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Exopolysaccharide (EPS) production by Ganoderma lucidum BCCM 31549 immobilised on polyurethane foam (PUF) cubes exhibited high ability for continuous EPS production in a repeated-batch fermentation (RBF). RBF by immobilised mycelium was optimised for broth replacement time point and broth replacement ratio. Interestingly, seven batches could be fermented consecutively in the shake flask for 55 days using 80% broth ratio and at day 13 time point, where this smart immobilised approach was deployed. Fermentation time was reduced from 13 days to 6 days, and the medium usage was also reduced due to the broth replacement volume procedure. Critically, immobilised G. lucidum mycelium grown on PUF cubes could be reused at least for seven successive cycles without any loss of EPS efficiencies with the PUF cubes retained the original structure, pointing out to be an ideal immobilisation matrix. The EPS productivity in immobilised culture at 0.045 g/L day −1 was higher than the value in a freely suspended culture (0.029 g/L day −1 ). The proposed immobilisation strategy proved that PUF as a physical carrier would provide an economical solution to enhance EPS production for extended liquid fermentation of mushrooms. [ABSTRACT FROM AUTHOR]

Exopolysaccharide (EPS) productivity of the slow-growing Ganoderma lucidum BCCM 31549 mushroom was enhanced in repeated batch fermentation (RBF) process by reducing the fermentation time. The results showed that by using 2.5-L STR unbaffled bioreactor, the fermentation time could be reduced from 10 days per batch to 5 days, due to lag-phase elimination by adopting a RBF strategy. RBF approach was also shown to achieve a semi-continuous mode of production by eliminating the inoculation time between each cycle. The fermentation period was shortened from 50 to 25 days in five consecutive cycles of RBF, with the productivity of EPS increased from 0.81 g/L day −1 to 1.32 g/L day −1 by using N-limiting medium. The presence of clamp connections in RBF ensures the reproducibility of this strategy. The proposed strategy proved to provide easy operation and thus can be adapted for EPS production. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Due to imprecise clinical staging, the finding of extravesical and node-positive disease at the time of radical cystectomy (RC) for patients with clinically localized bladder cancer is not uncommon. Circulating tumor cells (CTCs) have been shown to be present in the peripheral blood of patients with metastatic urothelial carcinoma. The object of this study was to evaluate the ability of CTCs to predict extravesical disease in bladder cancer patients prior to RC. Materials and methods: Peripheral blood samples from 43 patients with bladder cancer were evaluated using the CellSearch (Veridex, LLC, Raritan, NJ) CTC assay prior to RC. The sensitivity, specificity, and positive predictive value (PPV) of CTC status in predicting extravesical disease was calculated. Receiver operating characteristic (ROC) curves were generated to quantify the ability of CTCs to predict extravesical and node-positive disease. Results: CTCs were detected in 9 (21%) patients prior to RC. The sensitivity, specificity, and PPV of CTC status in predicting extravesical disease were 27%, 88% and 78%, respectively. The accuracy of CTC status in predicting extravesical (≥pT3 or node-positive) disease for the entire cohort was 0.576. In a model incorporating preoperative hydronephrosis, CTC status did not improve the predictive accuracy for extravesical disease (0.576 vs. 0.585, P = 0.915). Conclusion: CTCs were detected in low numbers in a small percentage (21%) of patients prior to undergoing RC at our institution. CTC status was not a robust predictor of extravesical or node-positive disease in this cohort. CTC status is not likely to be a clinically useful parameter for directing therapeutic decisions in patients with ≤cT2 bladder cancer. [Copyright &y& Elsevier]

Abstract: In the present study, we used a recombinant filamentous fungus strain, Aspergillus niger B1-D, as a model system, and investigated the antioxidant defences in this organism. Our findings indicate that pretreatment with low concentrations of H2O2 completely prevents killing by this oxidant at high concentrations. It shows that A. niger adapts to exposure to H2O2 by reducing growth and inducing a number of antioxidant enzyme activities, including superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, of which the induction of catalase is the most pronounced. Moreover the decline of these antioxidant enzymes activities after H2O2 detoxification, coincides with recommencement of growth. Results from monitoring the extracellular H2O2 concentration clearly indicate a very rapid detoxification rate for H2O2 in adapted A. niger cultures. A mathematical model predicts only very low concentrations of intracellular H2O2 accumulating in such cultures. Our results also show that glutathione plays a role in the oxidative defence against H2O2 in A. niger. On addition of H2O2, the intracellular pool of glutathione increases while the redox state of glutathione becomes more oxidized. [Copyright &y& Elsevier]

