Light-mediated biosynthesis of size-tuned silver nanoparticles using Saccharomyces cerevisiae extract.

Bio-based production of silver nanoparticles represents a sustainable alternative to commercially applied physicochemical manufacturing approaches and provides qualitatively highly valuable nanomaterials due to their narrow size dispersity, high stability and biocompatibility with broad application potentials. The intrinsic features of nanoparticles depend on size and shape, whereby the controlled synthesis is a challenging necessity. In the present study, the biosynthesis of size-tuned silver nanoparticles based on cell-free extracts of Saccharomyces cerevisiae DSM 1333 was investigated. Single parameter optimization strategies in phases of cultivation, extraction, and synthesis were performed to modify the nanoparticle scale and yield. Visible light was exploited as a tool in nanoparticle production. The influence of white light on the biosynthesis of silver nanoparticles was determined by using novel LED systems with the exposition of varying irradiation intensities and simultaneous performance of control experiments in the dark. Characterization of the resulting nanomaterials by spectrophotometric analysis, dynamic light scattering, scanning electron microscopy, and energy dispersive X-ray spectroscopy, revealed spherical silver nanoparticles with controlled, light-mediated size shifts in markedly increased quantities. Matching of irradiated and non-irradiated reaction mixtures mirrored the enormous functionality of photon input and the high sensitivity of the biosynthesis process. The silver nanoparticle yields increased by more than 90% with irradiation at 1.0 ± 0.2 mW cm - 2 and the reduction of particle dimensions was achieved with significant shifts of size-specific absorption maxima from 440 to 410 nm, corresponding to particle sizes of 130 nm and 100 nm, respectively. White light emerged as an excellent tool for nano-manufacturing with advantageous effects for modulating unique particle properties.

Growth, morphology, and formation of cinnabarin in Pycnoporus cinnabarinus in relation to different irradiation spectra.

BACKGROUND: The demand for natural pigments in general, and for fungi-derived pigments in particular, is constantly rising. Wood-decomposing fungi represent a promising source for natural pigments and they are usually easy to cultivate in pure culture. One of them, i.e., Pycnoporus cinnabarinus, offers a highly interesting spectrum of bioactivity, partly due to the formation of the orange-red pigment cinnabarin. However, apart from a few studies addressing its diverse potential biotechnological applications, there is still a large gap of knowledge concerning the influence of light on the formation of cinnabarin. The aim of this work was to investigate the effect of different irradiations on the cinnabarin content, the growth, and the morphology of three different P. cinnabarinus strains. We used highly standardized irradiation conditions and cultivation techniques in combination with newly developed methods for the extraction and direct quantification of cinnabarin. RESULTS: Red, green, blue, and UV-A irradiation (mean irradiance Ee = 1.5 ± 0.18 W m-2) had considerable effects on the growth and colony appearance of all three P. cinnabarinus strains tested. The cinnabarin content determined was, thus, dependent on the irradiation wavelength applied, allowing strain-specific thresholds to be defined. Irradiation with wavelengths below this strain-specific threshold corresponded to a lower cinnabarin content, at least at the intensity applied. The orange-red pigment appeared by light microscopy as incrusted extracellular plaques present on the hyphal walls. Highly efficient vegetative propagation occurred by arthroconidia, and we observed the tendency that this asexual reproduction was (i) most frequent in the dark but (ii) never occurred under UV-A exposure. CONCLUSION:  This study highlights a differential photo-dependence of growth, morphology, and cinnabarin formation in P. cinnabarinus. This confirms that it is advisable to consider the wavelength of the light used in future biotechnological productions of natural pigments.

Fungal Anthraquinone Photoantimicrobials Challenge the Dogma of Cationic Photosensitizers.

