Grape skins as a natural support for yeast immobilization

Grape skins were used to immobilize Saccharomyces cerevisiae. In repeated batch fermentations of grape by immobilized and free cells, the maximum specific rate of alcohol production on glucose decreased from 7.98 h^–1 at 25 °C to 0.7 h^–1 at 5 °C. The rate was approximately twice as high as that on fructose. The rates for free cells were very low. The maximum alcohol yield (0.45 g g^–1) was obtained at 5 °C when the immobilized biocatalyst was used.     

Production of mead by immobilized whole cells of Saccharomyces cerevisiae

Summary The continuous production of mead was achieved with whole cells of Saccharomyces cerevisiae immobilized in calcium alginate gels. The alcohol production was stable in the pH range of 2.5–6.0 and a temperature range of 18–30°C with a sharp increase at 35°C. The process reduced the problems of contamination and secondary fermentation which are associated with traditional mead production.     

Glucose oxidase and catalase activities ofPenicillium variabile P16 immobilized in polyurethane sponge

Conidia ofPenicillium variabile P16 were immobilized in polyurethane sponge and used in repeated-batch processes in a fluidized-bed reactor. Optimal conditions for production of glucose oxidase and catalase were: inoculum size, 10%; glucose concentration, 80 g L^–1; Ca-carbonate concentration, 15 g L^–1; temperature, 28°C and aeration rate, 4 VV^–1 min^–1. In an extended repeated-batch process, glucose oxidase activity was highest after the fourth batch and catalase activity was highest after the fifth batch. Scanning electron microscopy showed that the fungus grew only in the interior of carrier particles.     

Treatment of simulated wastewater containing toxic amides by immobilized <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> NHB-2 using a highly compact 5-stage plug flow reactor

Biodegradation of toxic amides by immobilized Rhodococcus rhodochrous NHB-2 has been studied to generate data for future development of reactors for the treatment of simulated wastewater containing various toxic amides. The whole resting cells were immobilized in different matrices like agar, polyacrylamide and alginate. Agar gel beads were selected for the treatment of simulated wastewater containing 100mM each acetamide, propionamide, and 10mM of acrylamide and packed in a highly compact five-stage plug flow reactor. The immobilized bacterium worked well in a broad pH range from 5 to 10, with an optimum at 8.7. The apparent K _m-value for the turnover of acetamide for the resting cells was determined to be around 40mM at pH 8.5 and 55°C, whereas the K _m-value of the purified amidase was predicted to be about 20 mM. This organism exhibited greater turnover of aliphatic amides as compared to aromatic amides. Although these cells showed maximal amide-degrading activity at 55°C, simulated wastewater treatment was carried out at 45°C, because of the greater stability of the amidase activity at that temperature. Of note, indices for overall temperature stability, based on the temperature dependence of apparent first order kinetic temperature denaturation constants, were determined to be –7.9±1.1×10^–4, and –13.7±1.3×10^–4, –14.5±0.7×10^–4, and –13.7±0.8×10^–4°Cmin, for free cells and cells immobilized in alginate, agar and polyacrylamide respectively. After 250min the reactor showed maximum degradation of acetamide, propionamide and acrylamide of about 97, 100 and 90%, respectively by using 883 enzyme activity units per reactor stage. The results of this investigation showed that R. rhodochrous NHB-2 expressing thermostable amidase could be used for the efficient treatment of wastewater containing toxic amides. Therefore, we suggest that this microbe has a very high potential for the detoxification of toxic amides from industrial effluents and other wastewaters.     

Enhanced conversion of sucrose to isomaltulose by a mutant of Erwinia rhapontici

Mutagenesis of Erwinia rhapontici was performed to enhance the production of isomaltulose from sucrose. A mutant strain, BN 68089, was obtained through a screening process involving automated and miniaturized cultivation in Bioscreen C. This high-throughput, miniaturized screening system was optimized to identify the mutant strain, which had a conversion yield (90%) and productivity (194 g l^–1 h^–1). The BN 68089 mutant cells were immobilized in sodium alginate and when operated in a packed bed reactor gave a yield of 89% and a productivity of 144 g l^–1 h^–1 of at 30 °C, the optimal temperature. Immobilized BN 68089 cells exhibited 8% and 15% higher yield and productivity, respectively, than those of the wild-type strain.     