Abstract: Dissolved carbon dioxide is one variable that changes between scales of bioreactor and may thus have a potential influence on the process as scale alters, and especially so in filamentous fungal cultures. The effects of dCO2 on growth and penicillin production by an industrial strain of Penicillium chrysogenum were examined systematically using a steam sterilisable dCO2 probe and a computerized image analysis system. Although low levels of CO2 stimulated growth (increased biomass, lower mean hyphal growth unit (M.H.G.U.) in lag phase) at the expense of antibiotic synthesis, higher levels led to elevated M.H.G.U.''s in the lag phase and were associated with increased clumping and severely reduced penicillin titre. The adverse effects of dCO2 in these cultures were largely mediated by inhibition of growth and substrate uptake in the lag phase, causing morphological changes which might also contribute to reduced penicillin levels. [Copyright &y& Elsevier]

Abstract: Gluconobacter suboxydans was used in the biotransformation of d-sorbitol to l-sorbose, a key step in the industrial manufacture of Vitamin C (ascorbic acid). However, despite evidence of the existence of a range of by-products in the bioprocess, relatively little scientific investigation has focused on the factors influencing by-product formation in this complex bioprocess. The aim of the present study was therefore to investigate systematically the extent of by-product formation in this economically important biotransformation process using batch and continuous cultivation methods and up to date analytical techniques and, thus, to arrive at a realistic assessment of the yield of sorbose via this route. The results presented here show that exogenous by-product addition to shake flask and batch cultures detrimentally affects sorbose production and yield, but can have some positive effects in continuous culture. Some inhibition of fructose accumulation by mannitol-1-phosphate addition was noted in shake flask cultures. [Copyright &y& Elsevier]

Inflammatory myofibroblastic tumor (IMT) is a tumor of unknown neoplastic potential that rarely arises in the genitourinary tract. We present the case of an otherwise healthy 16-year old boy with gross hematuria who underwent cystoscopy and transurethral resection of the bladder tumor without complication. Biopsy and immunohistochemistry were consistent with anaplastic lymphoma kinase-positive IMT. In addition to presenting this case of IMT, we review presentations, management and prognosis of bladder IMT. [ABSTRACT FROM AUTHOR]

Objectives: To assess the utility of genomic testing in risk-stratifying Black patients with low and intermediate risk prostate cancer.Methods: We retrospectively identified 63 Black men deemed eligible for active surveillance based on National Comprehensive Cancer Network (NCCN) guidelines, who underwent OncotypeDx Genomic Prostate Score testing between April 2016 and July 2020. Nonparametric statistical testing was used to compare relevant features between patients reclassified to a higher NCCN risk after genomic testing and those who were not reclassified.Results: The median age was 66 years and median pre-biopsy PSA was 7.3. Initial risk classifications were: very low risk: 7 (11.1%), low risk: 24(38.1%), favorable intermediate risk: 31(49.2%), and unfavorable intermediate risk: 1 (1.6%). Overall, NCCN risk classifications after Genomic Prostate Score testing were significantly higher than initial classifications (P=.003, Wilcoxon signed-rank). Among patients with discordant risk designations, 28(28/40, 70%) were reclassified to a higher NCCN risk after genomic testing. A pre-biopsy prostate specific antigen of greater than 10 did not have significantly higher odds of HBR (OR:2.16 [95% CI: 0.64,7.59, P=.2). Of favorable intermediate risk patients, 20(64.5%) were reclassified to a higher NCCN risk. Ultimately, 18 patients underwent definitive treatment.Conclusions: Incorporation of genomic testing in risk stratifying Black men with low and intermediate-risk prostate cancer resulted in overall higher NCCN risk classifications. Our findings suggest a role for increased utilization of genomic testing in refining risk-stratification within this patient population. These tests may better inform treatment decisions on an individualized basis. [ABSTRACT FROM AUTHOR]

