The membrane dialysis bioreactor with integrated radial-flow fixed bed —a new approach for continuous cultivation of animal cells

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran^® carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l^?1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran^® carriers with 3–5 mm diameter showed an advantage compared to those with 1–2 mm diameter. For the 3–5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s^?1 and 0.75 mm s^?1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.     

Biosynthesis of phloroglucinol compounds in microorganisms—review

Phloroglucinol derivatives are a major class of secondary metabolites of wide occurrence in biological systems. In the bacteria kingdom, these compounds can only be synthesized by some species of Pseudomonads. Pseudomonas spp. could produce 2,4-diacetylphloroglucinol (DAPG) that plays an important role in the biological control of many plant pathogens. In this review, we summarize knowledge about synthesis of phloroglucinol compounds based on the DAPG biosynthetic pathway. Recent advances that have been made in understanding phloroglucinol compound biosynthesis and regulation are highlighted. From these studies, researchers have identified the biosynthesis pathway of DAPG. Most of the genes involved in the biosynthesis pathway have been cloned and characterized. Additionally, heterologous systems of the model microorganism Escherichia coli are constructed to produce phloroglucinol. Although further work is still required, a full understanding of phloroglucinol compound biosynthesis is almost within reach. This review also suggests new directions and attempts to gain some insights for better understanding of the biosynthesis and regulation of DAPG. The combination of traditional biochemistry and molecular biology with new systems biology and synthetic biology tools will provide a better view of phloroglucinol compound biosynthesis and a greater potential of microbial production.     

Improved β-carotene production of Rhodotorula glutinis in fermented radish brine by continuous cultivation

Fermentation kinetics of growth and β-carotene production by Rhodotorula glutinis DM28 in batch and continuous cultures using fermented radish brine, a waste generated from fermented vegetable industry, as a cultivation medium were investigated. The suitable brine concentration for β-carotene production by R. glutinis DM28 was 30 g l^?1. Its growth and β-carotene production obtained by batch culture in shake flasks were 2.2 g l^?1 and 87 μg l^?1, respectively, while, in a bioreactor were 2.6 g l^?1 and 186 μg l^?1, respectively. Furthermore, its maximum growth rate and β-carotene productivity in continuous culture obtained at the dilution rate of 0.24 h^?1 were 0.3 g l^?1 h^?1 and 19 μg l^?1 h^?1, respectively, which were significantly higher than those in the batch. Therefore, improved growth rate and β-carotene productivity of R. glutinis in fermented radish brine could be accomplished by continuous cultivation.     

Effect of root exudates on beneficial microorganisms—evidence from a continuous soybean monoculture

Soybean (Glycine max) agriculture is characterized by a high proportion of mono-cropping which results in reduced crop production in the Northeast China. Among all biotic and abiotic factors, changes in soil microbial communities induced by root activities, especially root exudates, might play an important role in these effects. The aim of the present study was to investigate response of microbial biomass and two major beneficial microbial functional groups, ammonia-oxidizing bacteria (AOB), and arbuscular mycorrhizal fungi (AMF), to root exudates in an experimental field under continuous soybean monoculture for 13 years. The results showed that microbial biomass carbon changed significantly with years of mono-cropping and correlated with concentrations of genistein (r = 0.4399, P < 0.001) and daidzein (r = 0.4082, P < 0.05) in the rhizosphere. However, root exudates had little effect on the nitrifier community, but reduced nitrification in the rhizosphere. In contrast, total AMF hyphal length was significantly stimulated by genistein (r = 0.5252, P < 0.01). There was a trend that AMF spore density increased in the rhizosphere with increasing years of mono-cropping, while AMF infection was constant over time, which might be attributed to competition between AMF and soil-borne fungal pathogens, as the results of stimulatory effect of flavonoids on fungal community, especially fungal pathogens. Our results suggested that the yield reduction in the beginning of continuous soybean monoculture could be partially attributed to nitrogen availability and yield stabilization after few years to stimulatory effects on AMF. These results imply that some of plant root exudates play a crucial role in changing the soil microbial community, and that underground ecosystem functioning is also affected by interactions among microbial functional groups.     