Immobilization studies of whole microbial cells on transition metal activated inorganic supports

Summary Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial -d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.     

Root-zone temperature and salinity: Interacting effects on tillering,growth and element concentration in barley

The effects of root-zone salinity (0, 30, and 60 mmol L^–1 of NaCl) and root-zone temperature (10, 15, 20, and 25°C) and their interactions on the number of tillers, total dry matter production, and the concentration of nutrients in the roots and tops of barley (Hordeum vulgare L.) were studied. Experiments were conducted in growth chambers (day/night photoperiod of 16/8 h and constant air temperature of 20°C) and under water-culture conditions. Salinity and root temperature affected all the parameters tested. Interactions between salinity and temperature were significant (p<0.05) for the number of tillers, growth of tops and roots, and the concentration of Na, K, P in the tops and the concentration of P in the roots. Maximum number of tillers and the highest dry matter were produced when the root temperature was at the intermediate levels of 15 to 20°C. Effect of salinity on most parameters tested strongly depended on the prevailing root temperature. For example, at root temperature of 10°C addition of 30 mmol L^–1 NaCl to the nutrient solution stimulated the growth of barley roots; at root temperature of 25°C, however, the same NaCl concentration inhibited the root growth. At 60 mmol L^–1, root and shoot growth were maximum when root temperature was kept at the intermediate level of 15°C; most inhibition of salinity occurred at both low (10°C) and high (25°C) root temperatures. As the root temperature was raised from 10 to 25°C, the concentration of Na generally decreased in the tops and increased in the roots. At a given Na concentration in the tops or in the roots, respective growth of tops or roots was much less inhibited if the roots were grown at 15–20°C. It is concluded that the tolerance of barley plant to NaCl salinity of the rooting media appears to be altered by the root temperature and is highest if the root temperature is kept at 15 to 20°C.     

A solid-state bioreactor coupled with forced aeration and pressure oscillation

A novel design of a solid-state bioreactor, operated with periodic pressure oscillation coupled with forced aeration through the medium, gave efficient control of temperature. The evaluation of the bioreactor assembly with respect to temperature and cellulase production by Penicillium decumbens JUA10 showed that, at 4 atm and the bed depth of 6 cm, the maximal temperature variation in the reactor was +1.5 °C at a set value of 30 °C compared with +6.8 °C in a static tray system. The highest cellulase and -glucosidase activities were 15 IU g^–1 and 51 IU g^–1 substrate dry matter at 96 h, respectively, while only 10 IU g^–1 and 24 IU g^–1 were obtained in the static tray culture system.     

Effect of pH,aeration and sucrose feeding on the invertase activity of intactS. cerevisiae cells grown in sugarcane blackstrap molasses

S. cerevisiae was grown in a blackstrap molasses containing medium in batch and fed-batch cultures. The following parameters were varied: pH (from 4.0 to 6.5), dissolved oxygen (DO) (from 0 to 5.0 mg O₂L^–1) and sucrose feeding rate. When glucose concentration (S) was higher than 0.5 g L^–1 a reduction in the specific invertase activity of intact cells (v) and an oscillatory behavior of v values during fermentation were observed. Both the invertase reduction and the oscillatory behavior of v values could be related to the glucose inhibitory effect on invertase biosynthesis. The best culture conditions for attainingS. cerevisiae cells suitable for invertase production were: temperature=30°C; pH=5.0; DO=3.3 mg O₂L^–1; (S)=0.5 g L^–1 and sucrose added into the fermenter according to the equations: (V–V_o)=t²/16 or (V–V_o)=(V_f–V_o)·(e^0.6t–1)/10.This work was supported by FAPESP     

Fermentation of xylose and rice straw hydrolysate to ethanol byCandida shehatae NCL-3501