Abstract: Despite the importance of filamentous fungi in the biotechnology industry, little is known about their metabolism under the stressful conditions experienced in typical production fermenters. In the present study, oxygen enrichment was used to recreate an industrial batch process, and the effects of the increasing dissolved oxygen tension were studied as regards the cellular metabolism. It was found that elevated dissolved oxygen tension led to an oxidatively stressful environment, as detailed by rapid initial increases in reactive oxygen species (ROS) concentrations and antioxidant enzyme activities. Intracellular protein concentrations also decreased in oxygenated cultures; this appeared to be concomitant with a decrease in the adenosine-5′-triphosphate (ATP) pool in these cultures. Oxygenated cultures showed early senescence and death compared to aerated control cultures. Despite earlier studies proposing various mechanisms for such findings in fungal cultures subjected to oxidative stress, these findings can best be explained by the fact that in such cultures the activity of alternative NADH dehydrogenases was significantly increased, which served to maintain lower ROS concentrations throughout the duration of the process but in doing so also reduced the ability of the organism to create a proton motive force by which to drive ATP synthesis. The findings of the present study help further our understanding of the central roles of these highly conserved enzymes within fungal metabolism under oxidative stress. [Copyright &y& Elsevier]

Abstract: Thermophilic aerobic digestion (TAD) of heteroxylan waste was implemented at waste load of 30gL−1 with mineral nitrogen supplementation to study effect of the process on waste degradation, protein accretion and quality. Digestions were carried out at 45 50, 55, 60 and 65°C using Bacillus stearothermophilus in a CSTR under batch conditions at 1.0vvm aeration rate, pH 7.0 for a maximum of 120h. Amylase and xylanase activities appeared rapidly in the digest, while basal protease activity appeared early in the digestion and increased towards end of the processes. Highest degradation of volatile suspended solid, hemicellulose and fibre occurred at 55°C while highest degradation of total suspended solid occurred at 60°C. Highest protein accretion (258.8%) and assimilation of mineral nitrogen and soluble protein occurred at 55°C. The % content of amino acids of digest crude protein increased relative to raw waste and with digestion temperature. Quality of digest protein was comparable to the FAO standard for feed use. TAD has potentials for use in the protein enrichment of waste. [Copyright &y& Elsevier]

Abstract: It is now accepted that heat treatment can lead to oxidative stress events in microorganisms, but there are few detailed studies on protein oxidation and consequent recycling/elimination of oxidatively damaged proteins following such heat treatment. In batch cultures of a filamentous fungus, Aspergillus niger B1-D, raising culture temperature from 25°C to 30°C and 35°C led to a general enhancement of intracellular metabolism, a higher specific growth rate, increased consumption of carbon and nitrogen sources, and raised intracellular ATP content in the exponential phase. By contrast, there was a transient accumulation of protein carbonyls, a widely used biomarker of protein oxidation, following such temperature increases, which could indicate that cellular antioxidant defences were being temporarily overwhelmed under these circumstances, despite the fact that enhanced activities in antioxidant enzyme activities have been reported in microorganisms during such heat treatment. Protein carbonyls can only be removed by proteolysis. The intracellular proteolytic activity in batch cultures of A. niger was found generally enhanced by temperature elevation, suggesting the role of proteolytic activity in protein quality control during heat treatment was dependant on the culture temperature. [Copyright &y& Elsevier]

Abstract: Filamentous fungi are industrially important organisms because of their ability to produce secondary metabolites, and as expression systems for heterologous proteins. Since secretion of secondary metabolites in filamentous fungi is associated with the older, more vacuolated parts of the hyphae culture morphology is a crucial reflection of their physiology in submerged cultures. Therefore, any factor affecting fungal morphology will in turn affect secondary metabolite production. In this study we examine and report the effects of elevated levels of the potent morphogen, carbon dioxide upon morphology and antibiotic production in an industrially important fungal system. The effects of elevated CO2 levels on cephalosporin C (CPC) production, metabolic performance and morphological changes in batch cultures of Acremonium chrysogenum (ATCC 48272) were investigated. High levels of dCO2 led to reduction of CPC production by up to 36%. Both metabolic activity and substrate uptake were severely inhibited by elevated CO2, especially at 15 and 20% CO2. Both micro and macromorphology of A. chrysogenum were profoundly changed in bioprocesses sparged with CO2 especially at high levels, and these changes were a contributory cause of the reduction in CPC production. [Copyright &y& Elsevier]

Abstract: Oxygen enrichment (sparging bioreactors with oxygen-enriched air) ameliorates oxygen limitation in batch cultures of Aspergillus niger B1-D, and although nutrient utilization rates for both the carbon source and the nitrogen source are higher in oxygen-enriched cultures, excess oxygen does not enhance specific growth rate, instead the “extra” nutrient consumption is associated with defensive measures. Commencement of oxygen enrichment in early exponential phase leads to a transient rise in proteins showing oxidative damage (carbonylation), and to induction of enhanced proteolytic activity, which points to the antioxidant defense being temporarily overwhelmed, and that A. niger adapts to this oxidative environment by enhancing intracellular proteolytic activity to degrade damaged proteins which might otherwise accumulate under these conditions. Also the energy status of A. niger, reflected by intracellular ATP content, is found to be altered upon the commencement of oxygen enrichment and then reaches much lower levels than in the control. The reasons underlying this are discussed in the context of what is known regarding alternative respiration in industrial fungi. [Copyright &y& Elsevier]

Abstract: Maca (Lepidium meyenii walp.), a biennial herbaceous plant of the family Brassicae, which is cultivated mainly in the central Andes of Peru, has been used as both a food and a traditional medicine in the region for over 2000 years. The subterranean parts of the plant have long been used as a staple foodstuff by indigenous peoples in the Andean region, but the plant is also valued for its medicinal role. As is usual with many traditional “folk” medicines, many claims have been made regarding the efficacy of maca in treating a wide range of illnesses and medical conditions. However, in the 20th century most scientific attention has been focused in the areas where the pharmacological actions of maca seem most strongly attested, these include, enhancement of sexual drive in humans, increasing overall vigour and energy levels, and increasing sexual fertility in humans and domestic livestock. Since the early days of the 20th century numerous scientific studies have been carried out into the basis of its pharmacological action in these areas. In this review, the composition and pharmacological function of maca are systematically discussed. Additionally, the current discussion surrounding its mode of action in the areas listed above is also presented. [Copyright &y& Elsevier]

Abstract: The effects of agitation and aeration upon synthesis and molecular weight of the biopolymer gellan were systematically investigated in batch fermenter cultures of the bacterium, Sphingomonas paucimobilis. High aeration rates and vigorous agitation enhanced growth of S. paucimobilis. Although gellan formation occurred mainly in parallel with cell growth, the increase in cells able to synthesise gellan did not always lead to high gellan production. For example, at very high agitation rates (1000rpm) growth was stimulated at the expense of biopolymer synthesis. Maximal gellan concentration was obtained at 500rpm agitation and either 1 or 2vvm aeration (12.3 and 12.4g/l gellan, respectively). An increase in aeration (from 1 to 2vvm) enhanced gellan synthesis only at low agitation rates (250rpm). However, high aeration or dissolved oxygen was not necessary for high gellan synthesis, in fact oxygen limitation always preceded the phase of maximum gellan production and probably enhanced polysaccharide biosynthesis. Some gellan was formed even after glucose exhaustion. This was attributed to the intracellular accumulation of polyhydroxyalkanoates, (such as polyxydroxybutyrate) which were found in S. paucimobilis cells indicating the existence of a carbon storage system, which may contribute to gellan biosynthesis under glucose-limiting conditions. The autolysis of the culture, which occurred at the late stages of the process, seemed to be triggered mainly by limitations in mass (nutrient) transfer, due to the highly viscous process fluid that gradually develops. Rheological measurements generally gave a very good near real time estimate of maximum biopolymer concentration offering the possibility of improved process control relative to time consuming gravimetric assay methods. While mechanical depolymerisation of gellan did not occur, high aeration rates (2vvm) led to production of gellan of low molecular weight (at either 250 or 500rpm). This effect of aeration rate upon gellan molecular weight is reported here for the first time, and is important for the properties and applications of gellan. Mechanisms which may have led to this are discussed, but control of molecular weight of the biopolymers is clearly an area needing further research. [Copyright &y& Elsevier]

