Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting

Yeast population used in industrial production of fuel-ethanol may vary according to the plant process condition and to the environmental stresses imposed to yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as starter strain instead of less adapted commercial strains. This work reports the use of PCR-fingerprinting method based on microsatellite primer (GTG)₅ to characterize the yeast population dynamics along the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominate the yeast population and were more present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from fuel-ethanol producing process.     

Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting

Yeast populations used in industrial production of fuel-ethanol may vary according to the plant process conditions and to the environmental stresses imposed on yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as the starter strain instead of less adapted commercial strains. This work reports the use of a PCR-fingerprinting method based on microsatellite primer (GTG)₅ to characterize the yeast population dynamics during the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominated the yeast population and were more commonly present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from the fuel-ethanol producing process.     

Effect of carbon and nitrogen sources on growth and biological efficacy of Pseudomonas fluorescens and Bacillus subtilis against Rhizoctonia solani, the causal agent of bean damping-off

One of the most important environmental factors that regulate the growth and antagonistic efficacy of biocontrol agents is the medium. The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strains P-5 and P-6 (Pseudomonas fluorescens), B-3 and B-16 (Bacillus subtilis) and minimum cost of media, whilst maintaining biocontrol efficacy. All of the strains were grown in seven liquid media (pH=6.9) including: sucrose + yeast extract, molasses of sugar beet + yeast extract in 2:1 and 1:1 w/w ratios, molasses of sugar beet + urea, nutrient broth, molasses and malt extract, at an initial inoculation of 1 x 10(5) CFU ml(-1). Cells from over night cultures used to inoculate soil at 1 x 10(9) CFU cm(-3) soil. At the same time, fungal inoculum (infected millet seed with Rhizoctonia solani) was added to soil at the rate of 2 g kg(-1) soil. Results indicated that growth of P-6, B-3 and B-16 in molasses + yeast extract (1:1 w/w) medium was significantly higher than in the other media. Molasses + yeast extract (1:1 and 2:1 w/w) media supported rapid growth and high cell yields in P-5. In greenhouse condition, results indicated that the influence of the media on the biocontrol efficacy of P-5, P-6, B-3 and B-16 was the same and Pseudomonas fluorescens P-5 in molasses and malt extract media reduced the severity of disease up to 72.8 percent. On the other hand, there were observed significant differences on bean growth after one month in greenhouse. P-5 in molasses + yeast extract (1:1 w/w) medium had the most effects on bean growth promotion. In this study molasses media showed good yield efficacy in all of the strains. The high sucrose concentration in molasses justifies the high biomass in all of the strains. Also, the low cost of molasses allows its concentration to be increased in media. On the other hand, yeast extract was the best organic nitrogen source for antagonist bacteria but it is expensive for an industrial process. So it should be replaced by another industrial product instead of yeast extract, which confirm by an economic and technological study. The results obtained in this study could be used to provide a reliable basis to increase the population of biocontrol agents in fermentation process.     

Production of poly-beta-hydroxyalkanoates from soy molasses oligosaccharides by new, rapidly growing Bacillus species

AIMS: To isolate and characterize bacteria from nature capable of producing poly-beta-hydroxyalkanoates in high yields from soy molasses oligosaccharides. METHODS AND RESULTS: Several strains of bacteria were obtained from enrichment cultures employing raffinose as major carbon source and inoculated with soybean field soil, lake sediment, or lake water. Many of the isolates were Bacillus species and produced polyhydroxyalkanoates (PHAs) to high yield. The raffinose-degrading isolates produced endospores, were highly saccharolytic, and both respired and fermented a variety of mono-, di-, tri- and tetrasaccharides. Strain CL1 produced 90% of cell dry mass as PHA from various sugars, including raffinose, and did so without requiring a nutrient limitation. CONCLUSIONS: Strain CL1 could be the catalyst for an industrial fermentation converting soy molasses and other waste carbohydrates to PHAs. The properties of this organism that make it ideally suited for such a fermentation include (i) its ability to use a wide variety of plant-associated carbohydrates as PHA feedstocks; (ii) its rapid growth; (iii) its ability to grow under anoxic conditions; and (iv) its ability to produce spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of bacteria capable of making biodegradable plastics to high yield from soy molasses oligosaccharides.     

