Temperature affects the production,activity and stability of ligninolytic enzymes in<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and<Emphasis Type="Italic">Trametes versicolor</Emphasis>

Enzyme activity was determined in cultures of Pleurotus ostreatus and Trametes versicolor with cellulose as a sole C source and high C/N ratio. The fungi were able to grow and produce laccase and Mn-peroxidase (MnP) at 5-35 degrees C, the highest production being recorded at 25-30 degrees C in P. ostreatus and at 35 degrees C in T. versicolor. Production of both enzymes at 10 degrees C accounted only for 4-20% of the maximum value. Temperature optima for enzyme activity were 50 and 55 degrees C for P. ostreatus and T. versicolor laccases, respectively, and 60 degrees C for MnP. Temperatures causing 50% loss of activity after 24 h were 32 and 47 degrees C for laccases and 36 and 30 degrees C for MnP from P. ostreatus and T. versicolor, respectively.     

Thermoenergetics of pre-moulting and moulting kookaburras (<Emphasis Type="Italic">Dacelo novaeguineae</Emphasis>): they're laughing

We examined the effect of temperature on resting metabolic rate in seven field-captured laughing kookaburras (Dacelo novaeguineae) during late winter and early spring. Basal metabolic rate averaged 201+/-3.4 ml O(2) h(-1) (0.603 ml O(2) g(-1) h(-1)). Overall thermal conductance (K(o)) declined with ambient temperature ( T(a)) and averaged 0.026 ml O(2) g(-1) h(-1) degrees C(-1) at T(a)s<10 degrees C. Day-night differences in body temperatures (2.6 degrees C) and in alpha-phase versus rho-phase minimum metabolic rates were much greater (33%) than predicted for 340-g nonpasserine birds and suggest that these animals operate as low-metabolic intensity animals in their rest phase, but normal-metabolic intensity animals during their active phase. Metabolic rate was measured in four of the same birds undergoing moult. Thermal conductance increased to 60% above pre-moult values about 6 weeks after moult began. Basal metabolic rate of moulting birds showing peak thermal conductance readings averaged 17 ml O(2) h(-1) higher than pre-moult measurements. Although this increase was not statistically significant, we believe the moult costs of kookaburras are too low to overcome the inherent variability of BMR determination. We suggest that moult costs of kookaburras are only somewhat higher than the measured costs of protein synthesis of other endotherms.     

Mineralization of chlorpyrifos by co-culture of <Emphasis Type="Italic">Serratia</Emphasis> and <Emphasis Type="Italic">Trichosporon</Emphasis> spp.

A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l(-1) within 7 days. Co-cultures completely mineralized 50 mg chlorpyrifos l(-1) within 18 h at 30 degrees C and pH 8 using a total inocula of 0.15 g biomass l(-1).     

Novel chemolithotrophic,thermophilic, anaerobic bacteria <Emphasis Type="Italic">Thermolithobacter ferrireducens</Emphasis> gen. nov., sp. nov. and <Emphasis Type="Italic">Thermolithobacter carboxydivorans</Emphasis> sp. nov.

Three thermophilic strains of chemolithoautotrophic Fe(III)-reducers were isolated from mixed sediment and water samples (JW/KA-1 and JW/KA-2(T): Calcite Spring, Yellowstone N.P., WY, USA; JW/JH-Fiji-2: Savusavu, Vanu Levu, Fiji). All were Gram stain positive rods (approximately 0.5 x 1.8 microm). Cells occurred singly or in V-shaped pairs, and they formed long chains in complex media. All utilized H(2) to reduce amorphous iron (III) oxide/hydroxide to magnetite at temperatures from 50 to 75 degrees C (opt. approximately 73 degrees C). Growth occurred within the pH(60C) range of 6.5-8.5 (opt. pH(60C) 7.1-7.3). Magnetite production by resting cells occurred at pH(60C) 5.5-10.3 (opt. 7.3). The iron (III) reduction rate was 1.3 mumol Fe(II) produced x h(-1) x ml(-1) in a culture with 3 x 10(7) cells, one of the highest rates reported. In the presence or absence of H(2), JW/KA-2(T) did not utilize CO. The G + C content of the genomic DNA of the type strain is 52.7 +/- 0.3 mol%. Strains JW/KA-1 and JW/KA-2(T) each contain two different 16S rRNA gene sequences. The 16S rRNA gene sequences from JW/KA-1, JW/KA-2(T), or JW/JH-Fiji-2 possessed >99% similarity to each other but also 99% similarity to the 16S rRNA gene sequence from the anaerobic, thermophilic, hydrogenogenic CO-oxidizing bacterium 'Carboxydothermus restrictus' R1. DNA-DNA hybridization between strain JW/KA-2(T) and strain R1(T) yielded 35% similarity. Physiological characteristics and the 16S rRNA gene sequence analysis indicated that the strains represent two novel species and are placed into the novel genus Thermolithobacter within the phylum 'Firmicutes'. In addition, the levels of 16S rRNA gene sequence similarity between the lineage containing the Thermolithobacter and well-established members of the three existing classes of the 'Firmicutes' is less than 85%. Therefore, Thermolithobacter is proposed to constitute the first genus within a novel class of the 'Firmicutes', Thermolithobacteria. The Fe(III)-reducing Thermolithobacter ferrireducens gen. nov., sp. nov. is designated as the type species with strain JW/KA-2(T) (ATCC 700985(T), DSM 13639(T)) as its type strain. Strain R1(T) is the type strain for the hydrogenogenic, CO-oxidizing Thermolithobacter carboxydivorans sp. nov. (DSM 7242(T), VKM 2359(T)).     

