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.     

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     

Thermoactive extracellular proteases of <Emphasis Type="Italic">Geobacillus caldoproteolyticus</Emphasis>, sp. nov., from sewage sludge

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH₄Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.     

Effect of temperature,hydrogen ion concentration and osmotic potential on oospore germination of fivePythium spp. isolated from pond water

Oospore germination occurred over a temperature ranging of 15–35°C forPythium coloratum, 10–35°C forP. diclinum, 15–30°C forP. dissotocum, 7–30°C forP. monospermum, and 10–30°C forP. pleroticum. Optimum temperature was 25°C for all species tested. In case of pH, oospore germination occurred over a range of 4.76–8.55 with an optimum of 6.40–7.40. The least germination occurred at pH 4.76 forP. coloratum, P. diclinum, P. monospermum andP. pleroticum, whileP. dissotocum germinated from pH 5.02. Oospores of the all tested pythia were able to germinate at –0.13 to –1.65 MPa and could not germinate at –3.40 MPa, with the highest germination rate at –0.27 to –0.47 MPa. The effect of temperature, pH and osmotic potential on oospore germination was discussed in relation to pollution of pond water.     

Production and characterization of xylanase from Bacillus thermoalkalophilus grown on agricultural wastes

Summary Bacillus thermoalkalophilus isolated from termite-infested mound soils of the semi-arid zones of India had the ability to produce good amounts of xylanase(s) from cheap agricultural wastes. Of the two hemicellulosic substrates tested, bagasse was found to be the better inducer for xylanase production. Alkali treatment of bagasse and rice husk had varied effects on enzyme production. The enzyme preparation had activity optima at 60° C and 70° C and a half-life of 60 min at 65° C. The enzyme was stable for 24 h over a pH range of 4.0–6.0, while maximum activity was observed at pH 6.0–7.0. Enzyme production and activity were inhibited by the end-product of xylan hydrolysis, xylose. Offprint requests to: Ajit Varma     

Effect of temperature on intrinsic rates of natural increase (rm) of a coccinellid and its spider mite prey

The intrinsic rate of natural increase(r_m) is useful to estimate the populationgrowth potential of insects and mites, whichmay help predict the outcome of pest-naturalenemy interactions. This study was conductedto determine how 12 constant temperatureregimes between 10–38 °C (± 0.5 °C) may differentially affect ther_m of the McDaniel spider mite, Tetranychus mcdanieli McGregor (Acarina: Tetranychidea), a common pest of raspberry, andits coccinellid predator, Stethoruspunctillum Weise (Coleoptera: Coccinellidae). Tetranychus mcdanieli survived tomaturity in the 14–36 °C range, comparedto the 14–32 °C range for S.punctillum. Survival above 24 °Cremained high for the spider mite, butdecreased markedly for the coccinellid. Tetranychus mcdanieli's range forreproduction was similar to its survival range,but S. punctillum failed to reproduce at14 °C and reproduced only poorlyat 16 °C. Offspring production peakedat 24 °C for both T. mcdanieli(average 152 eggs per female), and S.punctillum (280 eggs per female). At alltemperatures suitable for reproduction, femalelongevity of the coccinellid was greater thanthe spider mite, which was characterized byearlier/faster reproduction than thecoccinellid. As temperature increased, ther_m followed a typical asymmetricaldome-shape pattern, with maximum values of0.196 d^–1 and 0.385 d^–1at 30 °C and 34 °C, for S.punctillum and T. mcdanieli,respectively. For each species, ther_m-temperature relation was successfullymodelled using a curvilinear regressionequation previously shown to predictdevelopment rate. In both species, thedevelopment rate response to temperature has amajor influence on the temperature-r_mrelationship. In the 16–32 °C rangesuitable for population growth of both species,the r_m of T. mcdanieliwas 1.9 (30 °C) to 8 (16 °C) times greaterthan S. punctillum. These growthpotential ratios are consistently in favor ofthe prey, suggesting a limitation of thecoccinellid with respect to its capacity totrack T. mcdanieli populations. However,under short season conditions, the inferiorreproductive dynamics of S. punctillum'svs. spider mite prey should not have aprevailing influence in determining impact, andmay be compensated by high voracity incombination with a strong aggregativeresponse.     

