Continuous cultivation of Trichoderma viride on cellulose

Using ball milled cellulose as the only carbon source Trichoderma viride was grown in a continuous flow culture at pH = 5.0 and T = 30°C. Steady-state values for cell protein, cellulose, and cellulase for different substrate concentrations (4–11 g/liter) and dilution rates (0.033–0.080 hr^?1) were obtained. Under steady-state conditions, 50–75% of the cellulose was consumed indicating a critical dilution rate on 0.17 hr^?1. Cellulase activity (U/ml) in the fermentation broth increased slightly with increasing substrate concentration and decreased with increasing dilution rate, while the specific cellulase productivity (U/mg cell protein·hr) was fairly independent of the dilution rate, with a maximum around D = 0.05 hr^?1. Following step changes in substrate concentration and dilution rate, new steady-state values were reached after three to five residence times (cell protein and cellulose) and four to six residence times (celullase activity).     

The influence of rope cultivation ofGraciliaria on a wildZostera japonica bed in the lagoons of Southern Primorye, Sea of Japan

Cultivation of the red agar-producing algaGracilaria verrucosa (Huds.) Papenf. on suspended ropes produced significant changes in water motion and irradiance in near-bottom waters. However, no significant changes were found in a wild bed ofZostera japonica Arch. et Iraebn in the initial period of cultivation ofGracilaria. There was only a reduction in the growth rate ofZ. japonica and increase in the abundance and diversity of invertebrates in the community located below the ropes with cultivatedGracilaria.     

Mammalian cell cultivation using nutrients extracted from microalgae

Mammalian cells have been used in various research fields. More recently, cultured cells have been used as the cell source of “cultured meat.” Cell cultivation requires media containing nutrients, of which glucose and amino acids are the essential ones. These nutrients are generally derived from grains or heterotrophic microorganisms, which also require various nutrients derived from grains. Grain culture, in turn, requires many chemical fertilizers and agrochemicals, which can cause greenhouse gas emission and environmental contamination. Furthermore, grain production is greatly influenced by environmental changes. In contrast, microalgae efficiently synthesize various nutrients using solar energy, water, and inorganic substances, which are widely used in the energy sector. In this study, we aimed to apply nutrients extracted from microalgae in the culture media for mammalian cell cultivation. Glucose was efficiently extracted from Chlorococcum littorale or Arthrospira platensis using sulfuric acid, whereas 18 of the 20 proteinogenic amino acids were efficiently extracted from Chlorella vulgaris using hydrochloric acid. We further investigated whether nutrients present in the algal extracts could be used in mammalian cell cultivation. Although almost all C2C12 mouse myoblasts died during cultivation in a glucose- and amino acid-free medium, the cell death was rescued by adding algal extract(s) into the nutrient-deficient media. This indicates that nutrients present in algal extracts can be used for mammalian cell cultivation. This study is the first step toward the establishment of a new cell culture system that can reduce environmental loads and remain unaffected by the impact of environmental changes.     

Microbial transformations of inorganic sulphur compounds in soil under conditions of heterocontinuous cultivation

Development and activity of the association of the sulphur cycle bacteria, represented byThiobacillus thioparus andDesulfovibrio sp., were followed in chernozem soil continuously supplemented with sodium thiosulphate. The technique of heterocontinuous cultivation made it possible (i) to determine changes in the individual components of microflora involved in successive metabolic steps, their time and space sequence, (ii) to follow changes in the transformations of substrate and formation of metabolic products, and (iii) to reach a steady state in the system. A possible use of this approach for the evaluation of the effect of ecological factors, for modelling microbiological processes of the sulphur cycle, for the investigation of trophic relationships among microorganisms in natural and artificial association and for the evaluation of the geochemical activity of sulphur bacteria is discussed.     

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor‐binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N‐glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser‐induced fluorescence detection (CGE‐LIF) based glycoanalysis (N‐glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N‐glycosylation pattern of Madin–Darby canine kidney (MDCK) cell‐derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum‐free growth has only a minor impact on the HA N‐glycosylation pattern. Only relative abundances of N‐glycan structures are affected. In contrast, host cell adaptation to serum‐free suspension growth resulted in significant changes in the HA N‐glycosylation pattern regarding the presence of specific N‐glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β‐propiolactone inactivation resulted—at best—only in minor changes in the relative N‐glycan structure abundances of the HA N‐glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N‐glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum‐free suspension growth significantly influenced HA N‐glycosylation regarding both, the type of attached glycan structures as well as their abundances. Biotechnol. Bioeng. 2013; 110: 1691–1703. © 2013 Wiley Periodicals, Inc.     

