Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

Biochemical and molecular characterization of Saccharomyces cerevisiae strains obtained from sugar-cane juice fermentations and their impact in cachaça production

Saccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H₂S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5′,5"-trifluor-d,l-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard.Cachaça (pronounced “kha-sha-ssa”), the sugar cane spirit, is the most popular distilled beverage produced in Brazil. The annual production reaches 1.3 billion liters, with 15% being produced in more than 8,500 distilleries in the state of Minas Gerais.Traditional cachaça production relies on a spontaneous fermentative process that is mediated by the microbiota present in the cane juice wort and on the surface of equipments used in the productive process. It has been already demonstrated that in such systems there occurs a succession of yeasts, with Saccharomyces cerevisiae being the predominant species. Cachaça quality depends on the ecology of the microbial populations during an initial spontaneous fermentation (18, 29, 31, 32, 39). The fermentative process occurs through a continuously open fermentative process which is completed within 24 h and generally takes place from May to November, corresponding to the sugar cane harvesting period.Considering the conditions of production usually found in the cachaça distilleries, fermenting yeast populations have to face different types of stress (osmotic, high temperature, and high ethanol concentration). Besides, they might also present some characteristics such as a good fermentative power, no H₂S production, killer activity, flocculation ability, and production of flavoring compounds. Taking all of these factors into account, we have developed a strategy to select yeast strains with appropriated characteristics to produce cachaça with potentially higher-quality standards (52).Parallel to the selection and development of S. cerevisiae strains toward ethanolic fermentations, molecular methods were developed and validated to study the evolution of yeast flora in spontaneous but also in inoculated fermentations. Chromosomal karyotyping by pulsed-field gel electrophoresis is a complex and time-consuming process but is very efficient for the delimitation of S. cerevisiae strains since it makes it possible to distinguish strains at both the intra- and the interspecies levels. Numerous other methods of typing based on DNA polymorphism allow differentiating closely related yeast strains (8, 36, 43, 50). Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) is faster and easier than other methods (23, 31). Digestion of mtDNA with restriction enzymes such as HinfI or RsaI is associated with a high rate of polymorphism and has been used to study the authenticity of commercial wine yeast strains (14, 40, 41). A PCR method based on variations found on the mitochondrial COX1 (a gene coding for cytochrome oxidase) intron number and position has been validated to distinguish S. cerevisiae strains allowing researchers to monitor the evolution of wine fermentations conducted by commercial active dry yeast (27). Moreover, in many other studies, RAPD [random(ly) amplified polymorphic DNA]-PCR with different primers such as EL1 and LA1 has been successfully used to discriminate between wine yeast strains (2) and for differentiation of Saccharomyces species isolated in Brazil (18). In the last few years, fingerprinting of microsatellite or simple sequence repeat loci, short (1 to 10 nucleotides) DNA tandem repeats dispersed throughout the genome and with a high degree of variability, has proven to be useful for distinguishing S. cerevisiae strains (16, 20, 34, 40, 45). These loci exhibit a substantial level of polymorphism and have been used in humans for paternity tests and forensic medicine but also for the demonstration of population structures among indigenous S. cerevisiae strains (24, 42, 43).We describe here the isolation and characterization of S. cerevisiae strains from cachaça and the use of two of these strains in cachaça production. The strains were isolated from local producers and characterized by growth in high-alcohol, high-sugar environments and in the presence of 5,5′,5"-trifluor-d,l-leucine (TFL) and cerulenin to detect overproducers of flavor compounds. We also used molecular methods to evaluate the polymorphisms of yeast strains in the fermentative process. Our results demonstrate that some methodologies based on DNA polymorphism are insufficient to evaluate the diversity of S. cerevisiae population during the cachaça production. The utilization of specific biochemical tests is necessary in order to permit a more precise characterization of the dynamics of the selected strains during the fermentative process. Only by combining biochemical and molecular methods were we able to select strains that showed a suitable profile to be used as starters. The cachaça produced with these strains was also evaluated, and the results demonstrate that the quality of the final product is better than that obtained with a commercial strain also used to produce cachaça.     

