Potential of carotenoids in aquatic yeasts as a phylogenetically reliable marker and natural colorant for aquaculture

Apart from Xanthophyllomyces dendrorhous, pink colony-forming yeasts have not been examined as a pigmentation source in captive animals. In this study, aquatic yeasts were screened with a view to abundances of carotenoids. Phylogenetic analyses of these caroetnoid-rich yeasts based on large subunit ribosomal RNA gene (LSU rDNA) partial sequences showed that all belonged to the order Sporidiobolales. Both the qualitative and the quantitative differences in carotenoids between the yeasts appeared to be consistent with their phylogenetic affiliations. This information might be useful in the selection of pigment-rich yeasts containing specific carotenoids from a large number of strains. We also found, for the first time, the potential of a pigment-rich Rhodotorula strain as a colorant for aquaculture. The integuments of tilapia and carp fed the alkali-treated cells of strain Rhodotorula dairenensis Sag 17 were pigmented after 3 months of cultivation. The fish integuments retained the yeast carotenes shortly after the start of feeding, and were converted to the fish-specific xanthophylls in vivo.     

Yeasts found on an ephemeral reproductive caste of the leaf-cutting ant Atta sexdens rubropilosa

Winged males of leaf-cutting ants are considered an ephemeral reproductive caste only produced before the mating flight season. Although much is known about the yeast diversity found in fungus gardens of attine ants, no study has focused on the yeasts associated with males of leaf-cutting ants. Here, we surveyed the yeasts on the integuments of males of Atta sexdens rubropilosa and assessed their potential role in the attine ant-microbe symbiosis. Using culture-dependent techniques, we found yeasts to be abundant on the integuments of males (54.5 %, n = 200 alates). A total of 242 yeast strains were obtained representing six orders, ten genera and 25 species. Strains of Aureobasidium, Cryptococcus, Hannaella and Rhodotorula were prevalent on the integuments and likely originated from the fungus garden of the parental nest or from the soil. The majority of strains (87.1 %) produced at least one of the evaluated enzymes: pectinase, polygalacturonase, cellulase, xylanase, ligninases and lipase. Aureobasidium pullulans accounted for the highest number of strains that produced all enzymes. In addition, yeasts showed the ability to assimilate the resulting oligosaccharides, supporting observations of other studies that yeasts may be involved in the plant biomass metabolism in the fungus gardens. Because winged males harbor several yeasts with putative functional roles, these fungi may take part and be beneficial in the microbial consortia of the new incipient nest.     

A Hypothetical Cyclic Pathway for the Metabolism of Odd-carbon n-Alkanes or Propionyl-CoA via Seven-carbon Tricarboxylic Acids in Yeasts

An extensive study has been undertaken to elucidate the physiological significance of threo-D_s-2-methylisocitric acid produced mainly from odd-carbon n-alkanes by a mutant strain of Candida lipolytica. The mutant strain showed slower growth responses to odd-carbon n-alkanes, especially of shorter chain-length, and failed to utilize this acid as sole carbon source, whereas the parent strain and many other yeasts tested were able to utilize this acid. About one half of yeasts tested accumulated this acid extracellularly. Under a thiamine-deficient condition, amounts of pyruvate produced by the parent strain from odd-carbon n-alkanes were ten times as large as those from even-carbon n-alkanes. A scheme for the partial oxidation of propionyl-CoA to pyruvate via C₇-tricarboxylic acid by yeasts was supposed. This scheme may offer suggestion on the metabolism of propionyl-CoA by other living organisms. A hypothetical pathway of citrate accumulation from odd-carbon n-alkane was also presented.     

