In situ NMR systems

In situ NMR is becoming an established technology for applications in bioprocessing and metabolic engineering. The in situ NMR biosensor acts as a noninvasive pH, ion, and concentration meter, with 31P and 13C as the two main isotopes of study. A substantial data base now exists for phosphorus and carbon spectra of bacteria and yeast. In situ NMR can provide many of the state variables needed for modeling glycolytic pathway function. NMR micro-reactor technology has improved significantly in the last decade. Several designs for immobilized cell reactors have been tested, and in particular, considerable gains have been made in the feasibility of studying aerobic, chemostat cultures with in situ NMR. Acquisition of 31P spectra from cell suspensions of 3-5% v/v under controlled conditions can be made in 3-7 minute time resolution in several systems.     

Biotransformation of the potato stress metabolite,solavetivone, by cell suspension cultures of two solanaceous and three non-solanaceous species

Cell suspension cultures of several plant species which were exogenously supplied solavetivone accumulated four hydroxylated derivatives. Only solanaceous cell cultures converted exogenously supplied solavetivone to sesquiterpenoid compounds identified with the plant response to biotic stress. Failure of potato cell cultures to make these compounds under biotic stress may be linked to their inability to produce adequate levels of solavetivone.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - TMV Tobacco Mosaic Virus - NAA 1-naphthaleneacetic acid - TLC thin layer chromatography - GC gas chromatography - GC/MS gas chromotography/mass spectrometry - NMR nuclear magnetic resonance     

Cultivator for NMR studies of suspended cell cultures

When nuclear magnetic resonance (NMR) spectroscopy is employed for physiological experiments with suspended cells, providing for adequate nutrient and oxygen delivery is particularly important, because the inherent insensitivity of NMR requires that concentrated cell suspensions be used. In addition, it is desirable to be able to manipulate the growth rate of cells during a NMR experiment. To address these concerns, a continuous cell cultivator that provides convective oxygen and nutrient transport has been constructed for NMR experiments. The NMR detector coil is located within the cultivator volume. The location is advantageous because the rapid exchange of cells in and out of the coil leads to a small apparent spin lattice relaxation time, thus allowing for rapid pulsing and fast signal averaging. In this article we present the physical principles on which the cultivator's design is based. (31)P spectra showing the response of continuously cultivated Saccharomyces cerevisiae cultures to a phosphate bolus and growth rate shift are then given. (c) 1992 John Wiley & Sons, Inc.     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.     

Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.)

Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0°C/min). A cooling rate of 0.5°C/min provided a satisfactory recovery rate (30%) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of 1°C/min. Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soil.Abbreviations DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) - MMS modified MS - NAA -naphthaleneacetic acid     

Direct observation of glycogenesis and glucagon-stimulated glycogenolysis in the rat liver in vivo by high-field carbon-13 surface coil NMR

High-field 13C surface coil nuclear magnetic resonance has been employed to investigate glucose and glycogen metabolism in rat liver in vivo. Natural abundance and isotopically enriched proton-decoupled 13C NMR experiments were conducted at 90.56 MHz on a standard commercial spectrometer utilizing a laboratory-built high-sensitivity double-resonance coaxial coil probe. At variance with a previous preliminary report, natural abundance spectra of the liver in vivo from a rat fed ad libitum reveal resonances of substantial intensity from hepatic glycogen with approximately 10 min of signal averaging. The response of hepatic glycogen levels to an intravenous injection of the hormone glucagon was continuously monitored through the glycogen C-1 carbon resonance intensity; this revealed an average 60% depletion of hepatic glycogen stores in vivo within approximately 1 h. In a complementary study utilizing fasted rats, 100 mg of D-[1-13C]glucose (90% enriched) was administered via a peripheral vein injection and continuously monitored by 13C NMR with 3-min time resolution as it was incorporated into hepatic glycogen. The C-1 carbon resonances of hepatic glucose and glycogen are well-resolved in vivo enabling the time course for the relative change in concentration for both metabolites to be established simultaneously. The 13C label incorporated into the glycogen pool reaches a steady-state level in approximately 40 min.     

