Applying dimorphic yeasts as model organisms to study mycelial growth: Part 1. Experimental investigation of the spatio-temporal development of filamentous yeast colonies

Colony development of the dimorphic yeasts Yarrowia lipolytica and Candida boidinii on solid agar substrates under glucose limitation served as a model system for mycelial development of higher filamentous fungi. Strong differences were observed in the behaviour of both yeasts: C. boidinii colonies reached a final colony extension which was small compared to the size of the growth field. They formed cell-density profiles which steeply declined along the colony radius and no biomass decay processes could be detected. The stop of colony extension coincided with the depletion of glucose from the growth substrate. These findings supported the hypothesis that glucose-limited C. boidinii colonies can be regarded as populations of single cells which grow according to a diffusion-limited growth mechanism. Y. lipolytica colonies continued to extend after the depletion of the primary nutrient resource, glucose, until the populations covered the entire growth field which was accomplished by utilization of mycelial biomass.     

Coordinated Development of Yeast Colonies: Quantitative Modeling of Diffusion‐Limited Growth – Part 2

A mathematical model was developed which described the growth of yeast colonies based on the assumptions that (i) these populations were built up of single cells whose proliferation was (ii) exclusively controlled by nutrient availability in the environment. The model was of a hybrid cellular automaton type and described discrete cells residing on a one‐dimensional lattice as well as on continuously distributed nutrients. Experimental results and numerical calculations were compared to elucidate under which cultivation conditions the diffusion‐limited growth (DLG) was the major construction principle in yeast colonies. Simulations were scaled to the growth of Yarrowia lipolytica and Candida boidinii colonies under carbon and nitrogen limitation. They showed that nutrient‐controlled growth of the individual cells resulted in DLG of the population. Quantitative predictions for the spatio‐temporal development of the cell‐density profile inside a growing yeast mycelium were compared to the growth characteristics of the model yeast mycelia. Only for the carbon‐limited growth of C. boidinii colonies on glucose as the limiting nutrient resource did the DLG model reproduce the cell‐density profile estimated at the end of the cultivation. Under all other cultivation conditions, strong discrepancies between calculations and experimental results were evident precluding DLG as the ruling regulatory mechanism. Thus, whether or not the development of a yeast population could be described by a DLG scenario, was strongly dependent on the particular cultivation conditions and the applied yeast species. In those cases for which the DLG hypothesis failed to explain the observed growth patterns, the underlying assumptions, i.e., the complete absence of nutrient translocation between the individual cells inside the yeast mycelia as well as the exclusively nutrient‐controlled proliferation of the cells, have to be reevaluated. The presented study demonstrated how the mathematical analysis of growth processes in yeast populations could assist the experimental identification of potential regulatory mechanisms.     

Species-specific phytoplankton responses to nutrients and zooplankton manipulations in enclosure experiments

1. In situ enclosure experiments were performed in the mesotrophic Bermejales reservoir to evaluate the algal response to changes in the nutrient supply and in the zooplankton size structure and density in a 2 × 2 factorial design. The experiments were conducted during the spring bloom of nanoplanktonic diatoms in 1989. 2. Nutrient enrichment promoted a great increase of phytoplankton biomass indicating a strong nutrient limitation on phytoplankton growth. Total phytoplankton biomass was significantly lower in the Daphina-added enclosures at a given nutrient level and strong direct an indirect effect of zooplankton on phytoplankton community structure and nutrient availability were observed. 3. Most of the nanoplanktonic species were effectively grazed but species with protective coverings and large size colonies were favoured by grazers and small chlorococcales were unaffected probably because of their compensatory high growth rates. The decrease in total biomass imposed by grazers is attributable mainly to the decrease of Cyclotella ocellata, the most abundant species. This taxon suffers two net effects of zooplankton: direct grazing and the indirect decrease of Si availability caused by the growth of C. ocellata which was promoted by P excretion by zooplankton. Indirect effects of grazers on Si availability should, therefore, be taken into account in explaining phytoplankton succession and community structure. 4. In this experiment grazers affected considerably the nanoplanktonic community in Bermejales reservoir. The extent which they were affected, however, depended not only on the algal size as a determinant of edibility but also greatly on the specific nutrient requirements and taxonomic features of the algal species.     

Expression of a codon-optimized Aspergillus niger pectin methylesterase gene in the methylotrophic yeast Candida boidinii

A codon-optimized Aspergillus niger pectin methylesterase (PME) gene was expressed in the methylotrophic yeast Canidia boidinii. The PME-producing strains showed better growth on pectin than the wild-type strains, suggesting that the PME-producing strains could efficiently utilize methyl ester moieties of pectin. On the other hand, overproduction of PME negatively affected the proliferation of C. boidinii on leaves of Arabidopsis thaliana.     