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation

AIMS: To identify and characterize the main contaminant yeast species detected in fuel-ethanol production plants in Northeast region of Brazil by using molecular methods. METHODS AND RESULTS: Total DNA from yeast colonies isolated from the fermentation must of industrial alcohol plants was submitted to PCR fingerprinting, D1/D2 28S rDNA sequencing and species-specific PCR analysis. The most frequent non-Saccharomyces cerevisiae isolates were identified as belonging to the species Dekkera bruxellensis, and several genetic strains could be discriminated among the isolates. The yeast population dynamics was followed on a daily basis during a whole crop harvesting period in a particular industry, showing the potential of D. bruxellensis to grow faster than S. cerevisiae in industrial conditions, causing recurrent and severe contamination episodes. CONCLUSIONS: The results showed that D. bruxellensis is one of the most important contaminant yeasts in distilleries producing fuel-ethanol from crude sugar cane juice, specially in continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Severe contamination of the industrial fermentation process by Dekkera yeasts has a negative impact on ethanol yield and productivity. Therefore, early detection of D. bruxellensis in industrial musts may avoid operational problems in alcohol-producing plants.     

Evaluation of yeast diversity during wine fermentations with direct inoculation and pied de cuve method at an industrial scale

The diversity and composition of yeast populations may greatly impact wine quality. This study investigated the yeast microbiota in two different types of wine fermentations: direct inoculation of a commercial starter versus pied de cuve method at an industrial scale. The pied de cuve fermentation entailed growth of the commercial inoculum used in the direct inoculation fermentation for further inoculation of additional fermentations. Yeast isolates were collected from different stages of wine fermentation and identified to the species level using Wallersterin Laboratory nutrient (WLN) agar followed by analysis of the 26S rDNA D1/D2 domain. Genetic characteristics of the Saccharomyces cerevisiae strains were assessed by a rapid PCR-based method, relying on the amplification of interdelta sequences. A total of 412 yeast colonies were obtained from all fermentations and eight different WL morphotypes were observed. Non-Saccharomyces yeast mainly appeared in the grape must and at the early stages of wine fermentation. S. cerevisiae was the dominant yeast species using both fermentation techniques. Seven distinguishing interdelta sequence patterns were found among S. cerevisiae strains, and the inoculated commercial starter, AWRI 796, dominated all stages in both direct inoculation and pied de cuve fermentations. This study revealed that S. cerevisiae was the dominant species and an inoculated starter could dominate fermentations with the pied de cuve method under controlled conditions.     

Characterization of yeast starter cultures used in household alcoholic beverage preparation by a few ethnic communities of Northeast India

In this study, we report the characterization of traditional household starter cultures from a few ethnic groups of northeast India. Pure cultures obtained from the study have been deposited in the Microbial Type Culture Collection (MTCC), India. These isolates have been analyzed for their growth characteristics, sensitivity to temperature and pH, alcohol tolerance, alcohol production, and alcohol dehydrogenase (ADH) content. The pure cultures obtained from different starter cultures revealed the presence of Debaryomyces, Wickerhamomyces, and Candida along with the fermenting yeast Saccharomyces. The growth behavior at different temperatures, pH and alcohol tolerance revealed numerous facts and behavior of the yeast strains associated with traditional alcoholic fermentation. All the isolates were found to be thermotolerant up to 37 °C, fairly pH-resistant, good in ADH secretion, and with appreciable alcohol production. For all the strains studied, the Saccharomyces cerevisiae MTCC 3976 strain from the Tea Tribes of Assam and the Wickerhamomyces anomalus MTCC 3979 from the Apatani Tribe of Arunachal Pradesh were found to be exceptional in terms of thermotolerance, alcohol tolerance, alcohol production, and ADH activity, and hence may be identified as potential strains for industrial fermentation.     

Improvement of maltotriose fermentation by Saccharomyces cerevisiae

AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts.     