Low Temperature Stimulates Cell Enlargement and Intracellular Calcification of Coccolithophorids

Temperature effect on growth, cell size, calcium uptake activity, coccolith production was studied in coccolith-producing haptophytes, Emiliania huxleyi (Lohmann) Hay & Mohler (strain EH2) and Gephyrocapsa oceanica Kamptner (strain GO1) (Coccolithophorales, Prymnesiophyceae). E. huxleyi grew at a wider temperature range (10°–25°C), while G. oceanica growth was limited to warmer temperatures (20°–25°C). Cell size was inversely correlated with temperature. At low temperature, the enlargement of chloroplasts and cells and the stimulation of coccolith production were morphologically confirmed under fluorescent and polarization microscopes, respectively. ⁴⁵ Ca uptake by E. huxleyi at 10°C was greatly increased after a 5-day lag and exceeded that at 20°C. These results clearly showed that low temperature suppressed coccolithophorid growth but induced cell enlargement and as stimulated the intracellular calcification that produces coccoliths.     

Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m³ fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4–30°C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70–75°C. The enzyme was almost thermostable (91–92%) at pH 6.5 and 9.0 after 41 h preincubation at 55°C and lost only 20–33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0. Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55°C and pH 6.5. Correspondence to: J. Gomes     

Microbiological degradation of the herbicide dicamba

Summary Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba byP. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100–2000 mg dicamba L^–1 (0.5–9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl^– per mol of dicamba degraded. The cultures had an optimum pH 6.5–7.0 for dicamba degradation. Growth studies at 10°C, 20°C, and 30°C yielded activation energy values in the range of 19–36 kcal mol^–1 and an average Q₁₀ value of 4.0. Compared with the pure cultureP. paucimobilis, the consortium was more active at the lower temperature.     

Growth and hyoscyamine production of ‘hairy root’ cultures of Datura stramonium in a modified stirred tank reactor

Summary The growth and hyoscyamine production of transformed roots of Datura stramonium have been examined in a modified 14-1 stirred tank reactor in both batch and continuous fermentations on media containing half or full strength Gamborg's B5 salts and at three different temperatures. Under a range of conditions, roots grown on half strength B5 salts with 3% w/v sucrose had a higher dry matter content (up to 8.3% w/w) and a higher hyoscyamine content (up to 0.52 mg·g^–1 wet weight) than roots grown on full strength B5 salts with the same level of sucrose (up to 4.6% w/w dry matter and up to 0.33 mg hyoscyamine g^–1 wet weight). Growth at 30°C was initially faster than at either 25°C or 35°C and by day 12, the drained weight of roots in the fermentor at 30°C was about fourfold greater than at 25°C and twice that at 35°C. The ultimate hyoscyamine levels attained (approximately 0.5 mg·g^–1 wet weight) were similar at both 25°C and 30°C but some 40% lower at 35°C. Final packing densities of 70% w/v were achieved for roots after 37 days growth at 25°C and the highest production rate of 8.2 mg hyoscyamine l^–1 per day was obtained for roots grown at 30°C. In continuous fermentation at 25°C, the release of hyoscyamine into the culture medium was low (less than 0.5% w/w of the total) but was up to sevenfold higher in fermentors operated at 30°C or 35°C. Offprint requests to: M. G. Hilton     

Isolation and partial characterization of bacteriocins from<Emphasis Type="Italic"> Pediococcus</Emphasis> species

Lactic acid bacteria have received increased attention as a potential food preservative due to their strong antagonistic activity against many food-spoilage and pathogenic organisms. Three Pediococcus species, P. acidilactici NCIM 2292, P. pentosaceous. NCIM 2296 and P. cervisiae NCIM 2171, were evaluated for bacteriocin production. Inhibitory substance were produced during the late growth phase and maximum production occurred at 37 °C after 36–48 h of incubation. Bacteriocins partially purified from these species by cold-acetone precipitation at 0 °C and cell adsorption desorption techniques have a broad inhibitory spectrum against microorganisms, including gram-negative bacteria such as Escherichia coli and Pseudomonas. Proteolytic enzymes inactivated these peptides, but amylase and lipase did not show any effect. The bacteriocins were stable over a wide pH range (3–8) and apparently most active at pH 4.0–5.0. They were heat-stable (1 h at ~80 °C and autoclaving) at pH 5.0. No loss in activity was observed when stored under refrigeration (4–8 °C). Tris-Tricine SDS-PAGE revealed the molecular masses of these peptides to be between 3.5 and 5.0 kDa.     