Candida shehatae NCL-3501 utilized glucose and xylose efficiently in batch cultures. The specific rate of ethanol production was higher with mixtures of glucose and xylose (0.64–0.83 g g^–1 cells d^–1) compared to that with individual sugars (0.38–0.58 g g^–1 cells d^–1). Although the optimum temperature for growth was 30°C, this strain grew and produced appreciable levels of ethanol at 45°C. A stable ethanol yield (0.40–0.43 g g^–1 substrate utilized) was obtained between 10 g L^–1 and 80 g L^–1 of initial xylose concentration. Conversion efficiency was further improved by immobilization of the cells in calcium alginate beads. Free or immobilized cells ofC. shehatae NCL-3501 efficiently utilized sugars present in rice straw hemicellulose hydrolysate, prepared by two different methods, within 48 h. Ethanol yields of 0.45 g g^–1 and 0.5 g g^–1 from autohydrolysate, and 0.37 g g^–1 from acid hydrolysate were produced by free and immobilized cells, respectively.     

A new control strategy for yeast production based on the L/A* approach

Summary We studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35–38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24–28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26–34° C) when the rate of temperature decrease was 0.2–0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32–34° C. The optimum temperature decrease was 0.8° C/h. Offprint requests to: J. Chaloupka     

Heat induction of reporter gene expression via the gadd153 promoter and its possible application to hyperthermia treatment of cancer

The use of the gadd153promoter to induce expression of a reporter geneunder heat stress conditions was investigated,since the results of previous studies have suggestedthat the gadd153promoter is likely to be activated by the indirecteffects of hyperthermia, that is, by DNA damage thatoccurs when reactive oxygen species are produced byheat stress. The optimum temperature for a significantinduction was found to be between 41 and 43 °C andincreased expression of the reporter gene was observedat about 24 h after the heat treatment. Under theseconditions, the cell integrity was not alteredmorphologically and the growth stopped temporarily,while the viability was maintained. A second increasein expression occurred at a later stage when the cellswere severely damaged at 43–45 °C. Atthese temperatures, the cellular morphology showedsignificant alteration and the growth was stronglyarrested. This is likely to be due to a differentmechanism which could involve DNA repair processes. Itis expected that this method of induction can beexploited to drive the production of a protein ofinterest in a cancer treatment program that includes hyperthermia.     

Amphora margalefii Tomàs var. lacustris P. Sánchez var. nova,a new brackish water diatom

The rotifer Brachionus plicatilis can grow in a wide range of salinities and temperatures, but rapid shifts in both salinity and temperature may result in immobilized, non-swimming rotifers. The goal of this study was to examine the effect of perturbations in temperature and salinity on the swimming pattern of the rotifer.Only slight changes in mobility were observed when rotifers were exposed to changes in temperature (from 20 °C to 8–30 °C) and to an increase in salinity (from 20% to 30%). When the salinity was reduced to 15% and 5%, the proportion of mobile rotifers was reduced to 50% and 5%, respectively. The rotifers were throughout more resistant to perturbations in temperature than to those of salinity.Combined temperature and salinity perturbations compared to perturbations in each factor separately suggested a synergetic effect of temperature and salinity on the rotifers locomotion. Transfer from cultivation conditions to low salinity (5%) and high temperature (28 °C) resulted in very low percent of mobile rotifers (0–10%). However, if the temperature was reduced to 8 °C concomitant with the changes in salinity, the percent of mobile rotifers was 85%.Rotifers use a high share of their metabolic energy for locomotion, and it is therefore not surprising that perturbations in salinity and temperature may result in partial or complete immobilization.     

Batch and continuous mead production with pectate immobilised, ethanol-tolerant yeast

Saccharomyces cerevisiae immobilised in calcium pectate gel optimally fermented honey mash to mead with a beads/medium ratio of 1:3, at 30 °C with `Vitamon Ultra' salt as the best commercial nitrogen/nutrient source. In continuous fermentation using a two-column system, the overall ethanol production rate was 5.7 g l^–1 h^–1.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Effects of acetic acid on the temperature profile of ethanol tolerance inSaccharomyces cerevisiae

Summary In a strain ofSaccharomyces cerevisiae, acetic acid at concentrations up to 1% (v/v) depressed the tolerance to added ethanol, from 11% (v/v) down to zero, and simultaneously narrowed the temperature range of growth from 3–42°C to 19–26°C. In addition, acetic acid shifted the associative temperature profile of growthand death to lower temperatures, and depressed the growth yield on glucose.     