In situ pulse respirometric methods for the estimation of kinetic and stoichiometric parameters in aerobic microbial communities

In situ pulse respirometry was applied in an activated sludge bubble column treating synthetic wastewater for the estimation of the (i) maximum specific oxygen consumption rate, (ii) substrate affinity constant, (iii) biomass growth yield, (iv) maintenance coefficient, and (v) specific endogenous respiration rate. Parameters obtained from respirometry were compared to those obtained by the chemostat method, based on substrate and biomass measurements, under several dilution rates. The low sensitivity of substrate measurement methods and the difficulties of sampling heterogeneous biomass suspension are critical issues limiting the applicability of the chemostat method. Additionally, the extensive time consuming nature of this method allows concluding that chemostat method presents several disadvantages in comparison with in situ pulse respirometric techniques. Parameters were obtained from respirograms by fitting ASM1 and ASM3 models, and from experiments performed by injecting pulses of increasing substrate concentration. The injection of pulses of increasing concentration was the most adequate method, with several advantages such as a simpler experimental data interpretation, and results with better confidence.Considering the assessment and comparison of the experimental and calculation methods presented, it is recommended that the estimation of kinetic and stoichiometric parameters in mixed aerobic cultures should preferentially be performed by using in situ respirometric techniques.     

The effect of thiosulphate and other inhibitors of autotrophic nitrification on heterotrophic nitrifiers

It has been found that heterotrophic nitrification by Thiosphaera pantotropha can be inhibited by thiosulphate in batch and chemostat cultures. Allythiourea and nitrapyrin, both classically considered to be specific inhibitors of autotrophic nitrification, inhibited nitrification by Tsa. pantotropha in short-term experiments with resting cell suspensions. Hydroxylamine inhibited ammonia oxidation in chemostat cultures, but was itself fully oxidized. Thus the total nitrification rate for the culture remained the same.Heterotrophic nitrification by another organism, a strain of Pseudomonas denitrificans has also been shown to be inhibited by thiosulphate in short term experiments and in the chemostat. During these experiments it became evident that this strain is able to grow mixotrophically (with acetate) and autotrophically in a chemostat with thiosulphate as the energy source.     

Development of an acetone/water fractional precipitation process for purification of paclitaxel from plant cell cultures

This study investigated the efficiency of acetone/water fractional precipitation for the purification of paclitaxel from plant cell cultures. Adding distilled water at room temperature into an acetone solution of dissolved crude extract until the acetone/water ratio became 40:60, 30:70, 20:80, and 10:90 (v/v) and stirring the mixture for 10 min at room temperature resulted in paclitaxel yields of 54.3, 89.1, 95.5, and 97.6%, respectively. With an acetone/water ratio of 40:60, v/v, a high yield of paclitaxel (84.8 ~ 86.0%) was produced by an additional 2 h storage at a low temperature (4oC) without additional mixing, or at room temperature with additional mixing. In contrast, preparing the 40:60 (v/v) acetone/water mixture at a low temperature (4oC) and mixing for 10 min at a low temperature, a similar high yield (~ 87.9%) of paclitaxel was obtained immediately. Thus, increasing the proportion of distilled water, or decreasing the temperature of the added water were confirmed as important for obtaining high yields of paclitaxel by acetone/water fractional precipitation.     

