Continuous cultures of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> mt-2 overcome catabolic function loss under real case operating conditions

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h⁻¹ under toluene loading rates of 259 ± 23 and 801 ± 78 g m⁻³ h⁻¹ (inlet toluene concentrations of 3.5 and 10.9 g m⁻³, respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol⁻ population at day 4 to approx. 100% Tol⁺ cells from day 13 onward, sustaining elimination capacities of 232 ± 10 g m⁻³ h⁻¹ at 3.5 g Tol m⁻³ and 377 ± 13 g m⁻³ h⁻¹ at 10.9 g Tol m⁻³, which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol⁻ variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.     

Natural history and burrow system architecture of the silvery mole-rat from Brachystegia woodland

The silvery mole-rat Heliophobius argenteocinereus (Bathyergidae) is a solitary subterranean rodent, widely distributed throughout eastern and south-eastern Africa in a variety of habitats. Here, we provide the first data on its biology in a typical natural habitat, the Brachystegia woodland. The population density of mole-rats was low (4.6 ha⁻¹) and its distribution across the study site was random. Contrary to subadults and pups, the sex ratio of adult mole-rats was highly female biased (1:5.75), probably due to the higher mortality of males as a consequence of their mating strategy. Reproduction of Heliophobius is seasonal and pups are born at the beginning of the hot dry season. Burrow systems of the silvery mole-rat were long, highly branched, reticulated and comparable in fractal dimension to systems of social bathyergids. Variability in burrow architecture was related to the body mass of the burrow occupants, soil hardness and food supply when tested together. Burrow systems with a higher fractal dimension had inhabitants that had a greater body mass. Longer systems were less branched. Nests were typically deeper than foraging tunnels and experienced negligible temperature fluctuations. The microenvironmental characteristics of the subterranean niche including temperature, humidity and soil characteristics are provided for purpose of comparison with other mole-rat species.     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Asparagus racemosus cell cultures: a source for enhanced production of shatavarins and sarsapogenin

Asparagus racemosus is an important monocot medicinal plant that is in great demand for its steroidal saponins called shatavarins. This study was initiated to optimize the conditions for production of shatavarins in cell cultures of A. racemosus in a modified Murashige and Skoog (MS) medium supplemented with six different combinations of growth regulators. Biomass accumulation was correlated with saponin production over a 30-d culture cycle. Biomass and saponin accumulation patterns were dependent on combinations of growth regulators and the pH of the medium. Maximum levels of saponin and biomass accumulation were recorded on day 25 of the culture cycle within a pH range of 3.4 to 5.6. Total saponin produced by the in vitro cultures was 20-fold higher than amounts produced by cultivated plants. Saponin accumulation was not a biomass-associated phenomenon; cultures which showed the highest biomass accumulation were not the highest saponin accumulators. Maximum biomass (28.30 ± 0.29 g l⁻¹) and maximum levels of shatavarin IV(11.48 ± 0.61 mg g⁻¹) accumulation was found using a medium containing 2.0 mg l⁻¹ 2,4-D, 2 g l⁻¹ casein hydrolysate and 0.005% pectinase. The highest levels of sarsapogenin, secreted and intracellular (4.02 ± 0.09 mg g⁻¹), accumulated using a medium containing 1.0 mg l⁻¹ NAA, 1.0 mg l⁻¹ 2,4-D, 0.5 mg l⁻¹ BAP, 2 g l⁻¹ casein hydrolysate and 0.005% pectinase, after 25 d. Shatavarins were secreted into the medium and can be isolated easily for further purification.     

A maze-lover's dream: Burrow architecture,natural history and habitat characteristics of Ansell's mole-rat (Fukomys anselli)

The Ansell's mole-rat (Fukomys anselli, Bathyergidae) is a small-sized social subterranean rodent whose distribution is confined to the Lusaka area in Zambia. It is an established model species for various laboratory studies, but until now the knowledge of its biology under natural conditions has been limited. Here, we provide the first comprehensive natural history and ecological data on a free living population from Miombo woodland. The Ansell's mole-rat lives in groups of up to 13 individuals (mean 9.7 ± 2.5; N = 9 complete, fully established family groups) with a single breeding pair per group. The family groups occupy very large and complex burrow systems consisting of 0.5–2.8 km of tunnels (mean = 1.2 ± 0.6 km, N = 15), which are densely branched and reticulated especially around nests. Burrow systems of neighbouring family groups were often interconnected by a freely passable tunnel, which has never before been documented in any African mole-rats. Mole-rat food density in the study area was relatively low (10.0 ± 8.4 m^?2) but its biomass was large (457 ± 889 g m^?2). This, together with a relatively workable soil (cone resistance = 529 ± 26 N cm^?2 at the end of June, with 10% soil humidity), indicates moderate ecological conditions, which disagrees with the aridity food-distribution hypothesis (AFDH) considering small body size and sociality in bathyergids adaptations to harsh environments.     