The photoantimicrobial potential of four mushroom species (i.e., Cortinarius cinnabarinus, C. holoxanthus, C. malicorius, and C. sanguineus) was explored by studying the minimal inhibitory concentrations (MIC) via a light-modified broth microdilution assay based on the recommended protocols of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The extracts were tested against Candida albicans, Escherichia coli, and Staphylococcus aureus under blue (λ = 428 and 478 nm, H = 30 J/cm2) and green light (λ = 528 nm, H = 30 J/cm2) irradiation. Three extracts showed significant photoantimicrobial effects at concentrations below 25 µg/mL. Targeted isolation of the major pigments from C. sanguineus led to the identification of two new potent photoantimicrobials, one of them (i.e., dermocybin) being active against S. aureus and C. albicans under green light irradiation [PhotoMIC530 = 39.5 µM (12.5 µg/mL) and 2.4 µM (0.75 µg/mL), respectively] and the other one (i.e., emodin) being in addition active against E. coli in a low micromolar range [PhotoMIC428 = 11.1 µM (3 µg/mL)]. Intriguingly, dermocybin was not (photo)cytotoxic against the three tested cell lines, adding an additional level of selectivity. Since both photoantimicrobials are not charged, this discovery shifts the paradigm of cationic photosensitizers.

Light in the box-photobiological examination chamber with light trap ventilation system for studying fungal surface cultures illustrated with Metarhizium brunneum and Beauveria brongniartii.

Due to their versatile way of life as saprophytes, endophytes, and entomopathogens, fungi of the genera Metarhizium and Beauveria are exposed to varying illumination conditions in their natural habitats, which makes a thorough adaptation to light very likely. While the few available studies for these genera support this assumption, research in this field is still in its infancy and the data material restricted to only a few fungal species. Thus, the aim of this work was to explore how light influences growth, conidial production and secondary metabolite formation of two industrial relevant strains of M. brunneum (MA 43, formerly M. anisopliae var. anisopliae BIPESCO 5/F52) and B. brongniartii (BIPESCO 2). To achieve this, we constructed an easily adjustable illumination device for highly standardized photophysiological studies of fungi on Petri dishes, the so-called LIGHT BOX. With the aid of this device, M. brunneum and B. brongniartii were grown on S4G or S2G agar at 25 °C for 14 days either in complete darkness or under constant illumination with red light (λpeak = 635 nm), green light (λpeak = 519 nm) or blue light (λpeak = 452 nm). In addition, for each wavelength the effect of different illumination intensities was tested, i.e., intensities of red light ranging from 22.1 ± 0.1 to 136.5 ± 0.3 µW cm-2, green light from 16.5 ± 0.1 to 96.2 ± 0.1 µW cm-2, and blue light from 56.1 ± 0.2 to 188.9 ± 0.6 µW cm-2. Both fungi strongly responded in terms of growth, conidial production, pigmentation and morphology to changes in the wavelength and irradiation intensity. The wavelength-dependent production of the well-known secondary metabolite oosporein which is secreted by the genus Beauveria in particular, was also increased under green and blue light exposure. The established LIGHT BOX system allows not only to optimize conidial production yields with these biotechnologically relevant fungi, but also allows the photobiological exploration of other fungi.

Xanthoepocin, a photolabile antibiotic of Penicillium ochrochloron CBS 123823 with high activity against multiresistant gram-positive bacteria.