An assessment of the importance of emergent and floating-leaved macrophytes to trophic status in the Loosdrecht lakes (The Netherlands)

The potential importance of the six major emergent and floating-leaved macrophyte species in recycling of sediment phosphorus in the Loosdrecht lakes was studied. Representative plant samples were collected at the time of maximum biomass, and analysed for biomass and carbon, nitrogen and phosphorus contents. Species cover was determined by aerial photography.Total cover in the seven lakes studied ranged between 2 and 26 percent. For the four main species, biomass per unit area increased with lake trophic status. Consistent differences in C, N and P contents per unit biomass were not observed. Although cover values were small, significant amounts of C, N and P were contained in the macrophytes when compared with maximum sestonic content.Potential P loads from macrophyte decay were calculated. In Lake Loosdrecht, the P load represented 15 percent of current external P inputs. The potential importance of macrophyte decay to P recycling in the other lakes is greater.Decay of macrophyte species at the end of the growing season appears to affect autumnal nutrient and chlorophyll a levels in the water column of some lakes. The re-establishment of submerged species following lake restoration may increase the importance of this pathway in the lakes.     

Influence of Glucose and Saturated Free-Fatty Acid Mixtures on Citric Acid and Lipid Production by Yarrowia lipolytica

In the present report, the effect of glucose and stearin (substrate composed by saturated free-fatty acids) on the production of biomass, reserve lipid, and citric acid by Yarrowia lipolytica ACA-DC 50109 was investigated in nitrogen-limited cultures. Numerical models that were used in order to quantify the kinetic behavior of the above Yarrowia lipolytica strain showed successful simulation, while the optimized parameter values were similar to those experimentally measured and the predictive ability of the models was satisfactory. In nitrogen-limited cultures in which glucose was used as the sole substrate, satisfactory growth and no glucose inhibition occurred, although in some cases the initial concentration of glucose was significantly high (150 g/l). Citric acid production was observed in all trials, which was in some cases notable (final concentration 42.9 g/l, yield 0.56 g per g of sugar consumed). The concentration of unsaturated cellular fatty acids was slightly lower when the quantity of sugar in the medium was elevated. In the cases in which stearin and glucose were used as co-substrates, in spite of the fact that the quantity of cellular lipid inside the yeast cells varied remarkably (from 0.3 to 2.0 g/l – 4 to 20% wt/wt), de novo fatty acid biosynthesis was observed. This activity increased when the yeast cells assimilated higher sugar quantities. The citric acid produced was mainly derived from the catabolism of sugar. Nevertheless, citric acid yield on sugar consumed and citrate specific production rate, as evaluated by the numerical model, presented substantially higher values in the fermentation in which no fat was used as glucose co-substrate compared with the cultures with stearin used as co-substrate.     

A new actinomycete from a Guadalupian vertebrate coprolite from Brazil

Coprolites (fossil feces) are important sources of evidence of ancient food webs and ecosystems. Actinomycetes are a fundamental component in the decay of organic matter, and serve as catalysts for nutrient cycles. Recently, gas vesicles filled with numerous verrucose colonies of substrate mycelium of an actinomycete were discovered inside a fossilized spiral amphipolar fish coprolite recovered from mid–Permian deposits of Brazil. These colonies are composed of masses of substrate hyphae, some of which are undergoing segmentation. Arising from the colonies are chains of spores separated by narrow, elongate connectives. The fossil actinomycete is described below as Palaeostromatus diairetus gen. et sp. nov. and represents the oldest known actinomycete associated with vertebrate deposits. Since the colonies occur only inside the coprolite, either Palaeostromatus diairetus gen. et sp. nov. was part of the gut flora or it was acquired from a food source. The only other remains in the coprolite are eighteen paleoniscoid fish scales, which suggests that the producer was a carnivorous/omnivorous fish. This is the oldest record of a direct interaction between vertebrates and actinomycetes.     

Influence of mineral nutrient availability on growth of tree seedlings from the tropical deciduous forest

The effects of different nutrient availabilities on growth and biomass partitioning in seedlings from the tropical deciduous forest in Mexico were compared. The tree species studied were Heliocarpus pallidus, a species associated with disturbed parts of the forest, and Caesalpinia eriotachys, Jacquinia pungens and Recchia mexicana, species from mature, undisturbed habitats. The tropical deciduous tree seedlings were grown in pure silica sand for 50 days inside growth chambers under four nutrient regimes; 5, 20, 100 and 200% Long Ashton nutrient solutions. Data showed contrasting responses among species to different nutrient availabilities. Except Jacquinia pungens, all species had increased growth and productivity as nutrient level increased from 5 to 100%; however, no significant differences in these parameters were detected between 100 and 200% in all species. Compared with mature forest species, pioneer species showed higher variations in biomass production, relative growth rate and net assimilation rate. In contrast to mature forest species, root/ shoot ratios in Heliocarpus pallidus were greater and thus showed higher biomass allocation to roots when nutrient supply was limited. This response suggests higher phenotypic plasticity in pioneer species. Species from mature parts of the forest (Caesalpinia eriostachys, Recchia mexicana) showed less dependency on nutrient supply than pioneer species. These responses appear to support observations from studies with temperate plants investigating growth responses to soil fertility.     

