Monitoring cell concentration and activity by multiple excitation fluorometry.

Four key cellular metabolic fluorophores--tryptophan, pyridoxine, NAD(P)H, and riboflavin--were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems--bakers' yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cerevisiae RTY110/pRB58 growing on glucose. The measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. The results indicate that the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for the monitoring of cell concentration in bakers' yeast and C. utilis fermentations. Pyridoxine fluoresce is the best signal for the monitoring of cell concentration in the S. cerevisiae RTY110/pRB58 fermentation. In bakers' yeast fermentations the pyridoxine fluorescence signal can be used to monitor cellular activity. The NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used to control ethanol feeding in a fed-batch process.     

灵芝液态发酵胞内外多糖结构特征及其活性研究进展

灵芝是一味传统的中药材,具有很高的药用价值,多糖是其主要的活性成分之一,可从其子实体、孢子粉、发酵菌丝体和胞外液中获得。近年来,从灵芝菌丝体和胞外液中发现的多糖越来越得到研究者们的关注。本文从发酵培养基成分及发酵条件对灵芝胞内外多糖的影响、灵芝胞内外多糖的结构、灵芝胞内外多糖生物活性3个方面进行综述,为发酵来源灵芝多糖的开发利用提供科学理论依据。     

Use of fluorometry for monitoring and control of a bioreactor

A new fluorescent bioreactor monitoring probe-multiple excitation fluorometric system (MEFS)-has been developed. This probe was compared to the commercially available BioChem Technology FluroMeasure system (NADH probe). In this task the fluorescence behavior of three model fermentation systems, ethanol fermentation by Candida utilis, phenol fermentation by Pseudomonas putida, and glucose fermentation by Saccharomyces cerevisiae, were examined. The results indicated that the fluorescence intensity and behavior of various cellular fluorophors vary significantly between the different fermentation systems. Monitoring a fermentation process using only NAD(P)H fluorescence provided limited information. The NAD(P)H fluorescence was found not to be the best fluorescence signal for monitoring cell concentrations. The best way of monitoring a bioreactor by fluorometry may be to monitor several fluorophors in the whole culture broth simultaneously and to relate these fluorescence signals to various biological parameters.     

Plant cell culture monitoring using an in situ multiwavelength fluorescence probe

A multiwavelength fluorescence probe is proposed for in situ monitoring of Eschscholtzia californica and Catharanthus roseus plant cell cultures. The potential of the probe as a tool for real-time estimation of biomass and production in secondary metabolites has been studied. The probe excitation range is 270-550 nm and the emission range is 310-590 nm, with a step of 20 nm for both excitation and emission filters. Many endogenous fluorophores such as NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine and tryptophan, and fluorescent secondary metabolites were analyzed simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an adequate signal for estimating cells activity. Riboflavins fluorescence signal (450/530 nm) followed C. roseus cell concentration both for the growth phase and after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered with NAD(P)H signal during the production phase. For C. roseus, tryptophan, tryptamine, ajmalicine and serpentine were monitored by the probe. For E. californica, fluorescence from alkaloids overlapped with riboflavins preventing from using the probe to follow cell growth but global alkaloids production could be followed using the probe.     

Fluorometric behavior of a phenol fermentation

Summary The fluorescence of a batch culture ofPseudomonas putida grown on phenol was investigated. A linear relationship was found between cell concentration and culture fluorescence during in the early exponential growth phase. Accumulation of a metabolite in the culture broth affects fluorescence by NADH consumption and inner filter effects. The fluorescence is also affected by changes in the metabolic activity of the cells. These effects during the course of the fermentation are discussed.     

In situ multi-wavelength fluorescence spectroscopy as effective tool to simultaneously monitor spore germination, metabolic activity and quantitative protein production in recombinant Aspergillus niger fed-batch cultures

The production of a mutant green fluorescent protein (S65TGFP), controlled by the maltose inducible glucoamylase promoter, was followed in situ in fed-batch cultures of recombinant Aspergillus niger using multi-wavelength fluorescence spectroscopy. Disturbance of quantitative product analysis by interfering fluorescence signals was resolved by using a set of defined combinations of excitation and emission wavelengths (lambda(ex)/lambda(em)). This technique resulted in excellent linearity between on-line signal and off-line determined S65TGFP concentrations. Spore germination was detectable in situ by monitoring the back scattered light intensity. Moreover, flavin-like fluorophores were identified as the dominating fungal host fluorophores. The time-dependent intensity of this fluorophore, potentially fungal flavin-containing oxidoreductase(s), did not correlate with the biomass concentration but correlated well with the fungal metabolic activity (e.g. respiratory activity). Other fluorophores commonly found in microbial cultures such NADH, pyridoxine and the aromatic amino acids, tryptophan, phenylalanine and tyrosine did not contribute significantly to the culture fluorescence of A. niger. Thus, multi-wavelength fluorescence spectroscopy has proven to be an effective tool for simultaneous on-line monitoring of the most relevant process variables in fungal cultures, e.g. spore germination, metabolic activity, and quantitative product formation.     