Optimization of alkaline protease production by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis> ES1 in <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> tuber powder using statistical experimental design

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett-Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R (2)=0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 degrees C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 degrees C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost-effective production of alkaline protease by the strain.     

Mannose production from fructose by free and immobilized <Emphasis Type="SmallCaps">d</Emphasis>-lyxose isomerases from <Emphasis Type="Italic">Providencia stuartii</Emphasis>

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of d-fructose to d-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45°C in the presence of 1 mM Mn²⁺. Enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35°C = 25% (w/w) yield with a productivity of 75 g mannose l⁻¹ h⁻¹ after 23 cycles.     

Effect of mutations in the pea genes <Emphasis Type="Italic">Sym33</Emphasis> and <Emphasis Type="Italic">Sym40</Emphasis>

Two symbiotic pea (Pisum sativum L.) mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread formation in symbiotic root nodules were characterised with respect to dynamics of arbuscule development at 15 degrees C and 24 degrees C. Mutation of sym33 decreased mycorrhiza colonisation at both temperatures and delayed arbuscule development at 15 degrees C, whereas mutation of sym40 accelerated mycorrhiza colonisation and arbuscule senescence at 24 degrees C. The differences between the mutants and the wild-type were more pronounced at 24 degrees C, a temperature close to the optimum for pea growth. The results demonstrate that both pea genes are important in the control of arbuscular mycorrhiza development and can be considered necessary for the tripartite symbiosis in pea.     

Transformation of Industrialized Strain <Emphasis Type="Italic">Candida glycerinogenes</Emphasis> with Resistant Gene <Emphasis Type="Italic">zeocin</Emphasis> via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Candida glycerinogenes WL2002-5 has a modest sugar tolerance and an extremely high glycerol productivity. Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, into the nuclear genome of a wide variety of host cells. In this study, we constructed the plasmid pZR and transferred it into A. tumefaciens LBA4404 to form the strain LBA4404-ZR. LBA4404-ZR was cocultivated with C. glycerologenesis, and putative transformants were identified by selection for zeocin resistance. Polymerase chain reaction and Southern blot analysis confirmed that the gene zeocin was integrated into the genome of engineered C. glycerologenesis. Optimization of the transformation condition was performed in darkness at 25 degrees C on induction medium for 24 h by cocultivation of C. glycerinogenes and LBA4404-ZR with a cell ratio of 1:500-1000. The transformation efficiency reached 2 transformants per 10(4) C. glycerologenesis cells. Our results demonstrated that A. tumefaciens-mediated transformation can be used for C. glycerinogenes. This transformation system can provide the basis for research of C. glycerologenesis in the future.     

Triacylglycerols determine the unusual storage physiology of<Emphasis Type="Italic"> Cuphea</Emphasis> seed

Many species within the genus Cuphea (Lythraceae) produce seed with high levels of medium-chain fatty acids. Seeds of some Cuphea species lose viability when placed into storage at -18 degrees C. These species tolerate significant drying to 0.05 g/g and may, therefore, be intermediate in their storage characteristics. The thermal properties of seed lipids were observed using differential scanning calorimetry. Species with peak lipid melting temperatures >/=27 degrees C were found to be sensitive to -18 degrees C exposure while those with melting temperatures <27 degrees C were able to tolerate low-temperature exposure. This relationship was determined by the triacylglycerol composition of the individual species. Sensitive species have high concentrations of lauric acid (C(12)) and/or myristic acid (C(14)). Species with high concentrations of capric (C(8)) or caprylic acid (C(10)) or with high concentrations of unsaturated fatty acids tolerate low temperature exposure. Potential damage caused by low temperature exposure can be avoided by exposing seeds to a brief heat pulse of 45 degrees C to melt solidified lipids prior to imbibition. The relationship between the behavior of triacylglycerols in vivo, seed storage behavior and sensitivity to imbibitional damage is previously unreported and may apply to other species with physiologies that make them difficult to store.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Metabolism and thermoregulation in the springhare (<Emphasis Type="Italic">Pedetes capensis</Emphasis>)