Microaerophilic growth of Wolinella recta ATCC 33238

Abstract The influence of oxygen on growth and fumarate-dependent respiration of Wolinella recta ATCC 33238 was studied in continuous culture. Steady states were obtained with formate-limited cultures grown at a specific growth rate of 0.1 h⁻¹ with different levels of oxygenation. The extent of aeration was regulated by means of a redox control system permitting reproducible cultivation at oxygen levels below the detection limit of conventional lead-silver probes. The ratio of succinate produced to that of formate consumed (Suc/For) decreased from 0.99 in strictly anaerobic cultures to 0.06–0.10 in aerated cultures. The growth yield did not change significantly with increasing redox readings: 4.9–5.2 g cell carbon/mol formate. The ability to use O₂ as the sole electron acceptor was demonstrated in a chemostat culture with formate as electron donor and succinate as carbon source. Washed cells from all chemostat cultures comsumed O₂ with formate as electron donor at a high rate (2.1–3.7 μmol/min per mg protein) and possessed b - and c -type cytochromes and CO-binding pigments. These results clearly indicated the microaerophilic nature of W. recta .     

The physiology and pathogenicity of Mycobacterium tuberculosis grown under controlled conditions in a defined medium

A chemically-defined culture medium was developed which supported batch growth of Mycobacterium tuberculosis, strain H37Rv, at a minimum doubling time of 14.7 h. This medium also facilitated chemostat culture of M. tuberculosis at a constant doubling time of 24 h. Chemostat growth was optimized at a dissolved oxygen tension of 20% (v/v) and 0.2% (v/v) Tween-80. Chemostat cultures were dispersed suspensions of single bacilli (1.5-3 microm long), or small aggregates, at a mean density of log10 8.3 cfu ml-1. A limited number of amino acids was utilized (alanine, asparagine, aspartate and serine were depleted by >50%; glycine, arginine, isoleucine, leucine and phenylalanine, by approximately 40%). Chemostat-grown cells were pathogenic in aerosol-infected guinea pigs, producing disseminated infection similar to that caused by plate-grown cells. Cells from chemostat culture were significantly more invasive for J774A.1 mouse macrophages than agar- or batch-grown cells. This study demonstrates the suitability of chemostat culture for the growth of pathogenic mycobacteria in a defined physiological state with potential applications for the controlled production of mycobacterial components for therapeutic and vaccine applications.     

Role of anaerobic spore-forming bacteria in the acidogenesis of glucose: Changes induced by discontinuous or low-rate feed supply

A mineral salts medium containing 1% (w/v) glucose providing carbon-limited growth conditions was subjected to anaerobic acidogenesis by mixed populations of bacteria in chemostat cultures. The formation of butyrate was shown to be dependent on the presence of saccharolytic anaerobic sporeformers in the acid-forming population. By the use of pasteurized activated sludge as an inoculum a culture was obtained consisting solely of anaerobic sporeformers that gave rise to the formation of butyrate, acetate, hydrogen and carbon dioxide as the main fermentation products. No formation of propionate could be detected. In this culture, the role of sporulation was investigated by applying periods of starvation and a single-step lowering of dilution rate (shift-down). In an experiment using a mineral salts medium supplemented with 1% (w/v) glucose and 0.5% (w/v) casein hydrolysate formation of refractile forespores as well as cell lysis could be demonstrated after 6 h starvation.In mixed cultures, initially inoculated with non-pasteurized activated sludge, a regular interruption of feed supply for 1 h per day resulted in selection of non-sporulatiog anaerobes. The fermentation pattern changed to a production of propionate and acetate, with a concomitant reduction of gas production. Similar results were obtained with shift-down in dilution rate.     

Kinetics and energetics of reduced sulfur oxidation by chemostat cultures of Thiobacillus ferrooxidans

Thiobacillus ferrooxidans was grown in chemostat cultures with thiosulfate and tetrathionate as the limiting substrates. The yields at steady state on both substrates at different dilution rates were calculated. In a few experiments the air supply was supplemented with 2% CO₂ (v/v). This resulted in a slightly increased yield.Cells from the chemostat cultures were used to study the kinetics of thiosulfate, tetrathionate, sulfite and sulfide oxidation. With all substrates mentioned the Ks values were in the micromolar range. The values for thiosulfate and tetrathionate were 2 orders of magnitude lower that those published previously.     