Endophytic microorganisms—promising applications in bioremediation of greenhouse gases

Bioremediation is a technique that uses microbial metabolism to remove pollutants. Various techniques and strategies of bioremediation (e.g., phytoremediation enhanced by endophytic microorganisms, rhizoremediation) can mainly be used to remove hazardous waste from the biosphere. During the last decade, this specific technique has emerged as a potential cleanup tool only for metal pollutants. This situation has changed recently as a possibility has appeared for bioremediation of other pollutants, for instance, volatile organic compounds, crude oils, and radionuclides. The mechanisms of bioremediation depend on the mobility, solubility, degradability, and bioavailability of contaminants. Biodegradation of pollutions is associated with microbial growth and metabolism, i.e., factors that have an impact on the process. Moreover, these factors have a great influence on degradation. As a result, recognition of natural microbial processes is indispensable for understanding the mechanisms of effective bioremediation. In this review, we have emphasized the occurrence of endophytic microorganisms and colonization of plants by endophytes. In addition, the role of enhanced bioremediation by endophytic bacteria and especially of phytoremediation is presented.     

Approaches to cultivation of “nonculturable” bacteria: Cyclic cultures

The work deals with more efficient procedures for the isolation and cultivation of “nonculturable” microorganisms (NM) from environmental sources. The techniques for NM cultivation in situ and under laboratory conditions are discussed. A new approach is considered, viz., cultivation under cyclically varying conditions with the cycle duration comparable to the duration of the cell cycle. Cyclic cultivation implies sequential changes of several cultivation phases with different growth conditions. An established sequence of growth phases provides for the competitiveness of the target microorganisms and for accumulation of their biomass. Cultivation of phosphate-accumulating bacteria, nonculturable microorganisms which have not been previously isolated in pure culture, in an SBR reactor is discussed as an example of cyclic cultures.     

Oxidative stress in microorganisms—I

Oxidative stress in microbial cells shares many similarities with other cell types but it has its specific features which may differe in prokaryotic and eukaryotic cells. We survey here the properties and actions of primary sources of oxidative stress, the role of transition metals in oxidative stress and cell protective machinery of microbial cells, and compare them with analogous features of other cell types. Other features to be compared are the action of reactive oxygen species (ROS) on cell constituents, secondary lipid-or protein-based radicals and other stress products. Repair of oxidative injury by microorganisms and proteolytic removal of irreparable cell constituents are briefly described. Oxidative damage of aerobically growing microbial cells by endogenously formed ROS mostly does not induce changes similar to the aging of multiplying mammalian cells. Rapid growth of bacteria and yeast prevents accumulation of impaired macromolecules which are repaired, diluted or eliminated. During growth some simple fungi, such as yeast orPodospora spp., exhibit aging whose primary cause seems to be fragmentation of the nucleolus or impairment of mitochondrial DNA integrity. Yeast cell aging seems to be accelerated by endogenous oxidative stress. Unlike most growing microbial cells, stationaryphase cells gradually lose their viability because of a continuous oxidative stress, in spite of an increased synthesis of antioxidant enzymes. Unlike in most microorganisms, in plant and animal cells a severe oxidative stress induces a specific programmed death pathway-apoptosis. The scant data on the microbial death mechanisms induced by oxidative stress indicate that in bacteria cell death can result from activation of autolytic enzymes (similarly to the programmed mother-cell death at the end of bacillar sporulation). Yeast and other simple eukaryotes contain components of a proapoptotic pathway which are silent under normal conditions but can be activated by oxidative stress or by manifestation of mammalian death genes, such asbak orbax. Other aspects, such as regulation of oxidative-stress response, role of defense enzymes and their control, acquisition of stress tolerance, stress signaling and its role in stress response, as well as cross-talk between different stress factors, will be the subject of a subsequent review.     

Are primary cultures realistic models of prostate cancer?

Primary cultures fill a unique niche among the repertoire of in vitro model systems available to investigate the biology of the normal and malignant human prostate. This review summarizes some of the properties of primary cultures, with special emphasis on two questions: are primary cultures from adenocarcinomas really comprised of cancer rather than normal cells, and do primary cultures faithfully retain characteristics of cells of origin? © 2003 Wiley‐Liss, Inc.     

Genotype — environment interaction in tissue cultures of birch

Summary In vitro culture experiments were carried out with three birch genotypes characterized by certain genealogical relationships which serve as indicators of genetic similarity or dissimilarity. Each genotype was grown in each of six different environments (medium types), and callus growth and colour were observed. The aim was to improve our understanding of the operation of genetic and environmental effects at the early stages of regeneration in vitro. For this purpose we tried to answer the question as to whether genetic differences exert effects that are consistently distinguishable under different environments or whether environmental differences exert effects that are consistently distinguishable between different genotypes. Since conventional analytical methods, such as the analysis of variance, are inappropriate for providing satisfactory answers to this question, we applied a new concept of interpretation. With the help of this concept we obtained the following results which appear to be unique among their kind. 1) For both characters, callus growth and callus colour, genetic differences are masked only slightly by the environments while environmental differences are almost completely masked by the genotypes. Thus, in the present experiment, interaction is one-sided in the sense that environmental effects interact strongly with genotypic effects but genotypic effects interact only slightly with the environmental ones. 2) Nuclear effects seem to be responsible for the differences observed in callus growth, while the differences in callus colour can be explained by the joint action of nuclear and extranuclear effects.     