BACKGROUND: With the steady increase of antibiotic resistance, several strategies have been proposed in the scientific community to overcome the crisis. One of many successful strategies is the re-evaluation of known compounds, which have been early discarded out of the pipeline, with state-of-the-art know-how. Xanthoepocin, a polyketide widespread among the genus Penicillium with an interesting bioactivity spectrum against gram-positive bacteria, is such a discarded antibiotic. The purpose of this work was to (i) isolate larger quantities of this metabolite and chemically re-evaluate it with modern technology, (ii) to explore which factors lead to xanthoepocin biosynthesis in P. ochrochloron, and (iii) to test if it is beside its known activity against methicillin-resistant Staphylococcus aureus (MRSA), also active against linezolid and vancomycin-resistant Enterococcus faecium (LVRE)-a very problematic resistant bacterium which is currently on the rise. RESULTS: In this work, we developed several new protocols to isolate, extract, and quantify xanthoepocin out of bioreactor batch and petri dish-grown mycelium of P. ochrochloron. The (photo)chemical re-evaluation with state-of-the-art techniques revealed that xanthoepocin is a photolabile molecule, which produces singlet oxygen under blue light irradiation. The intracellular xanthoepocin content, which was highest under ammonium-limited conditions, varied considerably with the applied irradiation conditions in petri dish and bioreactor batch cultures. Using light-protecting measures, we achieved MIC values against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), which were up to 5 times lower than previously published. In addition, xanthoepocin was highly active against a clinical isolate of linezolid and vancomycin-resistant Enterococcus faecium (LVRE). CONCLUSIONS: This interdisciplinary work underlines that the re-evaluation of known compounds with state-of-the-art techniques is an important strategy in the combat against multiresistant bacteria and that light is a crucial factor on many levels that needs to receive more attention. With appropriate light protecting measures in the susceptibility tests, xanthoepocin proved to be a powerful antibiotic against MRSA and LVRE. Exploring the light response of other polyketides may be pivotal for re-introducing previously discarded metabolites into the antibiotic pipeline and to identify photosensitizers which might be used for (antimicrobial) photodynamic therapies.

A New High-Throughput-Screening-Assay for Photoantimicrobials Based on EUCAST Revealed Unknown Photoantimicrobials in Cortinariaceae.

Antimicrobial resistance is one of the biggest health and subsequent economic threat humanity faces. Next to massive global awareness campaigns, governments and NGOs alike stress the need for new innovative strategies to treat microbial infections. One of such innovative strategies is the photodynamic antimicrobial chemotherapy (PACT) in which the synergistic effects of photons and drugs are exploited. While many promising reports are available, PACT - and especially the drug-design part behind - is still in its infancy. Common best-practice rules, such as the EUCAST or CLSI protocols for classic antibiotics as well as high-throughput screenings, are missing, and this, in turn, hampers the identification of hit structures. Hit-like structures might come from synthetic approaches or from natural sources. They are identified via activity-guided synthesis or isolation strategies. As source for new antimicrobials, fungi are highly ranked. They share the same ecological niche with many other microbes and consequently established chemical strategies to combat with the others. Recently, in members of the Cortinariaceae, especially of the subgenus Dermocybe, photoactive metabolites were detected. To study their putative photoantimicrobial effect, a photoantimicrobial high-throughput screening (HTS) based on The European Committee on Antimicrobial Susceptibility Testing (EUCAST) was established. After validation, the established HTS was used to evaluate a sample set containing six colorful representatives from the genus Cortinarius (i.e., Cortinarius callisteus, C. rufo-olivaceus, C. traganus, C. trivialis, C. venetus, and C. xanthophyllus). The assay is built on a uniform, light-emitting diode (LED)-based light irradiation across a 96-well microtiter plate, which was achieved by a pioneering arrangement of the LEDs. The validation of the assay was accomplished with well-known photoactive drugs, so-called photosensitizers, utilizing six distinct emission wavelengths (λexc = 428, 478, 523, 598, or 640 nm) and three microbial strains (Candida albicans, Staphylococcus aureus, and Escherichia coli). Evaluating the extracts of six Cortinarius species revealed two highly promising species, i.e., C. rufo-olivaceus and C. xanthophyllus. Extracts from the latter were photoactive against the Gram-positive S. aureus (c = 7.5 µg/ml, H = 30 J/cm2, λ = 478 nm) and the fungus C. albicans (c = 75 µg/ml, H = 30 J/cm2, λ = 478 nm).

Students' Experiences of Working With a Socio-Scientific Issues-Based Curriculum Unit Using Role-Playing to Negotiate Antibiotic Resistance.