Statistical medium optimization for enhanced azadirachtin production in hairy root culture of Azadirachta indica

Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg’s medium vitamins, and 30 g l⁻¹ sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g⁻¹) and azadirachtin production (∼44 mg l⁻¹) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l⁻¹ dry weight. To enhance the production of azadirachtin, a Plackett–Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l⁻¹ sucrose, 0.19 g l⁻¹ potassium dihydrogen phosphate, 3.1 g l⁻¹ potassium nitrate, and 0.41 g l⁻¹ magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l⁻¹ and azadirachtin accumulation of 5.2 mg g⁻¹, which was equivalent to an overall azadirachtin production of 73.84 mg l⁻¹, 68% more than that obtained under non-optimized conditions.     

Production of Cold-Adapted Amylase by Marine Bacterium <Emphasis Type="Italic">Wangia</Emphasis> sp. C52: Optimization,Modeling, and Partial Characterization

The aim of this work was to optimize the fermentation parameters in the shake-flask culture of marine bacterium Wangia sp. C52 to increase cold-adapted amylase production using two statistical experimental methods including Plackett–Burman design, which was applied to find the key ingredients for the best medium composition, and response surface methodology, which was used to determine the optimal concentrations of these components. The results showed starch, tryptone, and initial pH had significant effects on the cold-adapted amylase production. A central composite design was then employed to further optimize these three factors. The experimental results indicated that the optimized composition of medium was 6.38 g L⁻¹ starch, 33.84 g L⁻¹ tryptone, 3.00 g L⁻¹ yeast extract, 30 g L⁻¹ NaCl, 0.60 g L⁻¹ MgSO₄ and 0.56 g L⁻¹ CaCl₂. The optimized cultivation conditions for amylase production were pH 7.18, a temperature of 20°C, and a shaking speed of 180 rpm. Under the proposed optimized conditions, the amylase experimental yield (676.63 U mL⁻¹) closely matched the yield (685.60 U mL⁻¹) predicted by the statistical model. The optimization of the medium contributed to tenfold higher amylase production than that of the control in shake-flask experiments.     

Soil particle accumulation in termite (Macrotermes bellicosus) mounds and the implications for soil particle dynamics in a tropical savanna Ultisol

This study investigated the influence of mound-building termites on soil particle dynamics on the land surface and in soil-forming processes by examining the amount of soil particles in mound structures of Macrotermes bellicosus in a highly weathered Ultisol of tropical savanna. Soil particle turnover via the mounds was estimated using particle stock data and soil turnover data from previous studies. A 4-ha study plot with six mounds of relatively uniform shape and size was investigated. Soil mass constituting the mounds was 6,166 ± 1,581 kg mound⁻¹ within which the mound wall and nest body accounted for 5,002 ± 1,289 and 1,164 ± 293 kg, respectively. The mound wall contained a significantly larger amount of clay (252 ± 9.97 g kg⁻¹) balanced with a lower sand content (676 ± 26.5 g kg⁻¹) than in the adjacent surface (Ap1) horizon, (46.4 ± 12.8 g clay kg⁻¹; 866 ± 83.2 g sand kg⁻¹); the nest body had much higher clay content (559 ± 51.0 g kg⁻¹) but less sand (285 ± 79.2 g kg⁻¹) than the mound wall. As a result, the mounds of M. bellicosus accumulated clay of 2,874 ± 781 kg ha⁻¹ (corresponding to 2.52% of clay stock in the Ap1 horizon) along with an estimated clay turnover rate of 169 kg ha⁻¹ year⁻¹. These findings suggest a positive feedback effect from termite mound-building activity on soil particle dynamics in tropical savanna ecosystems: M. bellicosus preferentially use subsoil material for mound construction, resulting in relocation of illuvial clay in the subsoil to the land surface where clay eluviation from the surface soil and its illuviation in the subsoil are major soil-forming processes.     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis>