Identification of chilling and heat requirements of cherry trees—a statistical approach

Most trees from temperate climates require the accumulation of winter chill and subsequent heat during their dormant phase to resume growth and initiate flowering in the following spring. Global warming could reduce chill and hence hamper the cultivation of high-chill species such as cherries. Yet determining chilling and heat requirements requires large-scale controlled-forcing experiments, and estimates are thus often unavailable. Where long-term phenology datasets exist, partial least squares (PLS) regression can be used as an alternative, to determine climatic requirements statistically. Bloom dates of cherry cv. ‘Schneiders späte Knorpelkirsche’ trees in Klein-Altendorf, Germany, from 24 growing seasons were correlated with 11-day running means of daily mean temperature. Based on the output of the PLS regression, five candidate chilling periods ranging in length from 17 to 102 days, and one forcing phase of 66 days were delineated. Among three common chill models used to quantify chill, the Dynamic Model showed the lowest variation in chill, indicating that it may be more accurate than the Utah and Chilling Hours Models. Based on the longest candidate chilling phase with the earliest starting date, cv. ‘Schneiders späte Knorpelkirsche’ cherries at Bonn exhibited a chilling requirement of 68.6?±?5.7 chill portions (or 1,375?±?178 chilling hours or 1,410?±?238 Utah chill units) and a heat requirement of 3,473?±?1,236 growing degree hours. Closer investigation of the distinct chilling phases detected by PLS regression could contribute to our understanding of dormancy processes and thus help fruit and nut growers identify suitable tree cultivars for a future in which static climatic conditions can no longer be assumed. All procedures used in this study were bundled in an R package (‘chillR’) and are provided as Supplementary materials. The procedure was also applied to leaf emergence dates of walnut (cv. ‘Payne’) at Davis, California.     

α-Amylase production in batch and continuous cultures by Bacillus caldolyticus

Summary The concentration and productivity of -amylase increased remarkably, 15- and 11-fold respectively, in a continuous culture of Bacillus caldolyticus DSM 405 compared with batch culture, provided starch was used as the sugar source in a casitone medium. In the casitone medium with or without glucose hardly any improvement of enzyme production was observed in continuous culture. The addition of a small amount of starch to the glucose-casitone medium had a marked effect in stimulating amylase formation in continuous culture but no effect in batch culture.It was suggested that the higher production of -amylase in the continuous culture using starch as the inducer was partly related to the predominance of some conditional non-sporulating variants with a higher amylase forming activity and to derepression of the enzyme at a low glucose concentration.     

Analyzing electrical activities of pancreatic β cells using mathematical models

Bursts of repetitive action potentials are closely related to the regulation of glucose-induced insulin secretion in pancreatic β cells. Mathematical studies with simple β-cell models have established the central principle that the burst-interburst events are generated by the interaction between fast membrane excitation and slow cytosolic components. Recently, a number of detailed models have been developed to simulate more realistic β cell activity based on expanded findings on biophysical characteristics of cellular components. However, their complex structures hinder our intuitive understanding of the underlying mechanisms, and it is becoming more difficult to dissect the role of a specific component out of the complex network. We have recently developed a new detailed model by incorporating most of ion channels and transporters recorded experimentally (the Cha-Noma model), yet the model satisfies the charge conservation law and reversible responses to physiological stimuli. Here, we review the mechanisms underlying bursting activity by applying mathematical analysis tools to representative simple and detailed models. These analyses include time-based simulation, bifurcation analysis and lead potential analysis. In addition, we introduce a new steady-state I-V (ssI-V) curve analysis. We also discuss differences in electrical signals recorded from isolated single cells or from cells maintaining electrical connections within multi-cell preparations. Towards this end, we perform simulations with our detailed pancreatic β-cell model.     

Competition patterns among phytoplankton functional groups: How useful are the complex mathematical models?