Biodiversity and the mitigation of climate change through bioenergy: impacts of increased maize cultivation on farmland wildlife

The public promotion of renewable energies is expected to increase the number of biogas plants and stimulate energy crops cultivation (e.g. maize) in Germany. In order to assess the indirect effects of the resulting land‐use changes on biodiversity, we developed six land‐use scenarios and simulated the responses of six farmland wildlife species with the spatially explicit agent‐based model system ALMaSS. The scenarios differed in composition and spatial configuration of arable crops. We implemented scenarios where maize for energy production replaced 15% and 30% of the area covered by other cash crops. Biogas maize farms were either randomly distributed or located within small or large aggregation clusters. The animal species investigated were skylark (Alauda arvensis), grey partridge (Perdix perdix), European brown hare (Lepus europaeus), field vole (Microtus agrestis), a linyphiid spider (Erigone atra) and a carabid beetle (Bembidion lampros). The changes in crop composition had a negative effect on the population sizes of skylark, partridge and hare and a positive effect on the population sizes of spider and beetle and no effect on the population size of vole. An aggregated cultivation of maize amplified these effects for skylark. Species responses to changes in the crop composition were consistent across three differently structured landscapes. Our work suggests that with the compliance to some recommendations, negative effects of biogas‐related land‐use change on the populations of the six representative farmland species can largely be avoided.     

Collagen/hyaluronan membrane as a scaffold for chondrocytes cultivation

Rabbit chondrocytes were cultivated in vitro using the collagen/hyaluronan membrane. The membrane did not show any adverse effects on chondrocyte viability during in vitro cultivation. The inoculated cells grew without any negative changes. According to the histochemical analyses: (i) hematoxylin and eosin; (ii) safranin O; and (iii) rabbit anti-human collagen type II staining, the rabbit chondrocytes maintained their morphology and phenotype during in vitro cultivation. The collagen/hyaluronan membrane became more stable and stiffer after long time cultivation. The proliferation of the chondrocytes stabilised the structure of the membrane. The collagen/hyaluronan membrane is suitable material for the chondrocyte growth and could provide functional tissue-engineered scaffold for cartilage repair.     

Phytotron cultivation of early barley mutants

Conclusions The authors have tried to gather data which permit some information on the between and within locus reactions of induced early barley mutants to different photo- and thermoperiods. Eight mutant cases, showing rather drastic earliness in field cultivation and representing three different gene loci, were examined in phytotron experiments according to routine methods of cultivation. One of the mutants, mat-a ⁸, has been released as an original Swedish barley variety under the name of Svalöf's Mari. In a previous publication (Dormling et al., 1966) this mutant was compared to its parent variety, Svalöf's Bonus, under 30 different climatic conditions. In the present investigation three photoperiods (24, 16 and 8 hours of artificial light) were combined with three suitable thermoperiods (20-15°, 20-10° and 15-10°C).The results indicate that photoperiodic insensitivity, with regard to ear formation and heading capacity, as well as kernel production, is of rather frequent occurrence in connection with drastically early mutants in barley. Four out of eight induced mutants give a more or less pronounced insensitivity. Three of the four insensitive mutants represent locus a, one belongs to locus b. Of the two c-mutants none was insensitive; both were on the contrary pronounced long-day types.Photo- and thermoperiods interact in various ways. This is especially clear in the c-mutants just mentioned, which have a high generative productivity and efficiency at continuous light and high thermoperiods. They produce no grain but considerable vegetative matter at 8 hours of light, independently of thermoperiod, as well as at 16 hours of light with high temperatures. In fact, mutants of loci a and c differ strikingly with regard to their relations to the climatic conditions applied. The insensitive mutant b ¹³ is remarkably similar to the mutant a ¹², but its resemblance to the sensitive mutants b ⁷ and b ¹⁰ of the same locus is evidenced by its high average internode number.It ought to be pointed out here that the mutants of the three gene loci analysed in this study can be distinguished phenotypically with regard to morphological as well as physiological properties, in the field as well as in phytotron cultivation. The c-mutants are especially characteristic. However, there also seem to be clear differences in reaction between the allelic mutants of a locus. In fact, all eight mutants studied seem to react more or less differently.The insensitive mutant a ⁸, which has been released into practice, is also widely used in recombination work, and successful segregates have been isolated. The characteristics of a ⁸, which make the mutant valuable in practice, are also found in phytotron experimentation, specially with regard to earliness, generative efficiency and yield. Also the semidwarf habit and the insensitivity to changes in photo- and thermoperiods readily show up.This investigation has been generously supported by the Swedish Research Council of Forestry and Agriculture.     