Temperature aspects of ecological bioenergetics in Brachionas angularis (Rotatoria)

The influence of temperature and food quality was studied on the following energy balance parameters of B. angularis: ingestion, production, growth and mortality. The ingestion rate rises to an optimum at 15 and 20 °C and decreases at 25 °C. The other rates increase continuously over the 5–25 °C range. The Q₁₀-values of production rate are higher than those of ingestion rate. Temperature also modifies the relationship between food concentration and bioenergetic rates. They react according to a Monod function (production at all temperatures, growth at 10 °C) or decrease at high concentrations (growth at 15° and 20 °C.)     

Production of mead by immobilized cells of Hansenula anomala

Summary Mead was produced by immobilized cells of Hansenula anomala in calcium alginate gels. The immobilized cell beads of 3 mm diameter packed in column reactors of dimensions 2.2x60, 4x40 and 8x80 cm, produced mead containing maximum concentrations of ethanol and ethyl acetate of 70 g/l and 730 mg/l, respectively at a dilution rate of 0.1 h^–1. The maximum alcohol productivity achieved was 23.1 g/l·h at a dilution rate of 0.33 h^–1. With intermittent regenerations of the cells the reactor operated continuously for 110 days. This process enables the quick production of matured mead by a single culture and the elimination of the traditionally used long aging periods.     

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.     

Phytoplankton primary production in a eutrophic cooling water pond

The seasonal variation of phytoplankton photosynthesis was measured with ¹⁴C-method in a warmed ice-free pond in central Finland. Simultaneously with in situ measurements the photosynthesis was also measured in an incubator with different water temperatures and constant light (ca. 16 W m^–2). The total annual photosynthesis was 57.2 C m^–2 a^–1. The portion of the winter and spring production of the annual photosynthesis was 18.4%, that of the autumn production ws 17.4%. Thus 64.3% of the total annual phytoplankton photosynthesis occurred in the three summer months. The range of the daily integrated photosynthesis per unit area was 1.9—563 mg C m^–2d^–1. The photosynthetic rate per unit chlorophyll a varied in situ from 0.94 to 33.1 mg C (mg chl. a)^–1 d^–1. The highest value was measured in the beginning of July and the lowest in mid-January. The photosynthetic rate increased in situ exponentially with increasing water temperature. In the incubator the highest photosynthetic rate values were also found in July and August (at+20 °C) when the phytoplankton population was increasing and the minimum values occurred after every diatom maximum both in spring and autumn. Light was a limiting factor for photosynthesis from September to Mid-January, low water temperature was a limiting factor from late January through May. The efficiency of the photosynthesis varied between 0.1 and 0.7% of P.A.R. According to the incubator experiments the Q₁₀ values for the photosynthesis were 2.45 and 2.44 for the winter population between 1 and 10° C and for the summer population between 5 and 15° C, respectively, but the Q₁₀ values decrease at the higher temperatures. The main effect of the warm effluents on the yearly photosynthesis was the increase of production in spring months due to the lack of ice cover. However, the increase of total annual phytoplankton photosynthesis was only ca. 10–15%, because the water temperature was during the spring months below 10° C.     

Effects of temperature on the properties of primary auditory fibres of the spectacled caiman,Caiman crocodilus (L.)

Summary The effect of temperature on the response properties of primary auditory fibres in caiman was studied. The head temperature was varied over the range of 10–35 ° C while the body was kept at a standard temperature of 27 °C (T_s). The temperature effects observed on auditory afferents were fully reversible. Below 11 °C the neural firing ceased.The mean spontaneous firing rate increased nearly linearly with temperature. The slopes in different fibres ranged from 0.2–3.5 imp s^–1 °C^–1. A bimodal distribution of mean spontaneous firing rate was found (<20 imp s^–1 and >20 imp s^–1 at T_s) at all temperatures.The frequency-intensity response area of the primary fibres shifted uniformly with temperature. The characteristic frequency (CF) increased nearly linearly with temperature. The slopes in different fibres ranged from 3–90 Hz °C^–1. Expressed in octaves the CF-change varied in each fibre from about O.14oct °C^–1 at 15 °C to about 0.06 oct °C^–1 at 30 °C, irrespective of the fibre's CF at T_s. Thresholds were lowest near T_s. Below T_s the thresholds decreased on average by 2dB°C^–1, above T_s the thresholds rose rapidly with temperature. The sharpness of tuning (Q_10db) showed no major change in the temperature range tested.Comparison of these findings with those from other lower vertebrates and from mammals shows that only mammalian auditory afferents do not shift their CF with temperature, suggesting that a fundamental difference in mammalian and submammalian tuning mechanisms exists. This does not necessarily imply that there is a single unifying tuning mechanism for all mammals and another one for non-mammals.Abbreviations BF best frequency: frequency of maximal response at an intensity 10 dB above the CF-threshold - CF characteristic frequency - FTC frequency threshold curve, tuning curve - T _s standard temperature of 27 °C     