Simple models have significant contribution to the development of ecological theory. However, these minimalistic modeling approaches usually focus on a small subset of the causes of a phenomenon and neglect important aspects of system dynamics. In this study, we use a complex aquatic biogeochemical model to examine competition patterns and structural shifts in the phytoplankton community under nutrient enrichment conditions. Our model simulates multiple elemental cycles (org. C, N, P, Si, O), multiple functional phytoplankton (diatoms, green algae and cyanobacteria) and zooplankton (copepods and cladocerans) groups. It also takes into account recent advances in stoichiometric nutrient recycling theory, and the zooplankton grazing term is reformulated to include algal food quality effects on zooplankton assimilation efficiency. The model provided a realistic platform to examine the functional properties (e.g., kinetics, growth strategies, intracellular storage capacity) and the abiotic conditions (temperature, nutrient loading) under which the different phytoplankton groups can dominate or can be competitively excluded in oligo, meso and eutrophic environments. Based on the results of our analysis, the intergroup variability in the minimum cell quota and maximum transport rate at the cell surface for phosphorus along with the group-specific metabolic losses can shape the structure of plankton communities. We also use classification tree analysis to elucidate aspects (e.g., relative differences in the functional group properties, critical values of the abiotic conditions, levels of the other plankton community residents) of the complex interplay among physical, chemical and biological factors that drive epilimnetic plankton dynamics. Finally, our study highlights the importance of improving the mathematical representation of phytoplankton adaptive strategies for resources procurement (e.g., regulation of transport kinetics, effects of transport kinetics on the kinetics of assimilation, relationship between assimilation and growth) to effectively link variability at the organismal level with ecosystem-scale patterns.     

Nutrient retention function of a stream wetland complex—A high-frequency monitoring approach

The ability of riverine ecosystems to retain nutrients depends on different hydrological, chemical and biological conditions including exchange processes between streams and wetlands. We investigated nutrient retention in a stream wetland complex on the time scale of daily hydrological exchange between both systems. Daily mass balances of NO₃-N, NH₄-N, TP and SRP were calculated with data obtained by two automated measurement stations in a stream reach upstream and downstream of a wetland. The pattern of hydrological exchange between stream and wetland was used to classify characteristic hydrological periods like floods, base and low flows. The nutrient retention function of the stream wetland complex varied considerably during phases of similar hydrologic conditions. Despite re-wetting measures in the wetland, an overall net export of all nutrients except for NH₄-N characterised the whole growing season. Nitrate retention occurred during summer flood (retention in the wetland, 23 kg NO₃-N d^?1, 17% of the input load) and low flow (retention in the stream, 1 kg NO₃-N d^?1, 2% of the input load). TP retention during summer could be assigned to sedimentation (0.7 kg TP d^?1, 7% during flooding in the wetland, 0.2 kg TP d^?1, 4% during low flow in the stream). SRP retention was only intermittent. We concluded that the nutrient retention of streams and wetlands can only be optimised by restoration measures that regard both systems as one functional unit in terms of nutrient retention.     

Carbon budgets in batch and continuous cultures: How can we understand natural physiology of marine phytoplankton?

Monitoring of respiratory and organic losses from carbon assimilationfor the diatom Thalassiosira pseudonana shows very slight (about5%) total carbon losses in continuous culture. Although correlationbetween batch and continuous culture cellular chemistry wasless than ideal, batch culture exponential phase cells showedcarbon metabolism similar to that for the continuous culture.In both cases, growth rates were in excess of 1.5 divisionsd^–1 and total carbon losses in the batch exponential phasewere similar (<10%) to those in the continuous culture. Estimatesof growth rates from C-14 uptake and particulate carbon matchedthose from changes in cell numbers in the batch culture andthat from the dilution rate in the continuous culture. In batchculture, immediately after starting the culture and in stationaryphase, carbon losses from respiration, organic excretion, andcellular degradation were large (>50%). To understand phytoplanktonphysiology in nature, it is necessary to find what effects growthrate and population density have on carbon losses and to ascertainwhether or not steady state conditions really pertain to theocean.     

“Diffusion” of microorganisms in calcium alginate beads

Summary The leakage of Serratia marcescens cells immobilised in Ca-alginate beads was quantified and the experimental values fitted to the pore diffusion model in order to obtain the biomass diffusion coefficient (D _x). This coefficient was then determined, resulting a value of 0.45 10^–7 cm² s^–1 at the beginning of the fermentation process. The variation of this coefficient, together with the porosity of the particles (_i), were also determined all along the process.     

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.