Perstraction of intracellular pigments by submerged cultivation of <Emphasis Type="Italic">Monascus</Emphasis> in nonionic surfactant micelle aqueous solution

“Milking processing” describes the cultivation of microalgae in a water-organic solvent two-phase system that consists of simultaneous fermentation and secretion of intracellular product. It is usually limited by the conflict between the biocompatibility of the organic solvent to the microorganisms and the ability of the organic solvent to secret intracellular product into its extracellular broth. In the present work, submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle aqueous solution for pigment production is exploited, in which the fungus Monascus remains actively growing. Permeabilization of intracellular pigments across the cell membrane and extraction of the pigments to the nonionic surfactant micelles of its fermentation broth occur simultaneously. “Milking” the intracellular pigments in the submerged cultivation of Monascus is a perstraction process. The perstractive fermentation of intracellular pigments has the advantage of submerged cultivation by secretion of the intracellular pigments to its extracellular broth and the benefit of extractive microbial fermentation by solubilizing the pigments into nonionic surfactant micelles. It is shown as the marked increase of the extracellular pigment concentration by the submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle solution.     

O2 delivery and redox state are determinants of compartment-specific reactive O2 species in myocardial reperfusion

Reperfusion of the ischemic myocardium leads to a burst of reactive O(2) species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early O(2) delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 min and then reperfused with either 95% or 20% O(2)-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial O(2) delivery during the first 5 min of reperfusion was 84.7 +/- 4.2 ml O(2)/min with 20% O(2)-saturated buffer and 354.4 +/- 22.8 ml O(2)/min with 95% O(2) (n = 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O(2) compared with 20% O(2). However, the resorufin signal (extracellular ROS) was significantly increased with 20% O(2) compared with 95% O(2) during reperfusion. Perfusion of hearts with 20 mM glucose reduced the (.)NADH during ischemia (P < 0.001) and the (.)ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial O(2) delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O(2) delivery promotes intracellular ROS and low O(2) delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor for reperfusion ROS production.     

Enabler for process analytical technology implementation in Pichia pastoris fermentation: Fluorescence‐based soft sensors for rapid quantitation of product titer

Rapid quantitation of product titer is a critical input for control of any bioprocess. This measurement, however, is marred by the myriad components that are present in the fermentation broth, often requiring extensive sample pretreatment before analysis. Spectroscopy techniques such as fluorescence spectroscopy are widely recognized as potential monitoring tools. Here, we investigate the possibility of using fluorescence of the culture supernatant as a potential at‐line monitoring tool to measure the concentration of a recombinant therapeutic protein expressed in a Pichia pastoris fed‐batch fermentation. We propose an integrated method wherein both the target protein and total protein concentrations are predicted using intrinsic riboflavin fluorescence and extrinsic fluorescence, respectively. The root mean square error for estimating the concentrations of the target protein (using riboflavin fluorescence) and total protein (using extrinsic fluorescence) have been estimated to be <0.1 and <0.2, respectively. The proposed approach has been validated for two different biotherapeutic products, human serum albumin and granulocyte colony stimulating factor, that were expressed using Mut⁺ and Mut^s strains of P. pastoris, respectively. The proposed approach is rapid (1 min analysis time, 10 min total with at line sampling) and thus could be a significant enabler for process analytical technology implementation in Pichia fermentation.     

Extra Nutritional Requirements of Artificially Aposymbiotic Crithidia deanei*

SYNOPSIS. Chloramphenicol cured Crithidia deanei of its endosymbiote. The derived aposymbiotic strain had additional growth requirements: purin (as adenine), heme, arginine, histidine, isoleucine, leucine, phenylalanine, threonine, tryptophan, valine, pyridoxine, riboflavin, and pantothenate and liver infusion (replaceable by high nicotinamide).     

Study on the effect of thiamine on the metabolism of yeast by intrinsic fluorescence.

Thiamine in living human bodies exists mainly as diphosphate, which works as a co-enzyme of the sugar metabolism system (active vitamin B1). Thiamine deficiency brings many clinically significant problems, such as dysphoria, quadriplegia and dyspepsia. Intrinsic fluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex systems such as biological cells and tissues. Cellular fluorescence provides a sensitive index of the functional state of a living cell (1). Different amounts of thiamine were added to culture medium and the fluorescence of tryptophan and NADH from yeast was determined. When the thiamine concentration was greater than 0-0.16 microg/mL, the intensity of tryptophan fluorescence increased linearly, whereas the NADH fluorescence decreased. When the thiamine concentration was above 0.24 microg/mL, the fluorescence of tryptophan and NADH was almost unchanged. We concluded that low thiamine concentration in culture medium had a large effect on the growth of Saccharomyces cerevisiae and possible reasons are discussed.     

Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells

Monitoring fluorescence properties of endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in normal and cancerous cells provide substantial information noninvasively on biochemical and biophysical aspects of metabolic dysfunction of cancerous cells. Time‐resolved spectral profiles and fluorescence lifetime images of NADH and FAD were obtained in human lung nonsmall carcinomas (H661 and A549) and normal lung cells (MRC‐5). Both fluorophores show the fast and slowly decaying emission components upon pulsed excitation, and fluorescence spectra of NADH and FAD show blue‐ and red‐shifts, respectively, during their decay. All identified lifetime components of NADH and FAD were found to be shorter in cancerous cells than in normal cells, no matter how they were measured under different extra‐cellular conditions (cells suspended in cuvette and cells attached on glass substrate), indicating that the changes in metabolism likely altered the subcellular milieu and potentially also affected the interaction of NADH and FAD with enzymes to which these cofactors were bound. The intensity ratio of NADH and FAD of cancerous cells was also shown to be larger than that of normal cells.     

Solubilization capacity of nonionic surfactant micelles exhibiting strong influence on export of intracellular pigments in Monascus fermentation

In this study, perstractive fermentation of intracellular Monascus pigments in nonionic surfactant micelle aqueous solution had been studied. The permeability of cell membrane modified by nonionic surfactant might have influence on the rate of export of intracellular pigments into its extracellular broth while nearly no effect on the final extracellular pigment concentration. However, the solubilization of pigments in nonionic surfactant micelles strongly affected the final extracellular pigment concentration. The solubilization capacity of micelles depended on the kind of nonionic surfactant, the super‐molecule assembly structure of nonionic surfactant in an aqueous solution, and the nonionic surfactant concentration. Elimination of pigment degradation by export of intracellular Monascus pigments and solubilizing them into nonionic surfactant micelles was also confirmed experimentally. Thus, nonionic surfactant micelle aqueous solution is potential for replacement of organic solvent for perstractive fermentation of intracellular product.     

Autofluorescence of viable cultured mammalian cells.

The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.     

低强度超声波刺激对阿氏假囊酵母细胞次生代谢的影响

一定强度的超声波能够促进细胞的生长和代谢,细胞膜是抑制微生物次生代谢产物产量的主要原因。以阿氏假囊酵母(E．ashbyii)为实验材料,选择功率6W、频率24kHz的超声加载于摇瓶发酵过程中,检测了加载对菌丝体干重和核黄素分泌的变化情况。结果表明,一定强度的超声间断加载对E．ashbyii的生长有一定的促进作用,并使核黄素的开始分泌时间从96h缩短到60h;同时发现,发酵96h后加载,能有效提高核黄素的产量至0．346mg／L,非常显地高于对照组;发酵后的104～112h是超声处理的最佳时间段,为维持较高的产率,需每隔1．5h再次超声处理。     

不同碳源和生长因子条件下粒毛盘菌代谢产物初步研究

采用GC-MS法对一种粒毛盘菌(Lachnum sp.)在不同碳源、生长因子条件下发酵代谢产物的挥发性成分组成与差异进行分析。结果显示,不同碳源和生长因子条件下产生的代谢产物不同,主要包括有机酸、胺类、烷烃类、酯类、醇类、吡咯等物质。分别以20 g/L的葡萄糖、蔗糖、淀粉为碳源的发酵液中检测到的挥发性代谢产物为7、7、10种;添加1 mg/L的V_C、V_(B1)、甘氨酸、色氨酸作为不同生长因子的发酵液中检测到的挥发性代谢产物分别为6、7、7、12种。结果显示粒毛盘菌YM406发酵代谢产物具有丰富的多样性,并且在不同的培养条件下产生的代谢产物存在一定的差异。     

On-line fluorescence measurements in assessing culture metabolic activities

Summary NADH fluorescence aided by a stoichiometric metabolic pathway model and culture dynamics was used to elucidate the unobservable intracellular physiological state in two metabolically different phases during culture of Clostridium acetobutylicum. The validity of the theoretical model was examined over a range of culture pH regimes and initial sugar concentrations. The H₂/CO₂ gas concentration ratio was found to be an important process parameter. NADH fluorescence detection was compared with simultaneous enzymatic measurements. The specific fluorescence (fluorescence per biomass, F/X) provided a distinction between oxidative and reductive culture metabolism independent of the pH or substrate concentration changes. A good indicator of the type of culture activity proved to be the dF/dt parameter. The net fluorescence measurements correlated with butanol accumulation under all growth conditions suggesting the possible use of the fluorescence probe as a butanol probe in this fermentation.     

Observing protein synthesis, export, and tryptophan incorporation by front-surface fluorescence

Front-surface detection of emission from fluorophores in the presence and absence of light-scattering particles was contrasted to right-angle and wave-guide detection. We found that front-surface detection was the least prone to the reabsorption, inner-filtering, and scattering effects that can plague fluorescent measurements. Front-surface detection was thus used to assess the use of protein and ANS fluorescence as a means of monitoring events in bacterial fermentations. Protein fluorescence appeared to track well changes in optical density during balanced growth. However, during the lag associated with diauxic growth and after exposure to ampicillin, protein fluorescence became decoupled from cellular growth in a manner consistent with prior observations and the known effect of ampicillin on cells. ANS proved to be nontoxic and capable of reporting the occurrence of protein release from cells. The spectral shifts of tryptophan indicated that the incorporation of tryptophan into cellular protein can be monitored.     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.