Strategies to select yeast starters cultures for production of flavor compounds in cachaça fermentations

In this work, we have used classical genetics techniques to find improved starter strains to produce cachaça with superior sensorial quality. Our strategy included the selection of yeast strains resistant to 5,5′,5″-trifluor-d,l-leucine (TLF) and cerulenin, since these strains produce higher levels of higher alcohols and esters than parental strains. However, no clear relationship was observed when levels of flavoring compounds were compared with the levels expression of the genes (BAT1, BAT2, ATF2, EEB1 genes) involved with the biosynthesis of flavoring compounds. Furthermore, we determined the stability of phenotypes considered as the best indicators of the quality of the cachaça for a parental strain and its segregants. By applying the principal component analysis, a cluster of segregants, showing a high number of characteristics similar to the parental strain, was recognized. One segregant, that was resistant to TLF and cerulenin, also showed growth stability after six consecutive replications on plates containing high concentrations of sugar and ethanol. “Cachaça” produced at laboratory scale using a parental strain and this segregant showed a higher level of flavoring compounds. Both strains predominated in an open fermentative process through seven cycles, as was shown by mitochondrial restriction fragment length polymorphisms analysis. Based on the physical chemical composition of the obtained products, the results demonstrate the usefulness of the developed strategies for the selection of yeast strains to be used as starters in “cachaça” production.     

Structure,ecology and physiology of root clusters – a review

Hairy rootlets, aggregated in longitudinal rows to form distinct clusters, are a major part of the root system in some species. These root clusters are almost universal (1600 species) in the family Proteaceae (proteoid roots), with fewer species in another seven families. There may be 10–1000 rootlets per cm length of parent root in 2–7 rows. Proteoid roots may increase the surface area by over 140× and soil volume explored by 300× that per length of an equivalent non-proteoid root. This greatly enhances exudation of carboxylates, phenolics and water, solubilisation of mineral and organic nutrients and uptake of inorganic nutrients, amino acids and water per unit root mass. Root cluster production peaks at soil nutrient levels (P, N, Fe) suboptimal for growth of the rest of the root system, and may cease when shoot mass peaks. As with other root types, root cluster production is controlled by the interplay between external and internal nutrient levels, and mediated by auxin and other hormones to which the process is particularly sensitive. Proteoid roots are concentrated in the humus-rich surface soil horizons, by 800× in Banksia scrub-heath. Compared with an equal mass of the B horizon, the A₁ horizon has much higher levels of N, P, K and Ca in soils where species with proteoid root clusters are prominent, and the concentration of root clusters in that region ensures that uptake is optimal where supply is maximal. Both proteoid and non-proteoid root growth are promoted wherever the humus-rich layer is located in the soil profile, with 4× more proteoid roots per root length in Hakea laurina. Proteoid root production near the soil surface is favoured among hakeas, even in uniform soil, but to a lesser extent, while addition of dilute N or P solutions in split-root system studies promotes non-proteoid, but inhibits proteoid, root production. Local or seasonal applications of water to hakeas initiate non-proteoid, then proteoid, root production, while waterlogging inhibits non-proteoid, but promotes proteoid, root production near the soil surface. A chemical stimulus, probably of bacterial origin, may be associated with root cluster initiation, but most experiments have alternative interpretations. It is possible that the bacterial component of soil pockets rich in organic matter, rather than their nutrient component, could be responsible for the proliferation of proteoid roots there, but much more research on root cluster microbiology is needed.     

Physiological diversity and trehalose accumulation in Schizosaccharomyces pombe strains isolated from spontaneous fermentations during the production of the artisanal Brazilian cachaça

Twenty-seven Schizosaccharomyces pombe isolates from seven cacha?a distilleries were tested for maximum temperature of growth and fermentation, osmotolerance, ethanol resistance, invertase production, and trehalose accumulation. Two isolates were selected for studies of trehalose accumulation under heat shock and ethanol stress. The S. pombe isolates were also characterized by RAPD-PCR. The isolates were able to grow and ferment at 41 degrees C, resisted concentrations of 10% ethanol, and grew on 50% glucose medium. Four isolates yielded invertase activity of more than 100 micromol of reducing sugar x mg(-1) x min(-1). The S. pombe isolates were able to accumulate trehalose during stationary phase. Two isolates, strains UFMG-A533 and UFMG-A1000, submitted to a 15 min heat shock, were able to accumulate high trehalose levels. Strain UFMG-A533 had a marked reduction in viability during heat shock, but strain UFMG-A1000 preserved a viability rate of almost 20% after 15 min at 48 degrees C. No clear correlation was observed between trehalose accumulation and cell survival during ethanol stress. Strain UFMG-A1000 had higher trehalose accumulation levels than strain UFMG-A533 under conditions of combined heat treatment and ethanol stress. Molecular analysis showed that some strains are maintained during the whole cacha?a production period; using the RAPD-PCR profiles, it was possible to group the isolates according to their isolation sites.     