Cryptococcus nodaensis sp nov, a yeast isolated from soil in Japan that produces a salt-tolerant and thermostable glutaminase

An anamorphic basidiomycetous yeast, which produced a salt-tolerant and thermostable glutaminase, was isolated from soil in Japan and classified in the genus Cryptococcus. Its substrate specificity suggests that this enzyme is an L-glutaminase asparaginase (EC 3.5.1.38). The strain, G60, resembles Cryptococcus laurentii in the taxonomic criteria traditionally employed for yeasts, however it can be distinguished as a separate species based on DNA–DNA reassociation experiments and sequence analysis of the large sub-unit rDNA. Phenotypically, the isolate can be differentiated from C. laurentii by the inability to utilize arbutin as a sole source of carbon. Based on sequence analysis, the strain is related to a group of hymenomycetous yeasts including Bulleromyces albus, Bullera unica, C. laurentii and C. skinneri. The strain, which is formally described as Cryptococcus nodaensis, is industrially important for the formation of the umami taste during production of proteolytic seasonings. Received 28 July 1998/ Accepted in revised form 04 February 1999     

Carotenoid production in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> achieved by metabolic engineering

The carotenoid synthetic genes, crtM and crtN, derived from Staphylococcus aureus, were introduced into B. subtilis, resulting in yellow pigmentation. Absorption maxima of pigments and MALDI-TOF mass spectrometry demonstrated that the pigmented strain accumulated two C₃₀ carotenoids, 4,4′-diapolycopene and 4,4′-diaponeurosporene. A survival test using H₂O₂ revealed that the pigmented strain was more resistant to oxidative stress than the strain harboring an empty-vector. These findings indicate that B. subtilis can produce carotenoids, and the strain accumulating the carotenoids, CarotenoBacillus, will become a basal host for production of C₃₀ carotenoids and evaluation of their antioxidative effects.     

Marine yeasts and bacteria as biological control agents against anthracnose on mango

The potential of four yeasts (Debaryomyces hansenii, Rhodotorula minuta, Cryptococcus laurentii and Cryptococcus diffluens) and three bacteria (Bacillus amyloliquefaciens, Bacillus subtilis and Stenotrophomonas rhizophila) with antagonistic capacity against anthracnose caused by Colletotrichum gloeosporioides in mango cv. Ataulfo fruit was investigated. Germination of C. gloeosporioides spores was significantly inhibited by all marine yeasts and bacteria strains of an in vitro test. When yeasts and bacteria were tested on mango fruit, the marine yeast D. hansenii 1R11CB strain and marine bacteria S. rhizophilaKM02 strain were the best antagonists to anthracnose (C. gloeosporioides), which significantly decreased disease incidence by 56% and 89%, respectively, and reduced lesion diameter by 91% and 92%, respectively. All the isolated strains of the phytopathogen, yeasts and bacteria were molecularly identified. Our results from in vitro and in vivo experiments suggest that marine yeasts and bacteria strains can be used as some effective biological control agents for anthracnose in mango.     

A new improved strategy for the selection of cold-adapted antagonist yeasts to control postharvest pear diseases

Postharvest diseases cause considerable losses of harvested fruits during transportation and storage. Many yeast species have been reported as good antagonists against postharvest pear pathogens. In this work, we used a novel selection strategy that involves the isolation of yeasts from washing fluids, showing biocontrol activity against a regional Penicillium expansum strain (primary screening), originally obtained from fruit wounds after long time storage at ?1/0°C. About 26 isolates representative of the 11 yeast species identified in the 27 selected washing waters were chosen to be evaluated in a secondary screening against a regional Botrytis cinerea strain on pear wounds. Among yeasts tested, 38% showed complete control of P. expansum, but only 15% reduced the decay incidence of B. cinerea to 60–80% at ?1/0°C. These results reveal that some of the yeasts found can be biological alternatives to fungicides in the control of P. expansum and B. cinerea infections. Based on the data obtained, our strategy seems to be much more effective than the previously reported methods in obtaining successful biocontrol agents.     

Chromosomal polymorphism and adaptation to specific industrial environments of Saccharomyces strains

Several industrial Saccharomyces strains, including bakers', wine, brewers' and distillers' yeasts, have been characterized with regards to their DNA content, chromosomal polymorphism and homologies with the DNA of laboratory strains. Measurement of the DNA contents of cells suggested that most of the industrial yeasts were aneuploids. Polymorphisms in the electrophoretic chromosomal pattern were so large that each strain could be individually identified. However, no specific changes relating to a particular group were observed. Hybridization using different probes from laboratory strains was very strong in all cases, indicating that all industrial strains possess a high degree of DNA homology with laboratory yeasts. Probes URA3, CUP1, LEU2, TRP1, GAL4 or ADC1 demonstrated the presence of one or two bands, two especially in bakers' strains. Also, results indicate that all hybridized genes are located on the same chromosomes both in laboratory and industrial strains. Translocation from chromosome VIII to XVI seems to have occurred in a distillers' strain, judging by the location of the CUP1 probe. Finally, when the SUC2 probe is used, results indicate a very widespread presence of the SUC genes in only bakers' and molasses alcohol distillers' strains. This clearly suggests that amplification of SUC genes is an adaptive mechanism conferring better fitness upon the strains in their specific industrial conditions. The widespread presence of Ty1 and Ty2 elements as well as Y′ subtelomeric sequences could account for the inter- and intrachromosomal changes detected. Received: 21 July 1997 / Accepted: 25 August 1997     

Effects of stress on the composition of yeast lipids

Pigmented(Rhodotorula glutinis) and nonpigmented(Lipomyces starkeyi) yeasts were studied. Exogenous Stressors (UV irradiation and methylene blue) were shown to change the composition of yeast lipids (especially the ratio of unsaturated fatty acids) and to increase the content of lipid peroxidation products formed (particularly in nonpigmented yeasts). In carotene-synthesizing yeasts, these Stressors decreased the amount of carotenoids produced and did not affect the ratio between carotenoid pigments (β-carotene, torulene, and torularhodin).     

Microbial population and biochemical changes during production of protein-enriched fufu

Summary Candida utilis strain BKT4 and Saccharomyces cerevisiae strain BKT7 isolated from burukutu (a local wine brewed from sorghum) were used to enrich fufu. During the fermentation process, there were changes in the microbiological and biochemical characteristics of the cassava. The total viable counts increased with increasing fermentation time while the counts of the lactics and fungi increased at the later stages of the fermentation due to the acidity of the medium. Various bacteria (Bacillus, Staphylococcus, Klebsiella, Escherichia, Streptococcus, Lactobacillus, Leuconostoc, Corynebacterium), moulds (Penicillium, Aspergillus, Fusarium, Mucor, Rhizopus) and yeasts (Candida, Saccharomyces, Hansenula, Rhodotorula) were found to be associated with the fermentation process. The pH of the fermenting cassava increased from 4.2 to 5.7 after 72 h while the cyanide level decreased from 2.2 mg/kg to 0.7 mg/kg over the same period of fermentation. Fufu (prepared by crushing and sieving fermenting cassava roots) enriched with 0.5 g of C. utilis strain BKT4, S. cerevisiae BKT7 and a mixed culture of the two organisms revealed a crude protein of 7.90, 6.34 and 10.0% respectively as compared to 2% protein content of the enriched fufu. There was a corresponding increase in protein content of the product as the quantity of the enrichment yeast was increased from 0.5 to 3.0 g. The aroma of the enriched fufu was preferred to that of the commercial fufu. Generally, good acceptability and organoleptic qualities (colour, taste, texture and aroma) of the protein enriched fufu was best achieved within 48 h of enrichment. The results of this study suggest that fufu can be made more nutritious with yeasts particularly Candida utilis strain BKT4 and Saccharomyces cerevisiae strain BKT7.     