Carbon-13 nuclear magnetic resonance studies of myocardial glycogen metabolism in live guinea pigs

Myocardial glycogen metabolism was studied in live guinea pigs by 13C NMR at 20.19 MHz. Open-chest surgery was used to expose the heart, which was then positioned within a solenoidal radio frequency coil for NMR measurements. The time course of myocardial glycogen synthesis during 1-h infusions of 0.5 g of D-[1-13C]glucose (and insulin) into the jugular vein was investigated. The possible turnover of the 13C-labeled glycogen was also studied in vivo by following the labeled glucose infusion with a similar infusion of unlabeled glucose. The degree of 13C enrichment of the C-1 glycogen carbons during these infusions was measured in heart extracts by 1H NMR at 360 MHz. High-quality proton-decoupled 13C NMR spectra of the labeled C-1 carbons of myocardial glycogen in vivo were obtained in 1 min of data accumulation. This time resolution allowed measurement of the time course of glycogenolysis of the 13C-labeled glycogen during anoxia by 13C NMR in vivo. With the solenoidal coil used for 13C NMR, the spin-lattice relaxation time of the labeled C-1 carbons of myocardial glycogen could be measured in vivo. For a comparison, spin-lattice relaxation times of heart glycogen were measured in vitro at 90.55 MHz. Natural abundance 13C NMR studies of the quantitative hydrolysis of extracted heart glycogen in vitro at 90.55 MHz showed that virtually all the carbons in heart glycogen contribute to the 13C NMR signals. The same result was obtained in 13C NMR studies of glycogen hydrolysis in excised guinea pig heart.     

MG-63 human osteosarcoma cells grown in monolayer and as three-dimensional tumor spheroids present a different metabolic profile: a (1)H NMR study

High resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy was used to determine if the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as small (about 50-80 microm in diameter), three-dimensional tumor spheroids with no hypoxic center has different metabolic characteristics. Consequently, the (1)H NMR spectra were obtained from both types of cultures and then compared. The results indicate that the type of cellular spatial array determines specific changes in MG-63 cells. In particular, small but significant differences in lactate and alanine indicating a perturbation in energy metabolism were observed in the two cell models. In addition, although variations in CH(2) and CH(3) groups were also seen, it is not possible at this time to establish if lipid metabolism is truly different in cells and spheroids.     

The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

Azotobacter beijerinckii was grown in ammonia-free glucose-mineral salts media in batch culture and in chemostat cultures limited by the supply of glucose, oxygen or molecular nitrogen. In batch culture poly-beta-hydroxybutyrate was formed towards the end of exponential growth and accumulated to about 74% of the cell dry weight. In chemostat cultures little poly-beta-hydroxybutyrate accumulated in organisms that were nitrogen-limited, but when oxygen limited a much increased yield of cells per mol of glucose was observed, and the organisms contained up to 50% of their dry weight of poly-beta-hydroxybutyrate. In carbon-limited cultures (D, the dilution rate,=0.035-0.240h(-1)), the growth yield ranged from 13.1 to 19.8g/mol of glucose and the poly-beta-hydroxybutyrate content did not exceed 3.0% of the dry weight. In oxygen-limited cultures (D=0.049-0.252h(-1)) the growth yield ranged from 48.4 to 70.1g/mol of glucose and the poly-beta-hydroxybutyrate content was between 19.6 and 44.6% of dry weight. In nitrogen-limited cultures (D=0.053-0.255h(-1)) the growth yield ranged from 7.45 to 19.9g/mol of glucose and the poly-beta-hydroxybutyrate content was less than 1.5% of dry weight. The sudden imposition of oxygen limitation on a nitrogen-limited chemostat culture produced a rapid increase in poly-beta-hydroxybutyrate content and cell yield. Determinations on chemostat cultures revealed that during oxygen-limited steady states (D=0.1h(-1)) the oxygen uptake decreased to 100mul h(-1) per mg dry wt. compared with 675 for a glucose-limited culture (D=0.1h(-1)). Nitrogen-limited cultures had CO(2) production values in situ ranging from 660 to 1055mul h(-1) per mg dry wt. at growth rates of 0.053-0.234h(-1) and carbon-limited cultures exhibited a variation of CO(2) production between 185 and 1328mul h(-1) per mg dry wt. at growth rates between 0.035 and 0.240h(-1). These findings are discussed in relation to poly-beta-hydroxybutyrate formation, growth efficiency and growth yield during growth on glucose. We suggest that poly-beta-hydroxybutyrate is produced in response to oxygen limitation and represents not only a store of carbon and energy but also an electron sink into which excess of reducing power can be channelled.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