Taxonomy and distribution of the françois' langur (Presbytis francoisi)

This paper discusses the taxonomy and distribution ofPresbytis francoisi. We are especially concerned with the classification of the white-headed langur.T'an (1957) suggested that the white-headed langur be classified as a separate species,Presbytis leucocephalus. We suggest that the white-headed langur is a subspecies ofPresbytis francoisi and should be referred to asP. francoisi leucocephalus. The distribution of the six subspecies of the françois' langur,P. f. delacouri, P. f. francoisi, P. f. hatinhensis, P. f. laotum, P. f. leucocephalus, andP. f. poliocephalus, is limited to southeastern Asia.     

Ecological requirements of Ostracoda (Crustacea) in a heavily polluted shallow lake,Lake Yeniçağa (Bolu,Turkey)

Although the area of Lake Yeniça?a is a potential candidate for RAMSAR convention, several anthropogenic factors compromise its biological diversity. This is mostly due to nutrient-rich water released from both point and nonpoint sources. Thirteen ostracod taxa (Candona neglecta, C. candida, Ilyocypris bradyi, Darwinula stevensoni, Cypridopsis vidua, Physocypria kraepelini, Cypria ophtalmica, Prionocypris zenkeri, Eucypris virens, Herpetocypris reptans, Pseudocandona compressa, Fabaeformiscandona fabaeformis Potamocypris cf. fulva) were found during this study. Potamocypris cf. fulva is a new record for the Turkish freshwater ostracod fauna. The first nine of these species have broad geographic ranges, implying high tolerance levels to different environmental variables. Based on the estimated species optima and tolerance levels, two species exhibited higher effective number of occurrences (C. neglecta, and D. stevensoni, respectively) than the other species. Three species (C. neglecta, D. stevensoni, I. bradyi) did not show significant correlation with any environmental variable we used. Both Canonical correspondence (CCA) and Pearson correlation analyses showed that temperature was the most effective predictor of species occurrence, followed by electrical conductivity and redox potential. In contrast, pH and dissolved oxygen of water were the least effective predictors. Approximately 71% of the correlation between community composition and environmental variables was explained by the first axis of the CCA diagram, which had a relatively low (7.7%) cumulative variance of species. The lower (560 μg/l) and the upper (2030 μg/l) levels of ammonia (NH₃) exceeded the limits during winter season. The concentrations of total coliform and Escherichia coli bacteria were measured up to 10 × 10⁷ cfu/ml and 10 × 10³ cfu/ml, respectively. Results of physicochemical measurements, microbiological counts, and species data indicate that water quality of Lake Yeniça?a has been rapidly deteriorated by anthropogenic factors that are the main threat for not only the lake’s aquatic diversity but also human health around the lake.     

Cyniclomyces guttulatus (Saccharomycopsis guttulata) — culture,ultrastructure and physiology

Organisms that form an essential extra inner lining of selected areas of the stomach mucosa occur in mice, rats and some other animals. The yeast Cyniclomyces guttulatus (Saccharomycopsis guttulata) was shown in this study to line the stomach of domestic and feral rabbits, guinea pigs, and chinchillas. The layer of yeast cells formed a loose barrier between lumen contents and mucosal surface. A rapid rate of multiplication in the stomach provided yeast cells that blended in with stomach lumen contents, passed throught the gut, and were finally excreted in large numbers in fecal pellets. Ascospore formation occurred during passage through the large intestine. The layer of yeast cells lining the stomach had no evident salubrious nor deleterious effect on the animal. C. guttulatus grew rapidly from stomach contents or single fecal pellets in a new enriched semisolid medium. Growth was good at pH 1 through 8 on the solidified enriched medium. A very unusual characteristic of C. guttulatus is optimal growht at 38° C, and growth at 42° C, with failure to grow below 30° C. TEM demonstrated a very thick, laminated cell wall which had a thick, filamentous external coating. There were mitochondria, polyribosomes, lipid droplets, and an unusually large central nucleus. The developing spore nucleus became extremely electron dense and encapsulated, along with condensed mitochondria, ribosomes, short membrane sections and other organelles, in a dense lamellar covering.     