The emergence and widespread of antibiotic-resistant pathogenic microorganisms are of great individual and societal relevance. Due to the complex and multilayered nature of the topic, antibiotic resistance (ABR) is the object of concern for several scientific fields, such as microbiology or medicine, and encompasses a broad range of political, economic, and social aspects. Thus, the issue related to antibiotic-resistant bacterial diseases offers an excellent platform for designing and implementing the teaching and learning of socio-scientific issues (SSI). We created a SSI-based curriculum unit for use in secondary science classrooms by developing a collaborative partnership between education researchers and microbiologists. This classroom environment allows students to explore and negotiate ABR as a societal and scientific phenomenon. For this purpose, we leveraged role-playing within the SSI-based unit as a productive context for engaging students in learning opportunities that provide multiple perspectives on ABR and the complex interplay of its accelerators. This case-based paper describes Austrian school students' experiences from their participation in a SSI-embedded role-playing classroom environment and subsequent activities that included a mini congress with a poster presentation and a panel discussion. An open-ended questionnaire-based assessment tool was used to examine the situational characteristics of the students' work. To assess students' contributions, we applied a qualitative content analysis design and identified cognitive and affective outcomes. The students' learning experiences demonstrate that they considered the content - the social complexities of antibiotic-resistant bacteria and associated diseases - exciting and very topical. The students perceived that learning about ABR is relevant for their future and involves both individual and societal responsibility for action. Although the curriculum unit and its assignments were described as labor-intensive, it became apparent that the role-playing setting has the potential to inform students about multiple stakeholder positions concerning ABR. Concerning the promotion of science practices, almost all students claimed that they learned to organize, analyze, evaluate, and present relevant information. Moreover, the students affirmed that they learned to argue from the perspective of their assigned roles. However, the students did not clarify whether they learned more through this SSI-based classroom instruction than through conventional science teaching approaches.

Fungal Growth in Batch Culture - What We Could Benefit If We Start Looking Closer.

Since filamentous fungi rapidly adjust their metabolic properties to environmental changes, a rigorous standardization and characterization of cultivation conditions is necessary to obtain meaningful and reproducible results. In batch cultures, which are commonly characterized according to the classical growth curve in textbooks (i.e., lag, exponential, stationary, and declining phase), this is of special difficulty. Although various studies in literature report atypically shaped growth curves of filamentous fungi in batch culture, systematic investigations on this topic are scarce and deviations are barely mentioned in textbooks. Summarizing approximately a decade of observations of growth characteristics from bioreactor batch grown filamentous fungi - in particular two strains (CBS123.823 and CBS123.824) of Penicillium ochrochloron - we demonstrate with a series of highly standardized bioreactor batch culture experiments that the classical growth curve failed to describe growth dynamics of the studied fungi in this work. The nature of the first exhausted nutrient was of remarkable importance for the resulting shape of the growth curve. In all experiments, online respirometry proved to be a powerful tool to distinguish growth phases and revealed more physiological states than expected from the mere biomass curve. In this respect we discuss why "atypical" shaped growth curves often remain unrecognized and that they might be the rule rather than the exception. Acknowledging the importance of the correct presentation of this complex topic in textbooks, we also propose a modified growth curve scheme to sensitize students for potential alternative shaped growth curves.

Critical evaluation of a putative glucosamine excretion by Aspergillus niger CBS120.49 and Penicillium ochrochloron CBS123.824 under citric acid producing conditions.

As one of the most frequently occurring monomers in the biosphere, glucosamine is a valuable metabolite for several applications. Although microbial glucosamine production is still in its infancy, it offers the possibility to circumvent problems associated with traditional production by hydrolysis. Of particular interest is a study with Aspergillus niger, which reports for the first time high glucosamine excretion in the early phase of citric acid production. These results have relevance for both the commercial glucosamine production and deeper insight into the regulation of organic acid excretion in fungi. To investigate glucosamine excretion, we performed bioreactor batch cultivations with Penicillium ochrochloron CBS123.824 and A. niger CBS120.49 using cultivation conditions which are known to trigger the production of citric acid. Glucosamine detection in culture filtrates was achieved by two photometric methods, High performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and HPLC with mass spectrometry detection (HPLC-MS). Surprisingly, we detected no glucosamine at all. Based on a critical review of published data for A. niger, we conclude that the reported high levels of excreted glucosamine might be an experimental artifact. However, growth experiments with glucosamine as a combined or single source for carbon or nitrogen showed that both organisms are in principle able to transport glucosamine across their plasma membrane, which is a prerequisite for the excretion of glucosamine.