Growth, copper-tolerant cells, and extracellular protein production in copper-stressed chemostat cultures of Vibrio alginolyticus.

The influence of elevated copper concentrations on cell numbers and extracellular protein production was investigated in chemostat cultures of Vibrio alginolyticus. High (20 microM) copper in the medium reservoir resulted in a dramatic drop in cell numbers which was overcome with time. The copper-stressed cultures established a new equilibrium cell concentration slightly (ca. 20%) lower than control cultures. Copper-stressed chemostat populations contained an increased number of copper-resistant cells, but these averaged only 26% of the copper-adapted population. Previously copper-stressed populations exhibited resistance to a second challenge with copper. Proteins with properties identical to those of copper-induced, copper-binding proteins (CuBPs) observed in batch cultures of V. alginolyticus were observed in the supernatants of copper-stressed chemostat cultures and not in controls. CuBPs from batch and chemostat cultures were identical in terms of their induction by copper, molecular weight, and retention volumes on both immobilized copper ion-affinity chromatography and reverse-phase high-performance liquid chromatography columns. The concentration of CuBP in the chemostat was dependent on copper concentration in the medium reservoir. Either one or two forms of CuBP were observed in various analyses from both batch and chemostat cultures. Gel-to-gel variability was implicated as a factor determining whether one or two forms were resolved in a given analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the anti-cancer drug adriamycin on the energy metabolism of rat heart as measured by in vivo 31P-NMR and implications for adriamycin-induced cardiotoxicity

In vivo 31P-NMR was used to measure the effects of the anti-tumor drug adriamycin on the energy metabolism of rat heart. The exclusive acquisition of NMR signal from cardiac muscle was assured by positioning a solenoidal radio-frequency NMR coil around the heart. Appropriate control experiments verified that 31P-NMR spectra solely originated from this organ. Acute effects occurring shortly after adriamycin administration are expressed in 31P spectra as a dose-dependent decline in the cardiac levels of phosphocreatine, after which stabilization at a new steady-state level occurs. These acute effects of a single dose are complete in 30-60 min and no significant further changes take place within 150 min after drug introduction. Longer-term effects of single high doses and of multiple lower doses were measured up to a week after the initiation of treatment. It seemed that at a total dose of 20 mg/kg, drug-induced interference with cardiac energy metabolism was more pronounced than at the same dose in the acute phase. These 31P-NMR data demonstrate that adriamycin treatment is accompanied by a decrease of the cardiac phosphocreatine/ATP ratio which might be an expression of the well-established cardiotoxicity of the drug.     

The cyclone column culture vessel for batch and continuous,synchronous or asynchronous,culture of microorganisms

A simple inexpensive apparatus with a working volume of 10 liters of culture is described. Details of construction and procedures for operation of the cyclone column vessel are given. The vessel is self-contained, so that experimental parameters of temperature and aeration are individually controlled; homogeneous mixing and representative sampling of the culture, besides control of foam without need for antifoam, are obtained. The vessel may be used in single or multistage systems for aerobic or anaerobic cultivation of organisms in batch, chemostat, or phased cultures.