An examination of the role of colonial <Emphasis Type="Italic">Phaeocystis antarctica</Emphasis> in the microbial food web of the Ross Sea

The extensive buildup of phytoplankton biomass in the Ross Sea conflicts with the view that high rates of herbivory occur in all regions of the Southern Ocean. Nano and microplanktonic consumers comprise a significant fraction of total plankton biomass; however, the importance of grazing remains uncertain in the Ross Sea. Microzooplankton ingestion of solitary and colonial cells of Phaeocystis antarctica were calculated using a novel live-staining fluorescently-labeled algae method. Different morphotypes of P. antarctica were stained different colors, mixed, and observed inside Euplotes to determine their feeding preference. The blue (7-aminocoumarin) (CMAC) stain was used on the colonies and the green (CMFDA) CellTracker Probe was used on solitary cells. Both morphotypes can be seen inside the food vacuoles of the ciliate, supporting the idea that microzooplankton are capable of ingesting cells within the colonial matrix. This suggests that P. antarctica colonies enter the microbial loop in the Ross Sea before sedimentation.     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen

Summary Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC 404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively, of prostate epithelial cells in vivo.     

Fitness consequences of nest infiltration by the mutualist‐exploiter Megalomyrmex adamsae

1. Fungus‐growing ants are obligate mutualists. Their nutrient‐rich fungus garden provides a valuable food store that sustains the ant hosts, but can also attract social parasites. 2. The ‘guest ant' Megalomyrmex adamsae Longino parasitises the fungus‐growing Trachymyrmex zeteki Weber queen just after nest founding. The parasitic queen infiltrates the incipient nest, builds a cavity in the fungal garden, and lays eggs that develop into workers and reproductive males and females. 3. This study compared young parasitised and non‐parasitised laboratory colonies by measuring garden growth and biomass, and the number of host workers and reproductives. Host queen survival and parasite colony growth were also monitored. 4. Parasitised Trachymyrmex colonies had reduced host worker and alate numbers, as well as lower garden biomass, compared with non‐parasitised control colonies, confirming that M. adamsae is a xenobiotic social parasite. Host queen survival was not significantly different between parasitised and control colonies. 5. This is the first study that experimentally infects host colonies with a xenobiotic social parasite to measure fitness cost to the host. The natural history of M. adamsae and the fungus‐growing ant mutualism are evaluated in the context of three general predictions of (Bronstein, Ecology Letters, 4 , 277–287, 2001a) regarding the cost of mutualism exploiters.     

Factors controlling the colonial structure of <Emphasis Type="Italic">Pediastrum tetras</Emphasis> (Chlorophyceae)

Accounting for morphological plasticity in phytoplankton populations is relevant for taxonomy, systematic/evolutionary, and ecological studies. In this work, the green alga Pediastrum tetras (Ehrenberg) Ralfs was used to describe the variation in population size structure over its growth cycle and to analyze responses to changes in biotic and abiotic factors. Pediastrum cultures reached a final stable concentration in approximately 10 days. This density (8 × 10⁵ cells ml⁻¹) remained stable for at least another 13 days and the intrinsic growth rate was 0.24 ± 0.01 day⁻¹. In the exponential phase, the relative number of single cells and the proportion of large cells (with vesicles inside) within colonies increased. When density peaked, a relative increase of single cells as well as small cells in new colonies took place. Finally, during the stationary phase, the trend reversed: fewer single cells and a larger cell size (without vesicles) were observed. Results indicated that nutrient supply could affect population structure, diminishing the proportion of eight-cell colonies. Daphnia magna Straus significantly reduced the Pediastrum population density due to predation, and this led to a significant decrease in the density of the largest colonies. In addition, info-chemicals induced a slight increase in the density of the largest colonies compared to the control treatment. Our study suggests a possible trade-off in P. tetras colonial size in natural environments: during the stationary growth period in a lake, Pediastrum populations tend to increase in size for efficient use of nutrients, while they decrease in size in the presence of herbivores. Handling editor: J. Padisak     