Springhares are large, nocturnally active, diurnally fossorial rodents that typically inhabit arid and semi-arid areas. This lifestyle means that they need to balance excessive heat loss when foraging at night against insufficient heat loss in a potentially warm, humid burrow and both of these against the need to minimize water turnover and energy requirements. In this study we investigated metabolism and thermoregulation in these animals. Basal metabolic rate averaged 8.62+/-1.37 J g(-1) h(-1) and minimum thermal conductance 0.386+/-0.062 J g(-1) h(-1) degrees C(-1). These were higher and lower than expected, respectively. This, along with a relatively low, lower critical temperature and broad thermal neutral zone indicate that springhares are physiologically well suited to the low night-time temperatures, which they typically encounter. Body temperatures were quite labile but springhares became hyperthermic at temperatures above 30 degrees C suggesting that they are poor thermoregulators at high temperatures. This is attributed to their seldom, if ever, encountering temperatures in this range. Insufficient heat loss under normal resting conditions does not appear to be a problem, as springhares inhabit deep burrows in which the temperature never exceeds the upper critical temperature. Excess heat generated during vigorous underground exercise is presumably stored and dissipated to the cool night air or the cooler soil when subsequently resting. Water turnover and energy expenditure are presumably adequately addressed by other physiological and behavioural characteristics.     

<Emphasis Type="Italic">Acinetobacter soli</Emphasis> sp. nov., isolated from forest soil

A non-motile and rod shaped bacterium, designated strain B1(T), was isolated from forest soil at Mt. Baekwoon, Republic of Korea. Cells were Gram-negative, catalase-positive, and oxidase-negative. The major fatty acids were 9-octadecenoic acid (C(18:1) omega9c; 42%) and hexadecanoic acid (C(16:0); 25.9%) and summed feature 3 (comprising iso-C(15:0) 2-OH and/or C(16:1) omega7c; 10.0%). The DNA G+C content was 44.1 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain B1(T) formed a lineage within the genus Acinetobacter and was closely related to A. baylyi DSM 14961(T) (98.6% sequence similarity), followed by A. baumannii DSM 30007(T) (97.4%), A. calcoaceticus DSM 30006(T) (97.0%) and 3 genomic species (96.8 approximately 7.6%). Phenotypic characteristics, gyrB gene sequence analysis and DNA-DNA relatedness data distinguished strain B1(T) from type strains of A. baylyi, A. baumannii, and A. calcoaceticus. On the basis of the evidence presented in this study, strain B1(T) represents a novel species of the genus Acinetobacter, for which the name Acinetobacter soli sp. nov. is proposed. The type strain is B1(T) (= KCTC 22184(T)= JCM 15062(T)).     

Inactivation of barotolerant strains of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 by ultra high pressure and <Emphasis Type="Italic">tert</Emphasis>-butylhydroquinone combination

Antimicrobial efficacy of ultra-high-pressure (UHP) can be enhanced by application of additional hurdles. The objective of this study was to systematically assess the enhancement in pressure lethality by TBHQ treatment, against barotolerant strains of Escherichia coli O157:H7 and Listeria monocytogenes. Two L. monocytogenes Scott A and the barotolerant OSY-328 strain, and two E. coli O157:H7 strains, EDL-933 and its barotolerant mutant, OSY-ASM, were tested. Cell suspensions containing TBHQ (50 ppm, dissolved in dimethyl sulfoxide) were pressurized at 200 to 500 MPa (23+/-2 degrees C) for 1 min, plated on tryptose agar and enumerated the survivors. The TBHQ-UHP combination resulted in synergistic inactivation of both pathogens, with different degrees of lethality among strains. The pressure lethality threshold, for the combination treatment, was lower for E. coli O157:H7 (> or = 200 MPa) than for L. monocytogenes (> 300 MPa). E. coli O157:H7 strains were extremely sensitive to the TBHQ-UHP treatment, compared to Listeria strains. Interestingly, a control treatment involving DMSO-UHP combination consistently resulted in higher inactivation than that achieved by UHP alone, against all strains tested. However, sensitization of the pathogens to UHP by the additives (TBHQ in DMSO) was prominently greater for UHP than DMSO. Differences in sensitivities to the treatment between these two pathogens may be attributed to discrepancies in cellular structure or physiological functions.     

Winter warming facilitates range expansion: cold tolerance of the butterfly<Emphasis Type="Italic"> Atalopedes campestris</Emphasis>

Our ability to predict ecological and evolutionary responses to climate change requires an understanding of the mechanistic links between climate and range limits. The warming trend over the past half-century has generated numerous opportunities to develop much-needed case studies of these links. Species that are only limited by climatic factors are likely to shift range quickly during periods of warming. Such species directly impact recipient communities and indicate trends that will become more widespread. Because minimum temperature (T (min)) is rising at twice the rate of maximum temperature, species with this range-limiting factor may be especially responsive to global warming. In this study, I test the hypothesis that rising T (min) has directly affected the range of a skipper butterfly. Atalopedes campestris has moved northward rapidly this century, recently colonizing eastern Washington where January T (min) has risen 3 degrees C in 50 years. The results show that: 1. A. campestris' range lies completely within the -4 degrees C January average minimum isotherm, and that recently colonized areas were below this threshold earlier this century. 2. In acute cold stress experiments, -4 to -7 degrees C proved to be a critical thermal limit: median supercooling point was -6.3 degrees C, and minimum lethal temperature (LT50 with 12-h exposure) was -5.7 degrees C. 3. In chronic cold stress experiments, survivorship declined sharply in diurnally fluctuating thermal regimes typical of the current range edge. High mortality occurred under constant 0 degrees C conditions as well as in fluctuating regimes, implying that thermal insulation from snow would not protect A. campestris. 4. There was no evidence of evolution in cold tolerance at the range margin, despite strong selection. Thus, winter warming was apparently a prerequisite for the range expansion. Characteristics of this species that seem to be associated with its rapid response are that it is an opportunistic species, it is not habitat or dispersal limited, and it is constrained by T (min).     

Survival and motility of diatoms<Emphasis Type="Italic">Navicula grimmei</Emphasis> and<Emphasis Type="Italic">Nitzschia palea</Emphasis> affected by some physical and chemical factors

Navicula grimmei and Nitzschia palea occurring almost equally in a mixed population on department moist garden soil surface when maintained in fresh supernatant (of soil-water medium) at pH 7.0, temperature of 26 +/- 1 degree C and under continuous light (intensity of approximately equal to 30 micromol m(-2) s(-1)) in a culture chamber exhibited a similar cell survival period (of 28 d) and percentage (at the beginning 100 % and mid of survival period 65 %) and stop gliding 11 d prior to cell death (with gliding speed reduced in both from 204-330 microm/min at the beginning to 82.5-99 microm/min at the mid of gliding period) irrespective of their size differences. However, a sharp fall in the cell gliding period, gliding cell percentage and speed occurred at various levels (different from cell survival period and percentage) in both diatoms in a similar extent under water stress (2, 4 and 6 % agarized supernatant, liquid supernatant with 0.2-1.0 mol/L NaCl, blot-dryness of cells for 5-15 min), pH extreme of liquid supernatant (< or =5.0, > or =9.0), temperature extremes in liquid supernatant (< or =15, > or =40 degrees C), UV exposure (0.96-5.76 kJ/m2), lack of all nutrients from the medium (double distilled water), darkness or low light intensities (2 and 10 micromol m(-2) s(-1)), presence of 'heavy' metals (Ni, Cu, Zn, Co, Fe, Hg; 1-200 ppm), organic substances in liquid supernatant (DDT, captan, urea, 2,4-D, 100-2000 ppm; thiourea, 50-1000 ppm). N. palea sway (turn around at either ends) or not only when gliding but independent of cell gliding speed, which decreased continuously under all conditions.     

Fecundity and feeding of <Emphasis Type="Italic">Galerucella calmariensis</Emphasis> and <Emphasis Type="Italic">G. pusilla</Emphasis> on <Emphasis Type="Italic">Lythrum salicaria</Emphasis>

