Indonesian Tapé Ketan Fermentation

Indonesian tapé ketan is a fermentation in which a mold, Amylomyces rouxii Calmette (Chlamydomucor oryzae Went and Prinsen Geerligs), in combination with one or more yeasts such as Endomycopsis burtonii converts steamed rice to a sweet-sour, slightly alcoholic paste. A study was made to determine the biochemical changes that occur in the substrate during fermentation. It was found that the product was ready for consumption after fermentation at 30°C for 36 to 48 h. A. rouxii used about 30% of the total rice solids, resulting in a crude protein of 12% in 96 h, whereas the combination of the mold with E. burtonii reduced total solids by 50% in 192 h, causing crude protein to increase to 16.5%. Soluble solids increased from 5 to about 67% in 36 h and decreased to 12% at 192 h with A. rouxii alone, whereas soluble solids fell to about 8% at 192 h in the fermentation with both the mold and the yeast. The mold, by itself, reduced the starch content of the rice from 78 to 10% in 48 h and to less than 2% in 144 h. The mold plus yeast reduced the starch content to about 18% in 48 h; however the “starch” content did not fall below 6% even at 192 h, presumably because the yeast was producing glycogen, which was determined along with the residual starch. With both the mold and the mold plus yeast fermentations, reducing sugars increased from less than 1% to approximately 5% in 24 h and reached maximum concentration, 16 to 17%, between 36 and 48 h. A. rouxii by itself produced a maximum of about 5.6% (vol/vol) ethanol at 96 h. The highest concentration of ethanol (8%, vol/vol) was produced by the mold plus E. burtonii at 144 h. The mold by itself reduced the starting pH from 6.3 to about 4.0 in 48 h. The combination of the mold and yeast reduced the pH to 4.1 in 144 h. The mold increased total acidity to approximately 6.2 meq of H⁺ per 100 ml, and the combination of the mold and yeast increased the total acidity to 7.8 meq of H⁺ per 100 ml in 192 h. At 48 h there was practically no difference in the volatile acidity (0.20) for the combined fermentation compared with 0.26 meq of H⁺ per 100 ml for the mold fermentation. The mold and at least one species of yeast were required to develop the rich aroma and flavor of typical Indonesian tapé.     

Concomitant changes in high temperature tolerance and heat-shock proteins in desert succulents

Raising the day/night air temperatures from 30°C/20°C to 50°C/40°C increases the high temperature tolerated by Agave deserti, Carnegiea gigantea, and Ferocactus acanthodes by 6°C to 8°C; the increase is about half completed in 3 days and fully completed in 10 days. A 25 to 27 kilodalton protein concomitantly accumulates for all three desert succulents upon transfer to 50°C/40°C, while accumulation of other heat “heat-shock” proteins is species specific. Some of the induced proteins are more abundant at 3 days, while others (including the 25-27 kilodalton protein) remain after completion of high temperature acclimation.     

Fermentation Temperature Modulates Phosphatidylethanolamine and Phosphatidylinositol Levels in the Cell Membrane of Saccharomyces cerevisiae

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner.     

Synchronous Activation of Cell Division by Light or Temperature Stimuli in the Dimorphic Yeast Schizosaccharomyces japonicus

Many fungi respond to light and regulate fungal development and behavior. A blue light-activated complex has been identified in Neurospora crassa as the product of the wc-1 and wc-2 genes. Orthologs of WC-1 and WC-2 have hitherto been found only in filamentous fungi and not in yeast, with the exception of the basidiomycete pathogenic yeast Cryptococcus. Here, we report that the fission yeast Schizosaccharomyces japonicus responds to blue light depending on Wcs1 and Wcs2, orthologs of components of the WC complex. Surprisingly, those of ascomycete S. japonicus are more closely related to those of the basidiomycete. S. japonicus reversibly changes from yeast to hyphae in response to environmental stresses. After incubation at 30°C, a colony of yeast was formed, and then hyphal cells extended from the periphery of the colony. When light cycles were applied, distinct dark- and bright-colored hyphal cell stripes were formed because the growing hyphal cells had synchronously activated cytokinesis. In addition, temperature cycles of 30°C for 12 h and 35°C for 12 h or of 25°C for 12 h and 30°C for 12 h during incubation in the dark induced a response in the hyphal cells similar to that of light. The stripe formation of the temperature cycles was independent of the wcs genes. Both light and temperature, which are daily external cues, have the same effect on growing hyphal cells. A dual sensing mechanism of external cues allows organisms to adapt to daily changes of environmental alteration.     

A Genetic Test of Sexual Size Dimorphism in Pre-Emergent Chinook Salmon

Sex differences in early development may play an important role in the expression of sexual size dimorphism at the adult stage. To test whether sexual size dimorphism is present in pre-emergent chinook salmon (Oncorhynchus tshawytscha), alevins were reared at two temperatures (10 °C and 15 °C) and sexed using the OtY1 marker on the Y-chromosome. Linear mixed models were used to test for sex differences in alevin size within families while controlling for the random effects of sire and dam nested within sire. Males and females did not differ in weight at 10 °C but males were heavier than females at 15 °C. Sex accounted for 2% of the within-family variance in weight. In addition, at 15°C, the relationship between weight and sex was greater in families with larger eggs. Whereas male-biased sexual size dimorphism was present at the juvenile stage, female-biased sexual size dimorphism was present at sexual maturity. Males were also younger than females at sexual maturity. A head start on growth by males may underlie their earlier maturation at a smaller size, thus leading to female-biased SSD at the adult stage.     

Influence of Water and Temperature Stress on the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination

The temperature dependence of the rate and magnitude of the reappearance of photosystem II (PSII) variable fluorescence following illumination has been used to determine plant temperature optima. The present study was designed to determine the effect of a plant's environmental history on the thermal dependency of the reappearance of PSII variable fluorescence. In addition, this study further evaluated the usefulness of this fluorescence technique in identifying plant temperature optima. Laboratory and greenhouse grown potato (Solanum tuberosum L. cv “Norgold M”) plants had a thermal kinetic window between 15 and 25°C. The minimum apparent K_m of NADH hydroxypyruvate reductase for NADH occurred at 20°C. This temperature was also the temperature providing maximal reappearance of variable fluorescence. Soybean (Glycine max [L.] Merrill cv “Wayne”) plants had a thermal kinetic window between 15 and 30°C with a minimum apparent K_m at 25°C. Maximal reappearance of variable fluorescence was seen between 20 and 30°C. To determine if increasing environmental temperatures increased the temperature optimum provided from the fluorescence response curves, potato and soybean leaves from irrigated and dryland field grown plants were evaluated. Although the absolute levels of PSII variable fluorescence declined with increasing thermal stress, the temperature optimum of the dryland plants did not increase with increased exposure to elevated temperatures. Because of variability in the daily period of high temperature stress in the field, studies were initiated with tobacco plants grown in controlled environment chambers. The reappearance of PSII variable fluorescence in tobacco (Nicotiana tabacum L. cv “Wisconsin 38”) leaves that had experienced continuous leaf temperatures of 35°C for 8 days had the same 20°C optima as leaves from plants grown at room temperature. The results of this study suggest that the temperature optimum for the reappearance of variable fluorescence following illumination is not altered by the plant's previous exposure to variable environmental temperatures. These findings support the usefulness of this procedure for the rapid identification of a plant's temperature optimum.     

Low base‐substitution mutation rate and predominance of insertion‐deletion events in the acidophilic bacterium Acidobacterium capsulatum

Analyses of spontaneous mutation have shown that total genome‐wide mutation rates are quantitatively similar for most prokaryotic organisms. However, this view is mainly based on organisms that grow best around neutral pH values (6.0–8.0). In particular, the whole‐genome mutation rate has not been determined for an acidophilic organism. Here, we have determined the genome‐wide rate of spontaneous mutation in the acidophilic Acidobacterium capsulatum using a direct and unbiased method: a mutation‐accumulation experiment followed by whole‐genome sequencing. Evaluation of 69 mutation accumulation lines of A. capsulatum after an average of ~2900 cell divisions yielded a base‐substitution mutation rate of 1.22 × 10⁻¹⁰ per site per generation or 4 × 10⁻⁴ per genome per generation, which is significantly lower than the consensus value (2.5−4.6 × 10⁻³) of mesothermophilic (~15–40°C) and neutrophilic (pH 6–8) prokaryotic organisms. However, the insertion‐deletion rate (0.43 × 10⁻¹⁰ per site per generation) is high relative to the base‐substitution mutation rate. Organisms with a similar effective population size and a similar expected effect of genetic drift should have similar mutation rates. Because selection operates on the total mutation rate, it is suggested that the relatively high insertion‐deletion rate may be balanced by a low base‐substitution rate in A. capsulatum, with selection operating on the total mutation rate.     

Cultured Ovules as Models for Cotton Fiber Development under Low Temperatures

Cotton fibers (Gossypium hirsutum L.) developing in vitro responded to cyclic temperature change similarly to those of field-grown plants under diumal temperature fluctuations. Absolute temperatures and rates of temperature change were similar under both conditions. In vitro fibers exhibited a “growth ring” for each time the temperature cycled to 22 or 15°C. Rings were rarely detected when the low point was 28°C. The rings seemed to correspond to alternating regions of high and low cellulose accumulation. Fibers developed in vitro under 34°C/22°C cycling developed similarly to constant 34°C controls, but 34°C/22°C and 34°C/15°C cycling caused delayed onset and prolonged periods of elongation and secondary wall thickening. Control fiber length and weight were finally achieved under 34°C/22°C cycling, but both parameters were reduced at the end of the experiment under 34°C/15°C cycling. Fibers developed under all conditions had equal bundle tensile strength. These results demonstrate that: (a) cool temperature effects on fiber development are at least partly fiber/ovule-specific events; they do not depend on whole-plant physiology; and (b) cultured ovules are valid models for research on the regulation of the field cool temperature response.     

Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10³ CFU of L. monocytogenes/ml and 10⁵ CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log₁₀ CFU of L. monocytogenes/cm²). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.     

Analysis of Dissimilatory Sulfite Reductase and 16S rRNA Gene Fragments from Deep-Sea Hydrothermal Sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific

This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5°C and natural vent fluids at 7°C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and γ- and -Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with “Nanoarchaeota.” The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300°C were affiliated with the δ-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4°C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.     

Temperature Compensation of Circadian Period Length in Clock Mutants of Neurospora crassa

Temperature compensation of circadian period length in 12 clock mutants of Neurospora crassa has been examined at temperatures between 16 and 34°C. In the wild-type strain, below 30°C (the “breakpoint” temperature), the clock is well-compensated (Q₁₀ = 1), while above 30°C, the clock is less well-compensated (Q₁₀ = 1.3). For mutants at the frq locus, mutations that shorten the circadian period length (frq-1, frq-2, frq-4, and frq-6) do not alter this temperature compensation response. In long period frq mutants (frq-3, frq-7, frq-8), however, the breakpoint temperature is lowered, and the longer the period length of the mutants the lower the breakpoint temperature. Long period mutants at other loci exhibit other types of alterations in temperature compensation—e.g. chr is well-compensated even above 30°C, while prd-3 has a Q₁₀ significantly less than 1 below 30°C. Prd-4, a short period mutant, has several breakpoint temperatures. Among four double mutants examined, the only unusual interaction between the individual mutations occurred with chr prd, which had an unusually low Q₁₀ value of 0.86 below 27°C. There was no correlation between circadian period length and growth rate. These strains should be useful tools to test models for the temperature compensation mechanism.     

Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected (“botrytized”) wine fermentations carried out at high (~30°C) and ambient (~20°C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10⁶ cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.     

Persistence of Viruses in Desert Soils Amended with Anaerobically Digested Sewage Sludge

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log₁₀ reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 × 10⁵ Pa for both soils) and temperatures of 15, 27, and 40°C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40°C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40°C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15°C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log₁₀ reduction per day in clay loam and sandy loam soils, respectively. At 27°C, this rate increased to 0.629 log₁₀ reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15°C (k = 0.064 log₁₀ reduction per day, clay loam; k = 0.095 log₁₀ reduction per day, sandy loam) and 27°C (k = 0.133 log₁₀ reduction per day, clay loam; k = 0.154 log₁₀ reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40°C. No reduction of PRD-1 was observed after 28 days at 15°C and after 16 days at 27°C. At 40°C, the inactivation rates were 0.208 log₁₀ reduction per day in amended clay loam soil and 0.282 log₁₀ reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15°C, within 3 days at 27°C, and within 2 days at 40°C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.