Influence of temperature and photoperiod on the re-attachment process ofGelidium sesquipedale (Clem.) Born. et Thur. (Gelidiales: Rhodophyta)

Thalli of the economically important rhodophyteGelidium sesquipedale were cultured for 8 weeks under laboratory conditions, and the influence of temperature and photoperiod on the re-attachment process were studied. Four different temperatures (16, 18, 20, 22 °C) and four different photoperiods (6:18, 12:12, 14:10, 16:8) were used and the results obtained in the thalli responses such as apical growth (measured as elongation of principal apex), rhizoidal cluster production and number of necrotic patches were tested.During the re-attachment process, the best results were obtained at temperatures of 16–18 °C, when rhizoidal cluster production was high and necrotic patch development was low (18 °C) or absent (16 °C). Temperatures of 20 and 22 °C favoured high rhizoidal cluster production, but also a high production of necrotic patches that finally led to death. The results suggest that long-day photoperiods (14:10, 16:8) produce a higher number of rhizoidal cluster bands than short-day photoperiods (6:18) at the same temperature.     

Phytate dephosphorylation by free and immobilized cells ofSaccharomyces cerevisiae

Summary Saccharomyces cerevisiae in the form of baker's yeast, cells cultivated on a yeast extract-peptone-glucose medium, as well as cells immobilized in 18% (w/v) polyacrylamide gel showed the ability to hydrolyze 1.727 mM sodium phytate solution at 45°C, pH 4.6, in a stirred tank reactor. Seventy percent yield of dephosphorylation was observed after 2 h using a baker's yeast concentration of 5.8 g dry matter per 100 ml. Hydrolytic activity at 1.8–2.0 M Pi min^–1 was observed between 1st and 3rd h of the reaction in cells cultured 24 or 48 h. No inhibition by the substrate was found at sodium phytate concentrations of 0.587–1.727 mM. After 1.5 h of hydrolysis a single, well distinguished peak ofmyo-inositol-triphosphate was the main product found. By means of immobilization the stability of the biocatalyst was enhanced 3.3-fold and reached its half-life at 64 ninety-minute runs.     

Effects of temperature and holding time on production of renewable fuels from pyrolysis of Chlorella protothecoides

To investigate the influence of temperature andholding time on the pyrolyzate yields of Chlorella protothecoides, the microalgal cells weresubjected to pyrolysis at 200, 300, 400, 500 and 600 ^°Cfor 5, 20, 60 and 120 min, separately. High oil yields above 40% dry weight cells wereobtained both at relatively low temperature (300 ^°C)with relatively long holding times (20–120min) and relatively high temperatures (400–500 ^°C)with relatively short holding times (5–20min). The maximum oil yield of 52.0% was achieved at500 ^°C for 5 min. The gas yield was generallyincreased with the increasing temperature and holdingtime. It could reach 63.3–76.0% at 600 ^°C.High pyrolytic rates of 72–87% were obtained at allexperiments except at 200 ^°C for 5–20 min or300 ^°C for 5 min. Thermogravimetric analysisindicated that the main thermal degradation of thismicroalga occurred at 200–520 ^°C. The resultsimply that C. protothecoides is a good candidatefor the production of renewable fuels by pyrolysis.     

Effect of temperature on bacterial gellan production

The effect of temperature on the production of the polysaccharide gellan by the bacterium Sphingomonas paucimobilis ATCC 31461 was studied in relation to carbon source. When glucose served as the carbon source, gellan formation by the strain was highest after 72 h of growth at an incubation temperature of 30–31 °C. Polysaccharide production by the sphingomonad cells grown on corn syrup for 72 h was maximal at an incubation temperature of 31 °C. The highest cellular productivity in elaborating gellan was observed at 31 °C after 72 h of growth independent of the carbon source utilized.