Occurrence and Diversity of Yeasts in the Mid-Atlantic Ridge Hydrothermal Fields Near the Azores Archipelago

The yeast community associated with deep-sea hydrothermal systems of the Mid-Atlantic Rift was surveyed for the first time. This study relied on a culture-based approach using two different growth media: a conventional culture medium for yeasts supplemented with sea salts (MYPss) and the same medium additionally supplemented with sulfur (MYPssS). For the evaluation of species diversity, a molecular approach involving minisatellite-primed polymerase chain reaction (MSP-PCR) strain typing and sequence analysis of the D1/D2 domains of the 26S rDNA was followed. In the seven water samples that were studied, the number of colony-forming units per liter (cfu/L) ranged from 0 to 5940. The nonpigmented yeasts were much more abundant than the pink-pigmented ones. This disproportion was not observed in studies of other marine systems and may be due to the unique conditions of hydrothermal vents, characterized by a rich animal and microbial diversity and therefore by the availability of organic compounds utilizable by yeasts. Higher counts of nonpigmented yeast were obtained using MYPss, whereas for pink yeasts, higher counts were obtained using MYPssS. Moreover, among pink yeasts, some of the MSP-PCR classes obtained were composed of isolates obtained only on MYPssS, which might be an indication that these isolates are adapted to the ecosystems of the hydrothermal vents. Twelve phylotypes belonged to the Ascomycota and seven phylotypes belonged to the Basidiomycota. The nonpigmented yeasts were identified as Candida atlantica, C. atmosphaerica, C. lodderae, C. parapsilosis, Exophiala dermatitidis, Pichia guilliermondii, and Trichosporon dermatis, whereas the pigmented yeasts were identified as Rhodosporidium diobovatum, R. sphaerocarpum, R. toruloides, and Rhodotorula mucilaginosa. Some of the yeasts that were found belong to phylogenetic groups that include species reported from other marine environments, and eight phylotypes represent undescribed species. The new phylotypes found at Mid-Atlantic Ridge hydrothermal fields represent 33% of the total number of yeast taxa that were found.     

Enumeration of gut associated extracellular enzyme‐producing yeasts in some freshwater fishes

Extracellular enzyme‐producing yeasts might be involved in the supplementation of enzymes within the gastrointestinal tract of fish. The present study was intended to detect yeasts in the intestine of three Indian major carps (Labeo rohita, Catla catla, Cirrhinus mrigala), three exotic carps (Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cyprinus carpio), as well as Nile tilapia (Oreochromis niloticus), and to identify the most promising extracellular enzyme‐producing (e.g. amylase, protease, lipase, cellulase, xylanase and phytase) yeast strains by 18S rDNA sequence analysis. Selected for qualitative enzyme assay were 121 yeast strains, from which 28 were further studied for quantitative enzyme assay. The strain CMH6A isolated from C. mrigala exhibited the best extracellular enzyme activities except for amylase and cellulase. The strain ONF19B isolated from O. niloticus was noted as the best extracellular enzyme producer among the strains that produced all of the extracellular enzymes studied. Sequencing of the 18S rDNA fragment followed by nucleotide blast in the National Centre for Biotechnology Information (NCBI) GenBank revealed that strains CMH6A and ONF19B were similar to Pichia kudriavzevii (Accession no. KF479403 ) and Candida rugosa (Accession no. KF479404 ), respectively. The test of antagonism (in vitro) revealed that the isolated yeasts could not affect the growth of the autochthonous gut bacteria. This might indicate likely co‐existence of autochthonous yeasts and bacteria in the fish gut. Further research is necessary to explore the possibilities of utilizing the extracellular enzyme‐producing yeasts detected in the present study for commercial aquaculture.     

Specificity of Yeast Sensitivity to the Mycocin of Tilletiopsis flava VKM Y-2823

The mycocinogenous strain Tilletiopsis flava VKM Y-2823 was found to possess fungicidal activity at pH 3.5–4.5, which was retained after curing the strain by eliminating the extrachromosomal genetic elements. The mycocin produced by the strain had a molecular mass of more than 10 kDa and was readily inactivated by heating and treatment with protease K. This mycocin was found to be active against species of the anamorphic genus Tilletiopsis. The overwhelming majority of other representatives of the order Tilletiales, as well as ascomycetous and basidiomycetous (including ballistosporous) yeasts of the orders Sporidiales and Tremellales, were resistant to it.     

An improved method for protoplast formation and its application in the fusion of Rhodotorula rubra with Saccharomyces cerevisiae

Protoplasts from various strains of red-pigmented yeasts were generated at high frequency using improved procedures. The use of sulphur-containing amino acids and 2-deoxyglucose in growth media led to impaired cell wall synthesis and rendered cells very susceptible to treatment with mercapto-ethanol and various lytic enzymes. Use of individual lytic enzymes separately resulted in relatively low frequencies of protoplasts from most of the red yeasts examined, whilst use of -glucoronidase, Novozyme and Zymolyase in series markedly increased stable protoplast formation. The latter effects were shown to be strain specific. The ability to generate large numbers of red yeast protoplasts prompted the attempt to examine intergeneric fusion between auxotrophs of a strain of Saccharomyces cerevisiae and Rhodotorula rubra. Putative hybrids were selected as variously-pigmented prototrophic colonies growing on minimal medium and stabilised by subculturing on the latter medium. Unusual cream, orange and yellow hybrid colonies were generated, composed of cells of varying morphologies (chains, multibudded). The majority of stable hybrids contained one nucleus, although several heterokaryons were also observed. Some hybrids possessed the phenotypes of both parents: fusant wcat41 grew as rapidly as the S. cerevisiae parent but also contained an inducible phenylalanine ammonia-lyase (PAL) which appeared to be more active than that of the Rhodotorula parent.     

Killer phenotype of indigenous yeasts isolated from Argentinian wine cellars and their potential starter cultures for winemaking

Of 31 yeasts, from different surfaces of two cellars from the northwest region of Argentina, 11 expressed killer activity against the sensitive strain Saccharomyces cerevisiae P351. Five of these killer yeasts were identified as S. cerevisiae by phenotypic tests and PCR-RFLP analysis. Two S. cerevisiae killer strains, Cf5 and Cf8, were selected based on their excellent kinetic and enological properties as potential autochthonous mixed starter cultures to be used during wine fermentation. They could dominate the natural microbiota in fermentation vats and keep the typical sensorial characteristics of the wine of this region.     

Effect of the carbon source on the carotenoid profiles of Phaffia rhodozyma strains

Phaffia rhodozyma strains ATCC 24202, ATCC 24203, ATCC 24228, ATCC 24229, ATCC 24261, NRRL Y-10921, NRRL Y-10922 and NRRL Y-17268 were grown on culture media containing glucose, sucrose or xylose as carbon sources. Carotenoids were extracted from biomass and analyzed by HPLC with diode-array detection. The carotenoid profiles depended on both the strain considered and the carbon source employed. Astaxanthin, the main pigment found in P. rhodozyma, accounted for 42–91% of total carotenoids. Other carotenoids such as canthaxanthin, echinenone, 3-hydroxyechinenone, lycopene, 4-hydroxy-3′, 4′-didehydro-β-ψ-carotene and phoenicoxanthin were detected. The highest volumetric carotenoid concentration (3.60 mg L⁻¹) was obtained with strain NRRL Y-17268 growing on xylose. In this case, astaxanthin accounted for 82% of total carotenoids. Received 29 May 1997/ Accepted in revised form 08 August 1997     

Novel strategy for yeast construction using δ-integration and cell fusion to efficiently produce ethanol from raw starch

We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining δ-integration and polyploidization through cell fusion. Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase/α-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The α-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts.     