Butanol, a four-carbon primary alcohol (C₄H₁₀O), is an important industrial chemical and has a good potential to be used as a superior biofuel. Bio-based production of butanol from renewable feedstock is a promising and sustainable alternative to substitute petroleum-based fuels. Here, we report the development of a process for butanol production from glycerol, which is abundantly available as a byproduct of biodiesel production. First, a hyper butanol producing strain of Clostridium pasteurianum was isolated by chemical mutagenesis. The best mutant strain, C. pasteurianum MBEL_GLY2, was able to produce 10.8 g l⁻¹ butanol from 80 g l⁻¹ glycerol as compared to 7.6 g l⁻¹ butanol produced by the parent strain. Next, the process parameters were optimized to maximize butanol production from glycerol. Under the optimized batch condition, the butanol concentration, yield, and productivity of 17.8 g l⁻¹, 0.30 g g⁻¹, and 0.43 g l⁻¹ h⁻¹ could be achieved. Finally, continuous fermentation of C. pasteurianum MBEL_GLY2 with cell recycling was carried out using glycerol as a major carbon source at several different dilution rates. The continuous fermentation was run for 710 h without strain degeneration. The acetone–butanol–ethanol productivity and the butanol productivity of 8.3 and 7.8 g l⁻¹ h⁻¹, respectively, could be achieved at the dilution rate of 0.9 h⁻¹. This study reports continuous production of butanol with reduced byproducts formation from glycerol using C. pasteurianum, and thus could help design a bioprocess for the improved production of butanol.     

Cardiac responses to salinity variations in two differently zoned Mediterranean limpets

Cardiac activity of two Mediterranean limpets was tested at different salinities. Patella caerulea inhabits the lower midlittoral where it is exposed to variations in salinity, while P. aspera experiences more stable salinity conditions in the infralittoral fringe. When exposed to moderate hypo- and hypersalinity (23 g l⁻¹ and 43 g l⁻¹) for 24 min, P. caerulea showed no significant variation in heart rate with respect to the control salinity (33 g l⁻¹), while P. aspera exhibited a significant increase in heart rate in both conditions. This suggests a rise in metabolic rate due to activation of behavioural responses or physiological regulation. When exposed to extremely low salinity (3 g l⁻¹) for 24 min, heart contractions ceased in most specimens of P. caerulea. A smaller number of specimens also displayed cessation of heart beat when exposed to extremely high salinity (63 g l⁻¹). The heart beat resumed quickly in all specimens when they were returned to control salinity conditions. In contrast, cardiac activity was not interrupted in any of the P. aspera specimens at the 3 g l⁻¹ and 63 g l⁻¹ salinity levels, but strong bradycardia was evident. Contractile activity of the heart ceased in all specimens of P. caerulea and P. aspera when they were exposed to prolonged hypo-osmotic stress (3 g l⁻¹ for 24 h). This acardia was largely reversible in P. caerulea, but most specimens of P. aspera did not recover from the treatment. Accepted: 3 July 1999     

Cultivation of the green alga, Codium fragile (Suringar) Hariot, by artificial seed production in Korea

Codium fragile (Suringar) Hariot is an edible green alga farmed in Korea using seed stock produced from regeneration of isolated utricles and medullary filaments. Experiments were conducted to reveal the optimal conditions for nursery culture and out-growing of C. fragile. Sampling and measurement of underwater irradiance were carried out at farms cultivating C. fragile at Wando, on the southwestern coast of Korea, from October 2004 to August 2005. Growth of erect thalli and underwater irradiance were measured over a range of depths for three culture stages. During the nursery cultivation stage (Stage I), growth rate was greatest at 0.5 m depth (0.055 ± 0.032 mm day⁻¹), where the average midday irradiance over 60 days was 924 ± 32 μmol photons m⁻² s⁻¹. During the pre-main cultivation stage (Stage II), the greatest growth rate occurred at a depth of 2 m (0.113 ± 0.003 mm day⁻¹) with an average irradiance of 248 ± 116 μmol photons m⁻² s⁻¹. For the main cultivation stage (Stage III) of the alga, thalli achieved the greatest increase in biomass at 1 m depth (7.2 ± 1.0 kg fresh wt m⁻¹). These results suggest that optimal growth at each cultivation stages of C. fragile could be controlled by depth of cultivation rope.     

Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris

We tested 10 different Chlorella and Parachlorella strains under lipid induction growth conditions in autotrophic laboratory cultures. Between tested strains, substantial differences in both biomass and lipid productivity as well as in the final content of lipids were found. The most productive strain (Chlorella vulgaris CCALA 256) was subsequently studied in detail. The availability of nitrates and/or phosphates strongly influenced growth and accumulation of lipids in cells by affecting cell division. Nutrient limitation substantially enhanced lipid productivity up to a maximal value of 1.5 g l⁻¹ day⁻¹. We also demonstrated the production of lipids through large-scale cultivation of C. vulgaris in a thin layer photobioreactor, even under suboptimal conditions. After 8 days of cultivation, maximal lipid productivity was 0.33 g l⁻¹ day⁻¹, biomass density was 5.7 g l⁻¹ dry weight and total lipid content was more than 30% dry weight. C. vulgaris lipids comprise fatty acids with a relatively high degree of saturation compared with canola oil offering a possible alternative to the use of higher plant oils.     

Application of oscillation for efficiency improvement of continuous ethanol fermentation with Saccharomyces cerevisiae under very-high-gravity conditions

Compared with steady state, oscillation in continuous very-high-gravity ethanol fermentation with Saccharomyces cerevisiae improved process productivity, which was thus introduced for the fermentation system composed of a tank fermentor followed by four-stage packed tubular bioreactors. When the very-high-gravity medium containing 280 g l⁻¹ glucose was fed at the dilution rate of 0.04 h⁻¹, the average ethanol of 15.8% (v/v) and residual glucose of 1.5 g l⁻¹ were achieved under the oscillatory state, with an average ethanol productivity of 2.14 g h⁻¹ l⁻¹. By contrast, only 14.8% (v/v) ethanol was achieved under the steady state at the same dilution rate, and the residual glucose was as high as 17.1 g l⁻¹, with an ethanol productivity of 2.00 g h⁻¹ l⁻¹, indicating a 7% improvement under the oscillatory state. When the fermentation system was operated under the steady state at the dilution rate of 0.027 h⁻¹ to extend the average fermentation time to 88 h from 59 h, the ethanol concentration increased slightly to 15.4% (v/v) and residual glucose decreased to 7.3 g l⁻¹, correspondingly, but the ethanol productivity was decreased drastically to 1.43 g h⁻¹ l⁻¹, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h⁻¹.     

Optimization of fermentation medium for triterpenoid production from <Emphasis Type="Italic">Antrodia camphorata</Emphasis> ATCC 200183 using artificial intelligence-based techniques

In this study, alteration in morphology of submergedly cultured Antrodia camphorata ATCC 200183 including arthroconidia, mycelia, external and internal structures of pellets was investigated. Two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) were built to optimize the inoculum size and medium components for intracellular triterpenoid production from A. camphorata. Root mean squares error, R ², and standard error of prediction given by ANN model were 0.31%, 0.99%, and 0.63%, respectively, while RSM model gave 1.02%, 0.98%, and 2.08%, which indicated that fitness and prediction accuracy of ANN model was higher when compared to RSM model. Furthermore, using genetic algorithm (GA), the input space of ANN model was optimized, and maximum triterpenoid production of 62.84 mg l⁻¹ was obtained at the GA-optimized concentrations of arthroconidia (1.78 × 10⁵ ml⁻¹) and medium components (glucose, 25.25 g l⁻¹; peptone, 4.48 g l⁻¹; and soybean flour, 2.74 g l⁻¹). The triterpenoid production experimentally obtained using the ANN–GA designed medium was 64.79 ± 2.32 mg l⁻¹ which was in agreement with the predicted value. The same optimization process may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoid production from A. camphorata and to improve the production of bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.     

Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique

An efficient procedure has been developed for inducing somatic embryogenesis and regeneration of plants from tissue cultures of oil palm (Elaeis guineensis Jacq.). Thin transverse sections (thin cell layer explants) of different position in the shoot apex and leaf sheath of oil palm were cultivated in Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented with 0–450 μM picloram and 2,4-D with 3.0% sucrose, 500 mg L⁻¹ glutamine, and 0.3 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel. Embryogenic calluses were evaluated 12 wk after inoculation. Picloram (450 μM) was effective in inducing embryogenic calluses in 41.5% of the basal explants. Embryogenic calluses were maintained on a maturation medium composed of basal media, plus 0.6 μM NAA and 12.30 μM 2iP, 0.3 g L⁻¹ activated charcoal, and 500 mg L⁻¹ glutamine, with subcultures at 4-wk intervals. Somatic embryos were converted to plants on MS medium with macro- and micronutrients at half-strength, 2% sucrose, and 1.0 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel.     

Rate of dehydration,state of subcellular organisation and nature of cryoprotection are critical factors contributing to the variable success of cryopreservation: studies on recalcitrant zygotic embryos of <Emphasis Type="Italic">Haemanthus montanus</Emphasis>

Effects of sequential procedures required for cryopreservation of embryos excised from the recalcitrant seeds of Haemanthus montanus were assessed ultrastructurally and in conjunction with respiratory activity and the rate of protein synthesis. Fresh material (water content, 5.05 ± 0.92 g g⁻¹ dry mass) afforded ultrastructural evidence of considerable metabolic activity, borne out by respiratory rates. Neither exposure to glycerol nor sucrose as penetrating and non-penetrating cryoprotectants, respectively, brought about degradative changes, although increased vacuolation and autophagy accompanied both, while respiratory and protein synthetic activity were not adversely affected. Glycerol-cryoprotected embryos flash dried to water contents >0.4 g g⁻¹ showed organised ultrastructural features and considerable autophagy consistent with metabolic activity, and although respiratory activity was lower, protein synthesis rate was enhanced relative to fresh material. However, at water contents <0.4 g g⁻¹, embryo tissue presented a mosaic of cells of variable density and ultrastructural status, but trends in rates of respiration and protein synthesis remained similar. Flash drying after sucrose exposure was accompanied by considerable ultrastructural abnormality particularly at water contents <0.4 g g⁻¹, lysis of individual and groups of cells and considerable depression of respiration, but not of protein synthesis. Success, assessed as ≥50% axes forming seedlings after cryogen exposure, was obtained only when glycerol-cryoprotected embryos at water contents >0.4 g g⁻¹—in which the degree of vacuolation remained moderate—were rapidly cooled. The outcomes of this study are considered particularly in terms of the stresses imposed by prolonged, relatively slow dehydration and ultimate water contents, on embryos showing considerable metabolic activity.     

Evidence of central and peripheral gravireception in the ciliate Loxodes striatus

Effects of the density of the external medium on gravireception in Loxodes striatus were investigated using Percoll solutions. With increasing density, the swimming rates changed from prevailing in the downward direction to prevailing in the upward direction. A cellular density of 1.036 g cm⁻³ was determined measuring direction and speed of sedimenting immobilized cells at different accelerations and medium densities. Viscosity increases by Percoll were measured and taken into account. At 30% air saturation Loxodes maintained a negative gravikinesis of approximately −27 μm s⁻¹ at external densities corresponding to cellular density (±0.02 g cm⁻³). Negative gravikinesis decreased gradually to −9 μm s⁻¹ with the density difference rising from 0.020 to 0.036 g cm⁻³ (=normal). The data indicate the existence of central gravireception, presumably by the Müller organelle, to generate in swimming Loxodes a constant value of gravikinesis and a bimodal gravitaxis. Peripheral gravireception occurs, in addition to central gravireception, when the transmembrane density difference exceeds 0.02 g cm⁻³. Peripheral gravireception can neutralize, in part, gravikinesis as raised by the central gravireceptor. We hypothesize that both central and peripheral gravireception of Loxodes guide vertical locomotion in gliding and swimming cells. Accepted: 26 May 1998     

Nitrifying granules cultivation in a sequencing batch reactor at a low organics-to-total nitrogen ratio in wastewater

It is possible to cultivate aerobic granular sludge at a low organic loading rate and organics-to-total nitrogen (COD/N) ratio in wastewater in the reactor with typical geometry (height/diameter = 2.1, superficial air velocity = 6 mm/s). The noted nitrification efficiency was very high (99%). At the highest applied ammonia load (0.3 ± 0.002 mg NH₄⁺–N g total suspended solids (TSS)⁻¹ day⁻¹, COD/N = 1), the dominating oxidized form of nitrogen was nitrite. Despite a constant aeration in the reactor, denitrification occurred in the structure of granules. Applied molecular techniques allowed the changes in the ammonia-oxidizing bacteria (AOB) community in granular sludge to be tracked. The major factor influencing AOB number and species composition was ammonia load. At the ammonia load of 0.3 ± 0.002 mg NH₄⁺–N g TSS⁻¹ day⁻¹, a highly diverse AOB community covering bacteria belonging to both the Nitrosospira and Nitrosomonas genera accounted for ca. 40% of the total bacteria in the biomass.