Thermoregulatory behavior of a tropical marine fish: Canthigaster jactator (Jenkins)

Ten Canthigaster, jactator, tropical marine puffers from Hawaii, were tested individually for 3-day periods in electronic shuttle-boxes (Ichthyotrons) to determine their ability to thermoregulate behaviorally. These fish thermoregulated with a degree of precision comparable to that of temperate freshwater fishes: range 23–31°C, S.D. 1.8–2.4°C, S.E. 0.3–0.5° C. The modal final thermal preferendum was 27°C, comparable to temperate warmwater fishes. The mean preferred temperature did not differ significantly between night (26.5°C) and day (26.9°C); the 24-hr mean was 26.7°C. Apparently at least some tropical marine fishes are capable of thermoregulatory behavior similar to that of temperate freshwater fishes.     

Energy budgets for Stentor coeruleus Ehrenberg (Ciliophora)

Summary Growth and feeding rates in Stentor coeruleus fed on Tetrahymena are described in relation to prey concentrations ranging from 10:1–50:1 at 15°C and 20°C. Feeding rate was independent of temperature, but governed by prey concentration. Stentor decreased in size where less than 1,000 J were consumed in 24 hrs. A maximum level of growth was attained at 15°C, but not at 20°C within the range of prey concentration offered. Energy budgets, constructed using respiratory data published elsewhere (Laybourn, 1975), are presented. Assimilation efficiencies ranged from 74.4–76.4% at 20°C and 65.1–83.2% at 15°C. Gross growth efficiencies were between 70.9–73.6% at 20°C and 63.7–81.6% at 15°C. Net growth efficiencies were very high, 95.6–96.3% at 20°C and 97.5–97.9% at 15°C. The results are explained and discussed in relation to other Protozoa.     

Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus

The production of extracellular inulinase (\-1,2-d-fructan fructanohydrolase, EC 3.2.1.7) was studied in fed-batch cultures of the yeast Kluyveromyces marxianus CBS 6556 at 30 and at 40° C. At both temperatures, the final biomass concentration exceeded 100 g·l^–1 and more than 2 g enzyme. L^–1 of culture supernatant was produced. The biomass yield on O₂ at 40° C was substantially lower than at 30°C. Nevertheless, at 40° C a growth rate of 0.20 h^–1 could be maintained for a longer period than at 30° C. The unexpected higher O₂-transfer rate at 40°C is probably due to a lower viscosity of the culture broth. The 40°C fermentation took only 33 h as compared to 42 h at 30° C. These results indicate that K. marxianus is a promising host for the extracellular production of heterologous proteins under the control of the inulinase promoter.     

Amylase and cellulase activities of thermophilic fungi causing deterioration of tobacco products in Nigeria

The conditions necessary for the production and activities of extracellular enzymes of thermophilic fungi obtained from tobacco products were determined. While amylases were produced in extractable amounts by all the fungi used in this study, extracellular cellulases were produced only by Chaetomium thermophile La Touche, Humicola insolens Cooney and Emerson, Malbranchea pulchella Sacc. et Penzig var. sulfurea (Miehe) Cooney and Emerson, and Mucor pusillus Lindt. A narrow range of pH 6–7 was best for the activities of the amylases of all the fungi used while a lower range of pH 5–6 was optimal for their cellulase activities. Peak activities were recorded for both enzymes at a temperature range of 45 °–50 °C. The possible use of the exo-enzymes of these fungi in industrial fermentation processes is suggested.