Suitability of Selected Bacteria and Yeasts for Growing the Estuarine Heterotrich Ciliate Fabrea salina (Henneguy)1

ABSTRACT. Twenty-six species of bacteria and seven species of yeasts were aseptically presented separately as potential food sources to the estuarine heterotrich ciliate, Fabrea salina, under standardized conditions of cell number, medium, and temperature. The bacteria and yeasts were classified according to their effect on the intrinsic growth rate of the ciliate: Nutritious bacteria: Photobacterium fisherii, Vibrio neresis, V. natriegens, Flavobacterium sp. strain ASN16, V. harveyi, Xanthomonas sp. strain ASN22; Maintainer bacteria: V. vulnificus, Vibrio sp. strain V344, V. alginolyticus, V. anguillarum, Bacillus sp. strain ST₃32B₂, Vibrio sp. strain V415, Acinetobacter sp. strain BHT8, Flectobacillus marinus, and Enterobacter aerogenes; Maintainer yeasts: Candida albicans and Cryptococcus marcerans strain 2; Nonnutriuous bacteria: V. parahaemolyticus, Planococcus sp. strain ASN13, P. mandapapanensis, Hyphomicrobium vulgari, Pseudomonas sp. strain CNS1, an unidentified gram-negative, yellow-pigmented, motile bacillus strain IG9A2, Thiobacillus thioparus, Escherichia coli, Corynebacterium sp. strain BR 17, Achromobacter sp. strain 23030, and Oceanospirillum beijerinckii; Nonnutriuous yeasts: C. marcerans strain 1, Saccharomyces cerevisiae, Debaryomyces hansenii, an unidentified black yeast, and C. laurentii. Under the conditions specified, bacteria appear to have a certain minimal value for the growth of Fabrea while yeast have little to none.     

A comparison of clinical and food Saccharomyces cerevisiae isolates on the basis of potential virulence factors

Saccharomyces cerevisiae is the most widely used yeast in industrial/commercial food and beverage production and is even consumed as a nutritional supplement. Various cases of fungemia caused by this yeast species in severely debilitated traumatized or immune-deficient patients have been reported in recent years, suggesting that this species could be an opportunistic pathogen in such patients. To determine whether the industrial S. cerevisiae strains can be included in this virulent group of strains, we carried out a comparative study between clinical and industrial yeasts based on the various phenotypic traits associated with pathogenicity in two other yeast species (Candida albicans and Cryptococcus neoformans). The majority of the clinical isolates were found to secrete higher levels of protease and phospholipase, grow better at 42°C and show strong pseudohyphal growth relative to industrial yeasts. However three industrial yeast strains, one commercial wine strain, baker’s yeast and one commercial strain of S. cerevisiae (var. boulardii), were exceptions and based on their physiological traits these yeasts would appear to be related to clinical strains.     

New carotenoids from the thermophilic green sulfur bacterium Chlorobium tepidum: 1′,2′-dihydro-γ-carotene, 1′,2′-dihydrochlorobactene, and OH-chlorobactene glucoside ester, and the carotenoid composition of different strains

The complete carotenoid composition of the thermophilic green sulfur bacterium Chlorobium tepidum strain TNO was determined by spectroscopic methods. Major carotenoids were four kinds of carotenes: γ-carotene, chlorobactene, and their 1′,2′-dihydro derivatives (1′,2′-dihydro-γ-carotene and 1′,2′-dihydrochlorobactene). In lesser amounts, hydroxyl γ-carotene, hydroxyl chlorobactene, and their glucoside fatty acid esters were found. The only esterified fatty acid present was laurate, and OH-chlorobactene glucoside laurate is a novel carotenoid. In other strains of C. tepidum, the same carotenoids were found, but the composition varied from strain to strain. The overall pigment composition in cells of strain TNO was 4 mol carotenoids and 40 mol bacteriochlorophyll c per mol bacteriochlorophyll a. The effects of nicotine on carotenoid biosynthesis in C. tepidum differed from those in the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus. Received: 3 February 1997 / Accepted: 6 June 1997