Comparative fermentation studies of industrial strains belonging to four species of solvent-producing clostridia

Industrial and culture collection strains of solvent-producing clostridia, classified as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum were utilised in a comparative study of fermentation performance in a laboratory fermentation medium, a molasses fermentation medium, and a maize fermentation medium under standardised culture conditions. At least one representative strain was selected from each of the sub-groups within the four species. Preliminary evaluations were first undertaken for the three different fermentation media to determine the most appropriate media formulations, carbohydrate concentrations, and culture conditions for comparison of the solvent-producing ability of these strains. Standardised fermentation media and culture conditions were then selected for each of the comparative fermentation studies. These included TYA medium containing 4% glucose, a supplemented molasses medium containing 6% fermentable sugars, and a supplemented maize mash medium containing 8% maize. Additional comparative fermentation studies on industrial strains belonging to two species of solvent-producing clostridia were carried out in molasses containing higher concentrations of fermentable sugars, and the sugar concentrations supporting maximum levels of solvent production were determined. Although all the strains tested grew in the maize fermentation medium and degraded starch, only a few strains produced consistently high solvent levels. Optimum starch utilisation and solvent production was obtained at a maize concentration of 80 g/l. Pretreatment of the maize by milling or saccharification decreased the buffering capacity of the medium and resulted in decreased solvent production. Decreasing the time used to gelatinise the starch had little effect. Solvent yields and concentrations obtained in this study were compared with various published data in the scientific and patent literature and appeared to closely simulate the results obtained in the industrial fermentation process. The fermentation performances of individual strains could provide useful comparative data for the selection and development of strains for use on various commercial fermentation substrates.     

A cost-effective cane molasses medium for enhanced cell-bound phytase production by Pichia anomala

AIM: Formulation of an inexpensive cane molasses medium for improved cell-bound phytase production by Pichia anomala. METHODS AND RESULTS: Cell-bound phytase production by Pichia anomala was compared in synthetic glucose-beef extract and cane molasses media. The yeast was cultivated in 250 ml flasks containing 50 ml of the medium, inoculated with a 12 h-old inoculum (3 x 10(6) CFU ml(-1)) and incubated at 25 degrees C for 24 h at 250 rev min(-1). Different cultural parameters were optimized in cane molasses medium in batch fermentation. The cell-bound phytase content increased significantly in cane molasses medium (176 U g(-1) dry biomass) when compared with the synthetic medium (100 U g(-1) dry biomass). In fed-batch fermentation, a marked increase in biomass (20 g l(-1)) and the phytase yield (3000 U l(-1)) were recorded in cane molasses medium. The cost of production in cane molasses medium was pound 0.006 per 1000 U, which is much lower when compared with that in synthetic medium (pound 0.25 per 1000 U). CONCLUSIONS: An overall 86.6% enhancement in phytase yield was attained in optimized cane molasses medium using fed-batch fermentation when compared with that in synthetic medium. Furthermore, the production in cane molasses medium is cost-effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase yield was improved in cane molasses when compared with the synthetic medium, and the cost of production was also significantly reduced. This enzyme can find application in the animal feed industry for improving the nutritional status of feed and combating environmental pollution.     

Prototrophic hybrids of bakers yeast. I. Selection of haploids and diploids of prototrophic yeast

153 haploids, including 8 prototrophs, 12 biotin prototrophs and 4 biotin auxotrophs were isolated from Y, F and FB strains of the baker's yeast Saccharomyces cerevisiae. Remaining haploids were dependent on vitamins of B group other than biotin. Obtained haploids were characterized by large cell sizes, a or α mating type, the ability to fermentation of sugars and the assimilation of non-fermented carbon sources. Haploids obtained from the yeast of Y and FB strains a good growth in the synthetic medium. Prototrophic haploids, prototrophic haploids with biotin auxotrophs and biotin prototrophs with biotin auxotrophs were crossed. 89 prototrophic hybrids capable of growth in synthetic medium without vitamins were selected out of 178 hybrids obtained. Hybrids are characterized by features typical for baker's yeast; however, not all of them are capable of sporulation. As result of selection, prototrophic hybrids and 16 hybrids characterized by a good increase of biomass in molasses medium were chosen. The efficiency of the biomass of hybrids designated as YY 3040/2, YY 3040/3 and FFB 1910/2 is considerably higher than one obtained from the cultivation of the industrial strain French Mautner (Mf). All hybrids possess adequate enzymatic activity in anaerobic metabolism of saccharose and maltose. Selected prototrophic hybrids were sent out to two yeast factories in the country for experimental propagation.     

