on the strain contribution to wine quality subordinate to stress condition. This investigation is of applicative interest for winemaking and processing industry that use high sugar concentration musts.     

Fermentation behaviour and volatile compound production by agave and grape must yeasts in high sugar Agave tequilana and grape must fermentations

Few studies have been performed on the characterization of yeasts involved in the production of agave distilled beverages and their individual fermentation properties. In this study, a comparison and evaluation of yeasts of different origins in the tequila and wine industries were carried out for technological traits. Fermentations were carried out in high (300 g l⁻¹) and low (30 g l⁻¹) sugar concentrations of Agave tequilana juice, in musts obtained from Fiano (white) and Aglianico (red) grapes and in YPD medium (with 270 g l⁻¹ of glucose added) as a control. Grape yeasts exhibited a reduced performance in high-sugar agave fermentation, while both agave and grape yeasts showed similar fermentation behaviour in grape musts. Production levels of volatile compounds by grape and agave yeasts differed in both fermentations.     

The application of air-lift fermenters to the cultivation of filamentous fungi

Summary An investigation of the performance of air-lift fermenters showed that the value of the oxygen mass transfer coefficient (K_La) increased with both the aeration rate and vessel size. Although some change in the liquid circulation pattern occurred with increasing superficial gas velocity, there was no transition from bubbly to slug flow over the range of superficial gas velocities studied. Increases in broth viscosity caused an increase in gas hold up and a reduction in the values for K_La, although this reduction was not as great as that observed in mechanically agitated fermenters. Under conditions of aeration and agitation which gave comparable K_La values similar biomass yields of Aspergillus oryzae were obtained in 7.25 l and 100 l air-lift fermenters, and in a 3.5 l mechanically agitated fermenter.     

A survey ofSaccharomyces populations associated with wine fermentations from the Apulia region (South Italy)

The aim of this paper was to investigate the genetic and phenotypic characteristics of yeasts isolated from samples of grape musts collected from four different areas of Apulia region. The 68 yeast isolates were identified asSaccharomyces cerevisiae by PCR-RFLP of 5.8S-ITS region of the rRNA gene. Individual isolates were differentiated by RAPD-PCR and AFLP. The following oenological traits were studied: fermentation power, resistance to cycloheximide, alcohol and SO₂, formation of SO₂ and H₂S, β-glucosidase activity, and production of biogenic amines and secondary compounds. Many phenotypes were common to several yeasts isolated from the four different areas, such as high SO₂ resistance and fermentation power. In addition, someS. cerevisiae isolates showed a β-glucosidase activity and others had a high resistance to cycloheximide. All the strains formed biogenic amines. Solid Phase Microextraction was used to determine secondary compounds produced in wine by the single yeast cultures.     

Lycopene production from synthetic medium by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> NRRL 2895 (+) and 2896 (−) in a stirred-tank fermenter

The dissolved oxygen tension of 20% of air saturation, pH-shift from 4.0 to 5.5 on day 3, and a moderate shear stress (calculated as an impeller tip speed, V_\texttip = 0. 9 2 6- 2. 1 6 1  \textm/\texts V_{\text{tip}} = 0. 9 2 6- 2. 1 6 1 \, {\text{m}}/{\text{s}} ) were identified to be the key factors in scaling-up the mated fermentation of Blakeslea trispora NRRL 2895 (+) and 2896 (−) for lycopene production from a shake flask to a stirred-tank fermenter. The maximal lycopene production of 183.3 mg/L was obtained in 7.5-L stirred-tank fermenter, and then the mated fermentation process was successfully step-wise scaled-up from 7.5- to 200-L stirred-tank fermenter. The comparability of the fermentation process was well controlled and the lycopene production was maintained during the process scale-up. Furthermore, with the integrated addition of 150 μmol/L abscisic acid on day 3, 0.5 g/L leucine and 0.1 g/L penicillin on day 4, the highest lycopene production of 270.3 mg/L was achieved in the mated fermentation of B. trispora in stirred-tank fermenter.     

A comparison of some methods of estimating volumetric mass-transfer coefficients in an external-loop airlift fermenter

Volumetric mass-transfer coefficients were measured in an 11-L external-loop airlift fermenter with deionized water, a fermentation medium, and during a fermentation. Both a Mackareth oxygen electrode and a novel rapid-response probe were used. When the conventional step-change dynamic method was used for water, the long, nonlinear response time of the Mackareth electrode made correction of its readings difficult; this problem did not occur when the rapid-response probe was used. A comparison was made with a method of mass-transfer coefficient determination which does not involve any assumptions about the gas residence time distribution. However, this method requires that the liquid phase is well-mixed and this requirement was not met in the airlift fermenter. Comparison of the present results with other K(L) a determinations for airlift fermenters showed that K(L) a in water depends on the active gas holdup, the value of K(L) a/epsilon at 20 degrees C being ca. 0.37 s(-1). Although higher gas holdups were obtained with the fermentation medium than for water, the values of K(L) a/epsilon were lower, ca. 0.22 s(-1) at 20 degrees C.