A convenient workflow to spot photosensitizers revealed photo-activity in basidiomycetes.

Photodynamic therapy (PDT) is an alternative approach for the treatment of neoplastic diseases employing photosensitizers activated by light. In order to discover new natural photosensitizers, a convenient workflow was established. To validate the workflow, fungi were selected, because we hypothesized that fruiting bodies and mycelia are an overlooked source. The results proved the hypothesis, as exorbitant high photo-cytotoxicity values were detected. For example, the acetone extract of Cortinarius croceus was characterized by an EC50, 9.3 J cm-2 of 1 µg mL-1 against cells of a lung cancer cell-line (A549). In sum, a low-cost workflow for the detection and biological evaluation of photosensitizers is presented and discussed. Furthermore, this paper provides the first experimental evidence for phototoxic metabolites in basidiomycetes. This hints towards a new assignable function of fungal pigments, i.e. photochemical defense.

The Dynamics of Plasma Membrane, Metabolism and Respiration (PM-M-R) in Penicillium ochrochloron CBS 123824 in Response to Different Nutrient Limitations-A Multi-level Approach to Study Organic Acid Excretion in Filamentous Fungi.

Filamentous fungi are important cell factories. In contrast, we do not understand well even basic physiological behavior in these organisms. This includes the widespread phenomenon of organic acid excretion. One strong hurdle to fully exploit the metabolic capacity of these organisms is the enormous, highly environment sensitive phenotypic plasticity. In this work we explored organic acid excretion in Penicillium ochrochloron from a new point of view by simultaneously investigating three essential metabolic levels: the plasma membrane H+-ATPase (PM); energy metabolism, in particular adenine and pyridine nucleotides (M); and respiration, in particular the alternative oxidase (R). This was done in strictly standardized chemostat culture with different nutrient limitations (glucose, ammonium, nitrate, and phosphate). These different nutrient limitations led to various quantitative phenotypes (as represented by organic acid excretion, oxygen consumption, glucose consumption, and biomass formation). Glucose-limited grown mycelia were used as the reference point (very low organic acid excretion). Both ammonium and phosphate grown mycelia showed increased organic acid excretion, although the patterns of excreted acids were different. In ammonium-limited grown mycelia amount and activity of the plasma membrane H+-ATPase was increased, nucleotide concentrations were decreased, energy charge (EC) and catabolic reduction charge (CRC) were unchanged and alternative respiration was present but not quantifiable. In phosphate-limited grown mycelia (no data on the H+-ATPase) nucleotide concentrations were still lower, EC was slightly decreased, CRC was distinctly decreased and alternative respiration was present and quantifiable. Main conclusions are: (i) the phenotypic plasticity of filamentous fungi demands adaptation of sample preparation and analytical methods at the phenotype level; (ii) each nutrient condition is unique and its metabolic situation must be considered separately; (iii) organic acid excretion is inversely related to nucleotide concentration (but not EC); (iv) excretion of organic acids is the outcome of a simultaneous adjustment of several metabolic levels to nutrient conditions.

Rapid sample processing for intracellular metabolite studies in Penicillium ochrochloron CBS 123.824: the FiltRes-device combines cold filtration of methanol quenched biomass with resuspension in extraction solution.