Nutrient recycling by two phosphorus-rich grazing catfish: the potential for phosphorus-limitation of fish growth

In ecosystems where excretion by fish is a major flux of nutrients, the nitrogen (N) to phosphorus (P) ratio released by fish can be important in shaping patterns of algal biomass, community composition, primary production, and nutrient limitation. Demand for N and P as well as energy influences N/P excretion ratios and has broad implications in ecosystems where nutrient recycling by fishes is substantial. Bioenergetics and stoichiometric models predict that natural fish populations are generally energy-limited and therefore N/P recycling by fishes is relatively invariant. Yet, the potential for P limitation of growth has not been examined in herbivorous fishes, which are common in many aquatic habitats. We examined N/P excretion ratios and P demand in two P-rich herbivorous catfishes of the family Loricariidae, Ancistrus triradiatus (hereafter Ancistrus) and Chaetostoma milesi (hereafter Chaetostoma). Both fishes are common grazers in the Andean piedmont region of Venezuela where we conducted this study. Mass balance (MB) models indicate that these fishes have a high P demand. In fact, our Ancistrus’ P MB model predicted negative P excretion rates, indicating that Ancistrus did not consume enough P to meet its P demand for growth. Direct measurement of excretion rates showed positive, but very low P excretion rates and high N/P excretion ratios for both taxa. To obtain measured P excretion rates of Ancistrus from the MB model, gross growth efficiency must be reduced by 90%. Our results suggest that growth rates of both of these herbivorous and P-rich fish are likely P-limited. If P limitation of growth is common among herbivorous fish populations, herbivorous fishes recycle likely at high N/P ratios and act to diminish the quality of their food.     

Colony pattering ofAspergillus oryzae on agar media

To clarity the effects of nutrient concentration and diffusion on the pattern formation of fungal colonies, the colony patterning ofAspergillus oryzae at various nutrient and agar levels was studied experimentally and was summarized in a colony morphology diagram. Roles of the nutrient content and the relaxation of nutrient distribution on the colony patterning were discussed based on a computer model of the mycelial growth. The colony morphology changed from compact to ramified as the nutrient and agar levels were lowered. No clear boundary was found between these two morphologies. The deterioration of substrate around the growing colony was detected when the morphic switching from homogeneous into splitting patterns emerged in the growth of ramified colonies. In the mycelial growth model, dense compact colonies developed at low growth rates and high nutrient influx into the colonized area. Under low nutrient levels, splitting colonies appeared at high growth rates as compared with the nutrient influx.     

FTIR spectra of algal species can be used as physiological fingerprints to assess their actual growth potential

Fourier transform infrared (FTIR) spectra were measured from cells of Microcystis aeruginosa and Protoceratium reticulatum, whose growth rates were manipulated by the availability of nutrients or light. As expected, the macromolecular composition changed in response to the treatments. These changes were species‐specific and depended on the type of perturbation applied to the growth regime. Microcystis aeruginosa showed an increase in the carbohydrate‐to‐protein ratio with decreased growth rates, under nutrient limitation, whereas light limitation induced a decrease of the carbohydrate‐to‐protein ratio with decreasing proliferation rates. The macromolecular pools of P. reticulatum showed a higher degree of compositional homeostasis. Only when the lowest light irradiance and nutrient availability were supplied, an increase of the carbohydrate‐to‐protein FTIR absorbance ratio was observed. A species‐specific partial least squares (PLS) model was developed using the whole FTIR spectra. This model afforded a very high correlation between the predicted and the measured growth rates, regardless of the growth conditions. On the contrary, the prediction based on absorption band ratios generally used in FTIR studies would strongly depend on growth conditions. This new computational method could constitute a substantial improvement in the early warning systems of algal blooms and, in general, for the study of algal growth, e.g. in biotechnology. Furthermore, these results confirm the suitability of FTIR spectroscopy as a tool to map complex biological processes like growth under different environmental conditions.     

Data consistency,yield, maintenance,and hysteresis in batch cultures of Candida lipolytica cultured on n-hexadecane

Candida lipolytica was cultured batchwise using n-hexadecane as the main carbon source. Biomass production, n-hexadecane consumption, oxygen consumption, and carbon dioxide evolution were measured to follow the fermentation. The consistency of the measured data was examined using integrated and instantaneous available electron and carbon balances. Values of the “true” growth yield, η_max, and maintenance coefficient, m_e were estimated using three different sets of data (biomass and n-hexadecane, oxygen and biomass, and CO₂ and biomass), and the results were compared with estimates obtained from literature data. Hysteresis patterns were observed in plots of specific rates of oxygen consumption and carbon dioxide evolution versus specific growth rate.