Fecundity and feeding of two introduced sibling biological control species, Galerucella calmariensis and G. pusilla (Coleoptera: Chrysomelidae) on purple loosestrife, Lythrum salicaria L. (Lythraceae) were compared at constant temperatures of 12.5, 15, 20, 25, and 27.5 °C. Larval feeding was also carried out at 30 °C, but at this temperature, larvae developed only to the L2 stage and none pupated. Thus, data for this temperature were not used in the analysis. There were significant species × temperature interactions in fecundity. Of the two species, Galerucella pusilla laid more eggs. Although egg production of both species was lowest at 12.5 °C and increased to 20 °C, at higher temperatures, the two species reacted differently. From 25 to 27.5 °C, egg production decreased for G. pusilla, but G. calmariensis fecundity peaked at 27.5 °C. Significant temperature × species × life-stage interactions were also observed in feeding. For each species, the amount of feeding varied with temperature and stage of development. Galerucella pusilla adults consumed more foliage at 15, 20, and 27.5 °C. However, at 12.5 °C G. calmariensis adults fed more than G. pusilla. G. pusilla larvae consumed an average of 25% less foliage than G. calmariensis. The lower larval consumption of G. pusilla suggests that when food is limited, G. pusilla larvae may have a higher survival rate because of its ability to complete larval development with less food and produce more progeny due to its greater fecundity. When food is not limited neither species would have a competitive advantage and both species could coexist temporally and spatially. However, since G. calmariensis larvae consumed more leaf material, the larval stage of this species would have a greater impact on purple loosestrife than G. pusilla.     

Anaerobic oxidation of 2-chloroethanol under denitrifying conditions by<Emphasis Type="Italic"> Pseudomonas stutzeri</Emphasis> strain JJ

A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the mu(max) was 0.31 h(-1). NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

<Emphasis Type="Italic">Candida kashinagacola</Emphasis> sp. nov., <Emphasis Type="Italic">C. pseudovanderkliftii</Emphasis> sp. nov. and <Emphasis Type="Italic">C. vanderkliftii</Emphasis> sp. nov., three new yeasts from ambrosia beetle-associated sources

Three new yeast species, Candida kashinagacola (JCM 15019(T) = CBS 10903(T)), C. pseudovanderkliftii (JCM 15025(T) = CBS 10904(T)), and C. vanderkliftii (JCM 15029(T) = CBS 10905(T)) are described on the basis of comparison of nucleotide sequences of large subunit ribosomal DNA D1/D2 region (LSU rDNA D1/D2). The nearest assigned species of the three new species was Candida llanquihuensis. Candida kashinagacola and C. pseudovanderkliftii differed from C. llanquihuensis by 3.8% nucleotide substitution of the region, while C. vanderkliftii did by 4.4%. Three new species differed in a number of physiological and growth characteristics from any previously assigned species and from one another. A phylogenetic tree based on the sequences of LSU rDNA D1/D2 showed that these new species together with Candida sp. ST-246, Candida sp. JW01-7-11-1-4-y2, Candida sp. BG02-7-20-001A-2-1 and C. llanquihuensis form a clade near Ambrosiozyma species. The new species did not assimilate methanol as a sole source of carbon, which supported the monophyly of these non methanol-assimilating species which are closely related to the methylotrophic yeasts. Candida kashinagacola was frequently isolated from the beetle galleries of Platypus quercivorus in three different host trees (Quercus serrata, Q. laurifolia and Castanopsis cuspidata) located in the sourthern part of Kyoto, Japan, thus indicating that this species may be a primary ambrosia fungus of P. quercivorus. On the other hand, C. pseudovanderkliftii and C. vanderkliftii were isolated only from beetle galleries in Q. laurifolia. Candida vanderkliftii was isolated from beetle gallery of Platypus lewisi as well as those of P. quercivorus. Candida pseudovanderkliftii and C. vanderkliftii are assumed to be auxiliary ambrosia fungi of P. quercivorus.     

Improved (<Emphasis Type="Italic">R</Emphasis>)-phenylacetylcarbinol production with <Emphasis Type="Italic">Candida utilis</Emphasis> pyruvate decarboxylase at decreased organic to aqueous phase volume ratios

The effect of decreasing the organic (octanol) to aqueous phase volume ratio was evaluated in a two-phase enzymatic process for (R)-phenylacetylcarbinol (PAC) production. Decreasing the ratio from 1:1 to 0.43:1 at 4°C increased PAC in the organic phase from 112 g/l to 183 g/l with a 10% improvement in overall productivity. Interestingly, the rate of enzyme (pyruvate decarboxylase) activity loss was unaffected by the reduced phase ratio over the reaction period (48 h). At 20°C and 0.43:1 phase ratio the organic phase PAC concentration increased to 212 g/l and the overall productivity increased by 30% although the PAC yield (based on pyruvate) declined by about 10% due to greater byproduct acetoin formation at the higher temperature. Product recovery in such a system is facilitated both by the higher PAC concentration and the reduced organic phase volume.