BACKGROUND: Many issues concerning sample processing for intracellular metabolite studies in filamentous fungi still need to be solved, e.g. how to reduce the contact time of the biomass to the quenching solution in order to minimize metabolite leakage. Since the required time to separate the biomass from the quenching solution determines the contact time, speeding up this step is thus of utmost interest. Recently, separation approaches based on cold-filtration were introduced as promising alternative to cold-centrifugation, which exhibit considerably reduced contact times. In previous works we were unable to obtain a compact pellet from cold methanol quenched samples of the filamentous fungus Penicillium ochrochloron CBS 123.824 via centrifugation. Therefore our aim was to establish for this organism a separation technique based on cold-filtration to determine intracellular levels of a selected set of nucleotides. RESULTS: We developed a cold-filtration based technique as part of our effort to revise the entire sample processing method and analytical procedure. The Filtration-Resuspension (FiltRes) device combined in a single apparatus (1) a rapid cold-filtration and (2) a rapid resuspension of the biomass in hot extraction solution. Unique to this is the injection of the extraction solution from below the membrane filter (FiltRes-principle). This caused the mycelial cake to detach completely from the filter membrane and to float upwards so that the biomass could easily be transferred into preheated tubes for metabolite extraction. The total contact time of glucose-limited chemostat mycelium to the quenching solution could be reduced to 15.7 ± 2.5 s, whereby each washing step added another 10-15 s. We evaluated critical steps like filtration time, temperature profile, reproducibility of results, and using the energy charge (EC) as a criterion, effectiveness of enzyme destruction during the transition in sample temperature from cold to hot. As control we used total broth samples quenched in hot ethanol. Averaged over all samples an EC of 0.93 ± 0.020 was determined with the FiltRes-principle compared to 0.89 ± 0.049 with heat stopped total broth samples. CONCLUSIONS: We concluded that for P. ochrochloron this technique is a reliable sample processing method for intracellular metabolite analysis, which might offer also other possible applications.

Adapting High-Resolution Respirometry to Glucose-Limited Steady State Mycelium of the Filamentous Fungus Penicillium ochrochloron: Method Development and Standardisation.

Fungal electron transport systems (ETS) are branched, involving alternative NADH dehydrogenases and an alternative terminal oxidase. These alternative respiratory enzymes were reported to play a role in pathogenesis, production of antibiotics and excretion of organic acids. The activity of these alternative respiratory enzymes strongly depends on environmental conditions. Functional analysis of fungal ETS under highly standardised conditions for cultivation, sample processing and respirometric assay are still lacking. We developed a highly standardised protocol to explore in vivo the ETS-and in particular the alternative oxidase-in Penicillium ochrochloron. This included cultivation in glucose-limited chemostat (to achieve a defined and reproducible physiological state), direct transfer without any manipulation of a broth sample to the respirometer (to maintain the physiological state in the respirometer as close as possible to that in the chemostat), and high-resolution respirometry (small sample volume and high measuring accuracy). This protocol was aimed at avoiding any changes in the physiological phenotype due to the high phenotypic plasticity of filamentous fungi. A stable oxygen consumption (< 5% change in 20 minutes) was only possible with glucose limited chemostat mycelium and a direct transfer of a broth sample into the respirometer. Steady state respiration was 29% below its maximum respiratory capacity. Additionally to a rotenone-sensitive complex I and most probably a functioning complex III, the ETS of P. ochrochloron also contained a cyanide-sensitive terminal oxidase (complex IV). Activity of alternative oxidase was present constitutively. The degree of inhibition strongly depended on the sequence of inhibitor addition. This suggested, as postulated for plants, that the alternative terminal oxidase was in dynamic equilibrium with complex IV-independent of the rate of electron flux. This means that the onset of activity does not depend on a complete saturation or inhibition of the cytochrome pathway.

Organic Acid Excretion in Penicillium ochrochloron Increases with Ambient pH.