A Model of Holocene Mangrove Development and Relative Sea-level Changes on the Bragança Peninsula (Northern Brazil)

Based on the integration of geological information, the pollen record, radiocarbon data, and modern mangrove distribution, this paper proposes a model of evolutionary development for mangroves on the Brazilian Bragança Peninsula driven by relative sea-level changes from the middle to late Holocene. After a postglacial relative sea-level rise (RSL), the mangrove habitats on the Bragança Peninsula began to develop at about 5100 years BP close to the current RSL, originating in the middle of the peninsula. Between 1800 and 1400 years BP, this littoral area underwent a maximum RSL fall of 1 m below the current one, followed by a gradual RSL rise until 1000 years BP, when the modern RSL was reached. Between 5100 and 1000 years BP, the RSL of the Bragança coastline was probably never higher than 0.6 m above the present level. The first Holocene mangrove forest (5100 years BP) in this region disappeared from the plains due to RSL fall and was replaced by herbaceous vegetation (Cyperaceae and Poaceae).     

Influence of ε-caprolactam on growth and physiology of environmental bacteria

ε-Caprolactam was found to have an effect on ecologically important soil bacteria. It inhibited the growth of several Bacillus sp. and Rhizobium sp. but cells of Arthrobacter sp. were able to grow in the presence of caprolactam. Sphingomonas sp. lost its inherent capacity to produce extracellular polymer (EPS) if grown in medium containing caprolactam. In the case of raw domestic sewage, the diversity of native bacteria was diminished in presence of caprolactam. Polluted sea water yielded predominantly one type of caprolactam-degrading bacteria of the genus Achromobacter. These cells efficiently utilized up to 10 g caprolactam/L as the sole source of carbon and nitrogen in synthetic medium even in the presence of 20 g NaCl/L. Compared to cells of Arthrobacter sp., cells of Achromobacter sp. accumulated high amount of 6-aminocaproic acid due to degradation of caprolactam. When using caprolactam as sole source of carbon and nitrogen, Achromobacter cells showed unique physiological ability to produce EPS upon prolonged incubation in solid medium and in broth with low phosphate (C:N:P ratio 100:20:0.05). Hydrolyzed cell-free EPS had glucose as its major component though the only substrate provided in the medium for growth was caprolactam.     

Triacylglycerol phase and ‘intermediate’ seed storage physiology: a study of Cuphea carthagenensis

Seeds with ‘intermediate’ storage physiology store poorly under cold and dry conditions. We tested whether the poor shelf life can be attributed to triacylglycerol phase changes using Cuphea carthagenensis (Jacq.) seeds. Viability remained high when seeds were stored at 25°C, but was lost quickly when seeds were stored at 5°C. Deterioration was fastest in seeds with high (≥0.10 g g⁻¹) and low (0.01 g g⁻¹) water contents (g H₂O g dry mass⁻¹), and slowest in seeds containing 0.04 g g⁻¹. A 45°C treatment before imbibition restored germination of dry seeds by melting crystallized triacylglycerols. Here, we show that the rate of deterioration in C. carthagenensis seeds stored at 5°C correlated with the rate that triacylglycerols crystallized within the seeds. Lipid crystallization, measured using differential scanning calorimetry, occurred at 6°C for this species and was fastest for seeds stored at 5°C that had high and very low water contents, and slowest for seeds containing 0.04 g g⁻¹. Germination decreased to 50% (P50) when between 16 and 38% of the triacylglycerols crystallized; complete crystallization took from 10 to over 200 days depending on water content. Our results demonstrate interactions between water and triacylglycerols in seeds: (1) water content affects the propensity of triacylglycerols to crystallize and (2) hydration of seed containing crystallized triacylglycerols is lethal. We suggest that these interactions form the basis of the syndrome of damage experienced when seeds with intermediate storage physiologies are placed in long-term storage.     

Anatomical and histological observations of the stomach of Françis' leaf monkeys (Presbytis francoisi)

The anatomical and histological structure of the stomach was examined in François' leaf monkeys (Presbytis francoisi). The stomachs consisted of three parts, the saccus, the tubus gastricus and pars pylorica, and had well-developed taeniae and many haustra. The cardiac gland region occupied the saccular forestomach and the anterior half of the tubus gastricus, while the fundic gland region occupied the posterior half of the tubus gastricus. The pyloric gland region occupied the entire pars pylorica. In the mucosa of the cardiac gland region, villus-like processes were observed. The mucosae of the cardiac and pyloric gland regions contained not only neutral mucins but also acid mucins.     