Rheological characteristic of a batch cultivation ofAgrobacterium radiobacter

During a batch cultivation on D-glucoseAgrobacterium radiobacter produces an extracellular polysaccharide agrobacteran. The rheological character of the culture broth is non-Newtonian and can be described by a power-law model or by the Hershel-Bulkley equation. Correlations were obtained between the parameters of these equations and the concentration of agrobacteran in the medium. For the given mixing system the rheological properties of the medium can be used to approximately estimate the qualitative changes in the intensity of mixing and to employ these data for regulating mixing during cultivation.     

Solid-state cultivation ofPenicillium capsulatum on beet pulp

Summary Extracellular enzyme production byP. capsulatum during solid-state fermentation on beet pulp is maximal at 30°C with initial moisture contents of 60–75% and when growth medium is supplemented with corn steep liquor, ammonium sulphate and yeast extract. On balance, solid-state co-cultures ofP. capsulatum withT. reesei orT. emersonii do not yield greater overall enzyme activity thanP. capsulatum alone.     

Influence of oxygen and substrate supply on the metabolism of Candida maltosa during cultivation on n-alkanes

Summary The influence of different states of oxygen and alkane substrate supply on the metabolism of Candida maltosa during cultivation on n-alkanes has been investigated. At sufficient oxygen and substrate supply a nearly equimolar ratio between the formation of biomass and alkane oxidation was observed. About 45% of the carbon source utilized was incorporated into the biomass. Strong oxygen limitation decreased protein formation and carbon incorporation into the biomass with a simultaneous increase in CO₂ formation, whereas periodic changes of oxygen supply only caused a decrease in carbon incorporation into the biomass and an increase in CO₂ formation. During cultivation in the presence of an inert hydrocarbon (pristane) it was found that carbon limitation and oxygen saturation diminished the formation of total and nitrogen-containing biomass, whereas carbon and oxygen limitation reduced the formation of total biomass.Offprint requests to: P. Riege     

Effect of aeration and mixing in various types of cultivation devices on the cell cycle of budding yeasts

When investigating the effect of aeration capacityK _L a of a cultivation device on the cell cycle of daughter cells ofCandida utilis it was found that the length of a phase (S + G₂) of the cell cycle is influenced by the rate of oxygen transfer. An increase ofK _L a, of a cultivation device achieved by increasing the specific output of mechanical energy for air dispersion and mixing may lead to cell damage and to changes in the cell cycle. The effect of high intensity of aeration and mixing is thus invalidated.     

Axenic cultivation of an Enchytraeid worm,Cognettia sphagnetorum

Summary Treatment with an antibiotic mixture of penicillin, streptomycin and framycetin freed approximately 60% of individuals of the enchytraeid worm, C. sphagnetorum from living microorganisms and caused no deleterious behavioural changes. Fungi accounted for half of the remaining contamination. Feeding experiments showed that living microorganisms are not an essential requirement for the worm since treated worms grew as well, or slightly better than untreated worms, on both sterile irradiated and non-sterile foods.     

A new principle of continuous flow cell cultivation

Summary A completely liquid-filled culture chamber with gas exchange across a synthetic membrane (Larsen andNilsson 1985) was incorporated into an automatic continuous flow system. The absence of an airliquid interface in the system permits removal of cell samples, and addition of fresh medium, under strictly sterile conditions. In this system,Tetrahymena pyriformis can be kept under optimal growth conditions in a rich nutrient medium and any defined cell density may be maintained for extended periods of time by varying the dilution rate of the culture. Furthermore, it has been possible to demonstrate, in the slope of the growth curve, even small changes which are difficult to detect in batch cultures since the duration of these changes is short. In the continuous flow system, the relative cell volume distribution and the food vacuole forming capacity of the cells were unaltered; however, all cells contained small refractive granules. The system permits the culture volume to be varied, but a standard volume of 20 ml was maintained in most experiments. Since the culture volume is small, the system requires less than one liter of fresh medium per week to maintain the cells in the exponentially multiplying growth phase.     

A modification of tooth germ cultivation in vitro and in ovo

Mandibular molar anlages excised from 17-day mouse foetuses were cultured in vitro or in ovo (on the chorioallantoic membrane). In both cases, the explants were underlain either with a Millipore filter or with a piece of fibrin foam. Tooth germs were harvested after 7 days of cultivation and processed histologically. Spatial arrangement was highly preserved in the tooth germs cultured in vitro on fibrin foam. In vitro cultures on Millipore filters revealed significant flattening of tooth germs, caused especially by the collapse of enamel organ and the pulp. The cytodifferentiation of tooth germs cultured in vitro on both substrates (Millipore filter, fibrin foam) was characterized by the presence of odontoblasts, polarizing ameloblasts and predentine. The cytodifferentiation of tooth germs cultured in ovo on Millipore filters placed on chorioallantoic membrane was characterized by the presence of odontoblasts, ameloblasts, predentine, dentine and enamel. However, the flattening of these explants was identical with the changes of the explants cultured on Millipore filters in vitro. In ovo cultivation on the fibrin foam failed to bring satisfactory results.     

How the apple escaped cultivation in North America

Domesticated plants have been transported around the globe through their association with humans and have undergone changes in response to their new environments. In many regions, farmers and, later, plant breeders have developed local landraces to deal with the new conditions or to satisfy the culinary needs of consumers, showing the versatility of these plants and the ingenuity of plant breeders, both ancient and modern. However, in some cases, plants leave behind their human associations and become feral in either the crop fields or natural landscape of the new region. The evolution of ferality has been studied in some crop systems, with many advances made in our understanding of annual crop ferality (e.g., Burger, Lee, & Ellstrand, 2006; Hegde et al., 2006). In contrast, very little is known about the genetics of feral perennial crops, and the study by Cronin, Kron, and Husband (2020) in this issue of Molecular Ecology sheds new light on this type of evolution, revealing the remarkable ability of domesticated apple (Malus domestica) to thrive and reproduce in North America without genetic input from local species.     

A photobioreactor system for computer controlled cultivation of microalgae

A bioreactor system was developed for the cultivation of the microalgae Synechocystis sp. PCC6803 under controlled physiological conditions. The determination of the actual physiological state of the microalgae was provided by inline recording of chlorophyll fluorescence parameters. A feed-back loop was employed to keep the microalgae in a defined physiological state. For the construction of this feed-back loop, the temporal behaviour of the system was investigated using changes in light conditions (as caused by modulated UVB radiation) as input signal and chlorophyll fluorescence as output signal. The reproducibility of the responses was high. Kinetic analysis based on curve fitting revealed two time constants in the UVB-induced responses. The knowledge of these time constants was utilised for the development of an efficient feed-back loop which allows the cultivation of the microalgae in a defined physiological state. This new process strategy (called physiostat) was successfully tested. The performance in a culture of growing microalgae is shown.     

Effect of Brestan on Saccharomyces cerevisiae during continuous cultivation

An increase in Brestan concentration in nutrient media decreased the content of protein, phosphorus, total ribonucleic acid, activity of pyruvate carboxylase and isocitrate lyase in cells ofSaccharomyces cerevisiae parent strain and respiratory deficient (RD) mutant while the trehalose content increased. The respiration quotient value for the RD mutant was higher than for the parent strain. The RD mutant lacked cytochromeaa ₃; cytochromec andb contents were lower than those of the parent strain.     

Continuous cultivation of fission yeast: Classical and flow microfluorometry observations

The fission yeast Schizosaccharomyces pombe was grown in glucose-limited medium in a steady-state continuous flow reactor. Changes in mean cell protein and RNA contents with growth rate are consistent with earlier observations under different conditions. Flow microfluorometry measurements of the frequency functions of DNA at different dilution rates show changes in coordination of DNA synthesis and cell separation. Shifting from batch growth to small dilution rates results in unusual cell aggregation which leads to multiple steady states at identical operating conditions.