Simultaneous ethanol and bacterial ice nuclei production from sugar beet molasses by a Zymomonas mobilis CP4 mutant expressing the inaZ gene of Pseudomonas syringae in continuous culture

AIM: The aim of this work was to construct a Zymomonas mobilis mutant capable of simultaneous ethanol and ice nuclei production from agricultural by-product such as sugar beet molasses, in steady-state continuous culture. METHODS AND RESULTS: A sucrose-hypertolerant mutant of Z. mobilis strain CP4, named suc40, capable of growing on 40% (w/v) sucrose medium was isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. Plasmid pDS3154 carrying the inaZ gene of Pseudomonas syringae was conjugally transferred and expressed in suc40. The potential for simultaneous ethanol and bacterial ice nuclei production was assessed in steady-state continuous cultures over a range of dilution rates from 0.04 to 0.13 h(-1). In addition, the fatty acid and phospholipid profile of the three strains was also investigated. Ethanol production up to 43 g l(-1) was achieved at dilution rates below 0.10 h(-1) in sugar beet molasses. Ice nucleation activity gradually increased with increasing dilution rate and the greatest activity, -3.4 log (ice nuclei per cell), was observed at the highest dilution rate (0.13 h(-1)). Both mutant strains displayed a different fatty acid and phospholipid profile compared with the wild-type strain. CONCLUSIONS: The ability of the mutant and recombinant plasmid-containing strains to grow on high sugar concentrations and in high osmotic pressure environments (molasses) can be attributed to their phospholipid and fatty acid contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account that sugar beet molasses is a low cost agricultural by-product, the simultaneous ethanol and bacterial ice nucleation production achieved under the studied conditions is considered very promising for industrial applications.     

Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts

Aims: Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. Methods and Results: PCR‐RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains’ diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Conclusions: Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery‐resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. Significance and Impact of the Study: The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine‐producing Appellation of Origin ‘Vinos de Madrid’ is shown.     

Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid Kluyveromyces marxianus strain improved product quality attributes

Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Engineering of protein secretion in yeast: strategies and impact on protein production

Yeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secrete and modify foreign proteins according to a general eukaryotic scheme. Their rapid growth, microbiological safety, and high-density fermentation in simplified medium have a high impact particularly in the large-scale industrial production of foreign proteins, where secretory expression is important for simplifying the downstream protein purification process. However, secretory expression of heterologous proteins in yeast is often subject to several bottlenecks that limit yield. Thus, many studies on yeast secretion systems have focused on the engineering of the fermentation process, vector systems, and host strains. Recently, strain engineering by genetic modification has been the most useful and effective method for overcoming the drawbacks in yeast secretion pathways. Such an approach is now being promoted strongly by current post-genomic technology and system biology tools. However, engineering of the yeast secretion system is complicated by the involvement of many cross-reacting factors. Tight interdependence of each of these factors makes genetic modification difficult. This indicates the necessity of developing a novel systematic modification strategy for genetic engineering of the yeast secretion system. This mini-review focuses on recent strategies and their advantages for systematic engineering of yeast strains for effective protein secretion.     

Evaluation of a potential starter culture for enhance quality of coffee fermentation

The coffee fermentation is characterized by the presence of different microorganisms belonging to the groups of bacteria, fungi and yeast. The objectives of this work were to select pectinolytic microorganisms isolated from coffee fermentations and evaluate their performance on coffee pulp culture medium. The yeasts and bacteria isolates were evaluated for their activity of polygalacturonase (PG), pectin lyase (PL) and pectin methylesterase (PME) and metabolites production. Among 127 yeasts isolates and 189 bacterial isolates, 15 were pre-selected based on their ability to produce PL and organic compounds. These isolates were strains identified as Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Candida parapsilosis, Pichia caribbica, Pichia guilliermondii and Saccharomyces cerevisiae. When cultivated in Coffee peel and pulp media in single culture or two by two mixed inocula, different behavior concerning to PME, PL and PG were found. The two principal components PC1 and PC2 accounted for 45.27 and 32.02 % of the total variance. UFLA CN727 and UFLA CN731 strains were grouped in the positive part of PC1 being characterized by 1,2-propanediol, hexanoic acid, decanoic acid, nonanoic acid and ethyl acetate. The UFLA CN448 and UFLA CN724 strains were grouped in the negative part of PC1 and were mainly characterized by guaiacol, butyric acid and citronellol. S. cerevisiae UFLACN727, P. guilliermondii UFLACN731 and C. parapsilosis UFLACN448 isolates are promising candidates to be tested in future studies as coffee starter cultures.