Litter in a first–order stream of a temperate deciduous forest (Margaraça Forest, central Portugal)

To evaluate the importance and fate of organic matter inputs in forested streams, we determined the litterfall inputs and the benthic coarse particulate organic matter (CPOM) in one headwater stream flowing through a mixed deciduous forest, during one year. Both vertical traps and the stream bottom were sampled monthly. The material collected was sorted into four main categories: leaves, fruits and flowers, twigs and debris. Litter production was 715 g m−2 y−1 and seasonal, with 73% of the annual total during October–December (autumn). Leaves comprised the largest litter component. Benthic organic matter was 1880 g m−2 y−1, and was also seasonal. Highest accumulation was attained in spring, and twigs and branches comprised the major component. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stratigraphy and palynology of the Pennsylvanian continental Buçaco Basin (NW Iberia)

The Buçaco Basin is a Pennsylvanian continental basin located along an important NNW–SSE strike structure (Porto-Tomar-Ferreira do Alentejo shear zone) that separates the Ossa-Morena and Central Iberian Zones of the Iberian Variscan Fold Belt in central western Portugal. The shear zone controlled the sedimentation in the basin and probably its post-sedimentary evolution. Sedimentation is initially alluvial with characteristic red sandstones, breccias and conglomerates. A gradual change to a fluvial (and probably lacustrine) type of sedimentation is observed with finning-upward cycles of gravel conglomerates, sandstones and organic-rich mudstones with occasional coal seams. Three representative sections were sampled for palynology and seventeen samples yielded sporomorphs with moderate to poor preservation. The palynological content from the alluvial sediments shows low diversity and poorly preserved assemblages dominated by Triquitrites spp., Densosporites spp., Laevigatosporites spp., and other taxa associated with siliciclastic environments or rheophytic mires. The fluvial and lacustrine sediments show a dramatic increase in diversity with an abundant, typical peatland microflora including sporomorphs such as Endosporites spp., Lycospora spp. and Monoletes spp., but also marginal peat and siliciclastic substrate taxa such as Densosporites spp., Latensina/Cordaitina spp., and Florinites spp. Other common taxa are Cheiledonites spp., Crassispora spp., Dictyotriletes-like miospores (mostly fragments), Potonieisporites spp., and Wilsonites spp. The presence and considerable abundance of Potonieisporites novicus and Cheiledonites cf. major is indicative of the middle to upper Potonieisporites novicus-bhardwajii–Cheiledonites major (NBM) miospore biozone of Western Europe, corresponding to the late Stephanian (early Gzhelian).     

Microbiology of a NaCl stalactite ‘salticle’ in Triassic halite

Large regions of Earth's surface are underlain by salt deposits that evaporated from ancient oceans and are populated by extreme halophilic microbes. Some of these halophiles may have been preserved over geological timescales within hypersaline fluid inclusions, but ingresses of water and/or anthropogenic activities can lead to the formation of alternative habitats, including NaCl stalactites or other speleothems. While the microbiology of ancient evaporites has been well studied, the ecology of these recently formed structures is less-well understood. Here, the microbiology of a NaCl stalactite (‘salticle’) in a Triassic halite mine is characterized. The specific aims were to determine the presence of fluid inclusions, determine the microbial structure of the salticle compared with a nearby brine-pool and surficial soil, and characterize the ecophysiological capabilities of this unique ecosystem. The salticle contained fluid inclusions, and their microbiome was composed of Euryarchaetota, Proteobacteria, and Actinobacteria, with Haloarchaea in greater abundance than brine-pool or soil microbiomes. The salticle metagenome exhibited a greater abundance of genes involved in osmoregulation, anaerobic respiration, UV resistance, oxidative stress, and stress-protein synthesis relative to the soil microbiome. We discuss the potential astrobiological implications of salticles as enclosed salt-saturated habitats that are protected from ionizing radiation and have a stable water activity.     

Exploring Local Perceptions of and Attitudes toward Endangered François’ Langurs (Trachypithecus francoisi) in a Human-Modified Habitat

International Journal of Primatology - Understanding local community attitudes toward wildlife is critical for making context-sensitive conservation planning and management decisions that may...