Despite being of high biotechnological relevance, many aspects of organic acid excretion in filamentous fungi like the influence of ambient pH are still insufficiently understood. While the excretion of an individual organic acid may peak at a certain pH value, the few available studies investigating a broader range of organic acids indicate that total organic acid excretion rises with increasing external pH. We hypothesized that this phenomenon might be a general response of filamentous fungi to increased ambient pH. If this is the case, the observation should be widely independent of the organism, growth conditions, or experimental design and might therefore be a crucial key point in understanding the function and mechanisms of organic acid excretion in filamentous fungi. In this study we explored this hypothesis using ammonium-limited chemostat cultivations (pH 2-7), and ammonium or phosphate-limited bioreactor batch cultivations (pH 5 and 7). Two strains of Penicillium ochrochloron were investigated differing in the spectrum of excreted organic acids. Confirming our hypothesis, the main result demonstrated that organic acid excretion in P. ochrochloron was enhanced at high external pH levels compared to low pH levels independent of the tested strain, nutrient limitation, and cultivation method. We discuss these findings against the background of three hypotheses explaining organic acid excretion in filamentous fungi, i.e., overflow metabolism, charge balance, and aggressive acidification hypothesis.

Dynamics of energy charge and adenine nucleotides during uncoupling of catabolism and anabolism in Penicillium ochrochloron.

Filamentous fungi are able to spill energy when exposed to energy excess by uncoupling catabolism from anabolism, e.g. via overflow metabolism. In current study we tested the hypothesis that overflow metabolism is regulated via the energetic status of the hyphae (i.e. energy charge, ATP concentration). This hypothesis was studied in Penicillium ochrochloron during the steady state of glucose- or ammonium-limited chemostat cultures as well as during three transient states ((i) glucose pulse to a glucose-limited chemostat, (ii) shift from glucose-limited to ammonium-limited conditions in a chemostat, and (iii) ammonium exhaustion in batch culture). Organic acids were excreted under all conditions, even during exponential growth in batch culture as well as under glucose-limited conditions in a chemostat. Partial uncoupling of catabolism and anabolism via overflow metabolism was thus constitutively present. Under all tested conditions, overflow metabolism was independent of the energy charge or the ATP concentration of the hyphae. There was a reciprocal correlation between glucose uptake rate and intracellular adenine nucleotide content. During all transients states a rapid decrease in energy charge and the concentrations of nucleotides was observed shortly after a change in glycolytic flux ("ATP paradoxon"). A possible connection between the change in adenine nucleotide concentrations and the purine salvage pathway is discussed.

Characteristics of glucose uptake by glucose- and NH4-limited grown Penicillium ochrochloron at low, medium and high glucose concentration.

Glucose uptake by Penicillium ochrochloron (formerly Penicillium simplicissimum) was studied from 0.01 to 400 mM glucose using chemostat culture and bioreactor batch culture. The characteristics of glucose uptake varied considerably with the conditions of growth, harvest and uptake assay. Glucose-limited grown mycelium showed one saturable transport system [K(S) below 0.01 mM; v(max) 1.1-1.2 mmol (g dry weight)(-1)h(-1)] plus a first order process (permeability P=1.2x10(-7)cm s(-1)). Ammonium-limited grown mycelium showed only one saturable transport system [K(S) 0.3-0.7 mM; v(max) 0.5-0.8 mmol (g dry weight)(-1)h(-1)]. During exponential growth at high glucose concentration (300-400 mM) a first order process was found with a P value of 5.6-9.3x10(-7)cm s(-1). After ammonium exhaustion a second first order phase showed a lower P value (6.1-9.3x10(-8)cm s(-1)). A similar change in permeability was also found after a re-evaluation of published data for Gibberella fujikuroi, Aspergillus niger, Aspergillus awamori and Saccharomycopsis lipolytica. For the first order processes simple diffusion was ruled out as a mechanism for glucose uptake. Glucose uptake by P. ochrochloron was controlled more strongly by metabolism than by transport and was not rate limiting for overflow metabolism.

Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography.

Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD < or = 0.7 ng on-column), repeatable (sigma(rel) < or = 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation < or = 9.4%, interday variation < or = 6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 degrees C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined.