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Physician burnout is linked to decreased job performance, increased medical errors, interpersonal conflicts and depression. Two recent multispecialty studies showed that urologists had the highest rate of burnout. However, these reports were limited by a low sample size of urologists (119). We aimed to establish the prevalence of urologist burnout and associated factors.

Prediction of soluble protein expression levels in Escherichia colibased on the nature of protein itself remains a challenge for bioprocess development (BD). This review will critically discuss the current efforts and achievements that employ computational approaches to develop prediction models for soluble protein expression in E. coli. The contrast between the remarkable progresses made on the predictive models achieved by bioinformatics and their relatively infrequent application in BD will be explained. The effects of process-relevant variables at four different levels on the expression of heterologous proteins, for example, gene, vector, host cell and cultivation process, and also a critical comparison of several established bioinformatics tools for predicting expression levels will be presented. The potential utility of this emergent technology to increase the efficiency of BD strategies and thereby to reduce the cost of establishing a process for soluble protein expression are critically examined.

Background: Although several cancer biomarkers are now in use, few have the necessary sensitivity and specificity to eliminate the need for invasive diagnostic procedures. With the expansion of proteomics, new technologies have been used to study various cancers, resulting in the discovery of several potential urine biomarkers. Urine is an ideal medium for the detection of biomarkers because of the non-invasive means of collecting samples and demonstrated shedding of cells, proteins, enzymes, nucleic acids and metabolic products into urine during various pathological processes. Objective: To review the contemporary literature regarding urinary protein markers of cancer. Methods: A PubMed search for ‘urinary protein biomarkers of cancer’ revealed 4679 scientific publications. The search was limited to studies published over the last 5 years and reviewed pertinent findings regarding biomarker evaluation and discovery. Results: Several urinary protein biomarkers have been described for urologic, gynecologic, gastrointestinal and neurologic tumors. Some have been selected for use in clinical practice, whereas others have been abandoned owing to inconclusive follow-up studies. Conclusion: Several potential urinary protein markers for cancer exist, yet multi-institutional, prospective trials are needed to validate results before implementation in clinical scenarios.

The magnitude and duration of the effects of botulinum toxin A on acetylcholine (ACh) and norepinephrine release from the bladder and urethra of rats were measured using a radiochemical method.

One of the key goals in bioprocess monitoring is to achieve real-time knowledge of conditions within the bioreactor, i.e., in-situ. Near-infrared spectroscopy (NIRS), with its ability to carry out multi-analyte quantification rapidly with little sample presentation, is potentially applicable in this role. In the present study, the application of NIRS to a complex, fed-batch industrial E. coli (RV308/PHKY531) process was investigated. This process undergoes a series of temperature changes and is vigorously agitated and aerated. These conditions can pose added challenges to in-situ NIRS. Using the measurement of a key analyte (biomass) as an illustration, the details of the relationship between the at-line and in-situ use of NIRS are considered from the viewpoint of both theory and practical application. This study shows that NIRS can be used both at-line and in-situ in order to achieve good predictive models for biomass. There are particular challenges imposed by in-situ operation (loss of wavelength regions and noise) which meant the need for signal optimisation studies. This showed that whilst the at-line modelling process may provide some useful information for the in-situ process, there were distinct differences. This study shows that the in-situ use of NIRS in a highly challenging matrix (similar to those encountered in current industrial practice) is possible, and thus extends previous works in the area. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 405–413, 2002.

Direct detection of intracellular superoxide anion radical (O2^·−) production is of critical importance for investigating the responses of filamentous fungi to oxidative stress in bioprocesses. The purpose of this study is to establish a reliable method to monitor the O2^·− production within pellets of Aspergillus niger. Addition of pure oxygen and the redox cycling agent paraquat to fungal pellet suspensions resulted in a considerable increase in lucigenin-derived chemiluminescence (LDCL). In the presence of exogenous superoxide dismutase (SOD), the LDCL of a disrupted cell solution was inhibited. In contrast, with addition of diethyldithiocarbamate and sodium azide, respectively, the inhibitors of Cu, Zn-SOD and Mn-SOD, an increased LDCL was observed. Further, as a probe, lucigenin can be absorbed and accumulated in fungal pellet within a few minutes. Various pretreatments of the bioreactor sample for the measurement of LDCL, were also investigated in the present study, and the use of intact pellets was adopted here rather than disrupting cells because the latter treatment led to difficulties in LDCL measurement. These results show that lucigenin may be used as a convenient chemiluminescent probe to monitor intracellular production of O2^·− in filamentous fungi, and thus to follow changes in the level of this stressor within fungi © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 204–211, 2001.

Access to real-time process information is desirable for consistent and efficient operation of bioprocesses. Near-infrared spectroscopy (NIRS) is known to have potential for providing real-time information on the quantitative levels of important bioprocess variables. However, given the fact that a typical NIR spectrum encompasses information regarding almost all the constituents of the sample matrix, there are few case studies that have investigated the spectral details for applications in bioprocess quality assessment or qualitative bioprocess monitoring. Such information would be invaluable in providing operator-level assistance on the progress of a bioprocess in industrial-scale productions. We investigated this aspect and report the results of our investigation. Near-infrared spectral information derived from scanning unprocessed culture fluid (broth) samples from a complex antibiotic production process was assessed for a data set that incorporated bioprocess variations. Principal component analysis was applied to the spectral data and the loadings and scores of the principal components studied. Changes in the spectral information that corresponded to variations in the bioprocess could be deciphered. Despite the complexity of the matrix, near-infrared spectra of the culture broth are shown to have valuable information that can be deconvoluted with the help of factor analysis techniques such as principal component analysis (PCA). Although complex to interpret, the loadings and score plots are shown to offer potential in process diagnosis that could be of value in the rapid assessment of process quality, and in data assessment prior to quantitative model development. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 376–388, 2001.

The robustness of models developed for the near-infrared spectroscopic prediction of mycelial biomass, total sugars, and ammonium in a submerged Penicillium chrysogenumbioprocess was assessed by rigorously challenging them with artificially introduced analyte and background matrix variations, so that analyte concentrations were varied in an invariant matrix and vice versa. The models were also challenged by using a data set from a process operated at a different scale from that used in the original model formulation. Simple univariate and bivariate linear regression models, and partial least-squares (PLS) models with as few factors as three and four, performed sufficiently well for predicting analyte concentrations and were robust with respect to the matrix variations tested. However, models based on relatively weaker absorptions, or those that were likely to be influenced by stronger absorbers present in the same matrix, were vulnerable to changes in the matrix. A change in the scale of operation affected models that would be influenced by biomass, possibly due to an influence of the morphology of the mycelial biomass. An analysis of the loading vectors of some PLS models revealed details that were useful in understanding the type of information modeled and the behavior of these models to the variations tested.

A recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme, was investigated with regard to its susceptibility to “oxidative stress” in submerged culture in bioreactor systems. The culture response to oxidative stress, produced either by addition of exogenous hydrogen peroxide or by high-dissolved oxygen tensions, was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). Batch cultures in the bioreactor were generally found to have maximum specific activities of CAT and SOD (Umg · protein⁻¹) in the stationary/early-decline phase. Continuous addition of H2O2 (16 mmole L⁻¹ h⁻¹), starting in the early exponential phase, induced CAT but did not increase SOD significantly. Gassing an early exponential-phase culture with O2 enriched (25 vol%) air resulted in increased activities of both SOD and CAT relative to control processes gassed continuously with air, while gassing the culture with 25 vol% O2 enriched air throughout the experiment, although inducing a higher base level of enzyme activities, did not increase the maximum SOD activity obtained relative to control processes gassed continuously with air. The profile of the specific activity of SOD (U mg CDW⁻¹) appeared to correlate with dissolved oxygen levels in processes where no H2O2 addition occurred. These findings indicate that it is unsound to use the term “oxidative stress” to encompass a stress response produced by addition of a chemical (H2O2) or by elevated dissolved oxygen levels because the response to each might be quite different. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 662–669, 2000.

Near‐infrared spectroscopy (NIRS) is known to have potential for cost‐effective monitoring of bioprocesses. Although this has been demonstrated in many instances and several models have been reported, information regarding the complexity of models required and their utility over extended periods of time is lacking. In the present study, the complexity of the models required for the NIRS prediction of substrate (oil) and product (tylosin) concentration in an industrial bioprocess that employs a physicochemically heterogeneous medium for antibiotic production was assessed. Measurements made by both the diffuse reflectance and transmittance modes were investigated. SEP values for the prediction of the analytes averaged 5% or less, for the successful models, when evaluated using an external validation set, 2 years after the initial model development exercise. Diffuse reflectance measurements showed poorer results, compared to transmittance measurements, especially for monitoring tylosin. In general, this investigation provides evidence to support the fact that models built for the prediction of analytes in a commercial bioprocess that employs a physicochemically complex production medium can be robust in performance over an extended period of time and that simple models based on fewer terms or latent variables can perform well, even in the context of matrices that are relatively complex. It also indicates that sample presentation is likely to be a critical factor in the successful application of NIRS in bioprocess monitoring, which merits further detailed investigation.

ABSTRACT: For decades microbial exopolysaccharides have been invaluable ingredients in the food industry, as well as having many attractive pharmaceutical and chemical applications. Gellan gum is a comparatively new gum elaborated by the Gram-negative bacterium Sphingomonas paucimobilis. Although its physico-chemical properties have been well characterized, the ecology and physiology of Sphingomonas, and the factors influencing the fermentation process for production of this gum have received much less attention. This review focuses on the metabolism and the enzymic activity of this bacterium, as well as the factors that influence gellan production, including process temperature, pH, stirring rate, oxygen transfer, and composition of the production medium. Potential strategies for improving the production process are discussed in the context of processes for the production of other microbial biopolymers, particularly exopolysaccharides. In addition, the importance and potential utility of gellan lyases in modification of gellan and in other applications is critically evaluated.

The response of steady state chemostat cultures of a recombinant Aspergillus niger (B1-D), secreting both a heterologous enzyme (Hen Egg White Lysozyme [HEWL]) and a native enzyme (Glucoamylase), to varying levels of O2 enrichment of the process gas was evaluated. Formation of both the native and the foreign enzyme increased with increasing O2 supply. Conversely, biomass levels and total extracellular protein levels were generally not increased under O2 enriched conditions. Two distinct micromorphologies were apparent in these cultures, one, typically seen under O2 limiting conditions (i. e. at 0 and 10% enrichment levels), tended to be represented by long, sparsely branched hyphal elements, with low percentages of “active” length (i. e. how much of the hypha is cytoplasm filled); whilst, a second micromorphology, typical of O2 enriched cultures at 30 and 50% O2 enrichment, was represented by shorter hyphal elements, with more branching and a higher % “active” length. At these higher O2 levels, formation of a yellow pigment occurred, and signs of culture autolysis were noted. At 50% enrichment, a “stranded” aggregate morphology was apparent, possibly as a response to a hyperoxidant state. Production of both the native enzyme and HEWL correlated well with a simple morphological measure (tip number) or, with % “active” length. It is proposed the morphological changes noted in the cultures were associated with the increased production of both HEWL and glucoamylase. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 416–424, 1999.

The influence of bioreactor operational conditions on the micromorphology of batch cultures of Aspergillus niger B1-D, containing a hen egg white lysozome (HEWL) marker protein, was examined using computerised image analysis. Significant differences in micromorphology were observed with increased stirrer speed, with shorter organisms with shorter hyphal elements occurring as agitation speed increased, even though mean tip numbers were similar. This may explain the observed increase in the total extracellular protein, since the ratio of synthetic (tip) to non-synthetic zones became increasingly favourable. HEWL concentrations fell above 500 rpm, probably due to the effects of DOT (dissolved oxygen tension) on the glucoamylase HEWL fusion. HEWL was susceptible to proteolytic degradation, by native proteases, during the autolytic phase. Such insights may indicate why a gene from one mould expressed in a close relative can give production levels equivalent to levels of native enzymes, while secretion of a gene from a ‘distant’ source, eg a higher eukaryotic gene, occurs at much lower levels. © 1999 Society of Chemical Industry

The near-infrared (NIR) spectrum of microbial biomass was studied for a range of microorganisms, with relevance to applicability in bioprocess monitoring and control. Three experiments were carried out to investigate the validity of measuring biomass using NIR spectroscopy (NIRS). (1) A comparative study of the NIR reflectance spectra of five representative microorganisms, of interest in a bioprocessing context, showed that the spectral signature of biomass, with respect to the wavelength regions, is essentially identical for all five microorganisms. The signature wavelengths are listed. (2) The spectral signature correlated well quantitatively with dry cell mass measurements, when the biomass of a filamentous microorganism was isolated from its matrix and analysed. The observation was clearer with reflectance measurements of dried biomass than with transmittance measurements of aqueous suspensions. (3) The biomass reflectance spectral signature remained the same at different culture ages of Penicillium chrysogenum, and showed quantitative correlation with dry cell mass measurements at the relevant wavelengths. This study therefore indicates those spectral regions which are likely to be the basis for quantitative modelling of microbial biomass in bioprocesses using NIRS, regardless of the microbial type and the culture age.

ABSTRACT: Monitoring is an important exercise in the operation of submerged bioprocesses and is a subject that has attracted intense research activity in recent years. In order to achieve optimal production or conversion, the factors that influence the performance of a bioprocess should be measured, preferably on-line, so that the process can be monitored in real-time and appropriate control or remedial action implemented. Such measurements can be achieved either in situor ex situ. Ideally, in situapproaches are desirable. Where such an option is difficult to implement, ex situapproaches with appropriate sample handling techniques can be employed. In cases where direct analytical information is not available, inferential approaches can be adopted. In addition, analytical information not yet accessible on-line can be obtained off-line. Computational techniques have enabled the compilation of measurement data before utilizing the information for process diagnosis. Current developments in the different measurement technologies are presented with a critical assessment of their potential application in industrial production or process development.

Cultures of filamentous fungi that secrete significant amounts of exopolysaccharides are among the most difficult of fermentation fluids, presenting difficulties in the areas of aeration, agitation, mixing, and control that may in turn impact the physiology of the microorganism in an undesirable manner. The fungus Sclerotium glucanicum, which produces a potentially useful exopolysaccharide known as scleroglucan, illustrates many such difficulties. This review discusses in detail the range of physiological studies on the producing microorganism itself, including those concerning formation of "undesirable" byproducts, principally oxalate, but also, under certain conditions, other TCA cycle acids. In addition, the bioreactor technology in use for production of this type of biopolymer is discussed in relation to the difficulties such fluid types present. The potential of pneumatically agitated reactors for such production is evaluated, and the lack of fundamental studies on such reactors and on the hydrodynamics and mixing behavior of such complex fluids is pointed out.

For numerical solution of the reaction-mass transfer equations for immobilised biocatalysts it may be better to start integration at the particle surface and proceed inwards: calculations are targetted on the region to which practically interesting changes are often confined (because concentrations are effectively zero in the interior); and during iterative solution wrong initial estimates may be rejected after detecting anomalies early in the integration.

The viscous nature of fermentation broths has a marked effect on many parameters and process conditions within the bioreactor, often resulting in changes in microbial behavior and product formation. Although fermentation processes that involve significant changes in broth rheology have attracted much research interest, significant problems still remain. Particular difficulties remain in relation to processes where the broth develops pronounced non-Newtonian characteristics.This article reviews some of the models developed to describe these fermentation fluids and their limitations; the bioreactor types employed and their mode of operation; the problems encountered because of the nature of the fluids, for example, monitoring and control; and the gradients found in such systems. To illustrate the above, four specific processes are described in detail - the production of filamentous biomass (SCP) and three extracellular microbial polysaccharides, xanthan, pullulan, and scleroglucan.

By use of near-infrared (NIR) spectroscopy, simultaneous, multiple-constituent estimation of important bioprocess parameters can be obtained in a time frame (<1 min assay) that was previously unattainable. Therefore, with NIR spectroscopy the opportunity exists to incorporate real-time chemical information into bioprocess monitoring or control strategies which will lead to significant bioprocess improvements. The NIR spectroscopic analysis of unmodified whole broth samples for acetate, ammonium, biomass, and glycerol is described for an industrial Escherichia colifed-batch fermentation bioprocess. For acetate and glycerol, suitable results were obtained from multiple linear least-squares regression (MLR) analysis. A more sophisticated partial least-squares (PLS) regression analysis was necessary to adequately model ammonium and biomass. The respective prediction errors (1σ) of 0.7 g/L, 1.4 g/L, 0.7 g/L, and 7 mmol/L for acetate, biomass, glycerol, and ammonium compare well with the error of the wet chemical reference methods used to derive the calibration algorithms.

The effect of a single pulse of ammonium sulphate or of citrate upon the progress and final outcome of a batch citric acid fermentation was studied. It was found that the optimum addition time for the supplemental N was in the range of 40 to 75 h. Final citric acid concentration achieved was significantly increased when the concentration of N source added was between 0.25 and 0.5 kg m−3. The mechanism of the observed stimulation seemed to be an indirect one. Addition of exogenous citric acid to the broth, led to an increase in citrate production by the culture. The optimum time for citric acid addition was around 90 h.