Thermophilic aerobic digestion (TAD) of a model agricultural waste slurry was carried out in a continuously stirred tank reactor for a total of 156 h at 55 °C. The pH of the digestion was either unregulated or regulated at 6.0, 7.0, 8.0, 9.0 and 9.5 at 0.5 volume of air per volume of waste slurry per minute (vvm) aeration rate. The effect of aeration rate was studied at 0.1, 0.25, 0.5 and 1.0 vvm at pH 7.0. Development of thermophilic populations and amylase and cellulase enzyme activities in the digesting slurry were studied. Thermophiles developed rapidly to reach peak populations in 24 h or earlier. After 96 h, thermophiles were present mostly as spores. They were only slightly affected by aeration rate, but more by digestion pH. Amylase developed rapidly to reach peak activity before declining as rapidly. activity Cellulase was considerably affected by aeration rate but not digestion pH. Amylase profile closely reflected development of microbial activity, and so the progress of TAD. It may be useful as a control tool, either alone or in combination with other process data, for monitoring the TAD of complex waste slurries. [Copyright &y& Elsevier]

A recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme (HEWL), was investigated with regard to its susceptibility to oxidative stress. The culture response to oxidative stress, produced either by menadione (MD) or by H2O2, was characterised in terms of the intracellular activities of two key defensive enzymes, catalase (CAT) and superoxide dismutase (SOD), in the presence or absence of a range of vitamin supplements.Addition of either ascorbate (Vit C) or α-tocopherol (Vit E) resulted in different responses of A. niger cultures to exogenous H2O2 (1 mM). In the presence of Vit C the activity of SOD was increased by 50% compared to control cultures, whereas incorporation of Vit E appeared to have a “protective” effect against reactive oxygen species (ROS).Continuous addition of menadione (1.2 mmole l−1 h−1) to submerged cultures of A. niger in the bioreactor resulted in an increase of CAT and SOD activities during the exponential phase of growth, whereas enzyme levels remained almost constant during that phase in control cultures. Comparison of these results with the response to oxidative stress caused by addition of exogenous H2O2 and elevated dissolved oxygen tension (DOT) shows that these three stressors are quite distinct. [Copyright &y& Elsevier]

Radical cystectomy (RC) is associated with significant morbidity. Neuraxial analgesia is recommended by enhanced recovery after surgery guidelines, but largely supported by evidence extrapolated from colorectal surgery outcomes. We synthesized current evidence regarding short- and long-term outcomes associated with neuraxial analgesia versus patient controlled non-neuraxial analgesia following RC. PubMed, Embase, and Cochrane databases were searched for relevant studies published up to May 2020. Studies reporting complications, length of stay (LOS), pain score, opioid usage within 72 hours, overall survival, cancer-specific survival, and recurrence rate were included. Of 550 identified studies, 9 met criteria for inclusion. Four studies demonstrated a higher percentage of 90-day complications in the neuraxial analgesia cohort. Out of 6 studies reporting information regarding LOS, 4 demonstrated no improvement in LOS in the neuraxial cohort. A reduction in 72 hours post-RC opioid usage was observed in 2 out of 3 studies with available data. Information regarding post-RC pain scores were variable up to 3 days post-RC. One out of 2 studies with available data reported a significant association between neuraxial analgesia and an earlier time to recurrence. No significant associations were seen with respect to overall survival or cancer-specific survival. A majority of low-to-moderate quality evidence demonstrates neuraxial analgesia is associated with a higher rate of complications, variable information regarding pain control, no improvements in LOS, and no significant association with long-term oncological outcomes. Further research regarding the incorporation of nonopiate-based analgesic modalities into RC ERAS protocols is warranted. [ABSTRACT FROM AUTHOR]

Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in T m . The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability. [ABSTRACT FROM AUTHOR]

Abstract: The effects of oxygen availability and induction culture biomass upon production of an industrially important monoamine oxidase (MAO) were investigated in fed-batch cultures of a recombinant E. coli. For each induction cell biomass 2 different oxygenation methods were used, aeration and oxygen enriched air. Induction at higher biomass levels increased the culture demand for oxygen, leading to fermentative metabolism and accumulation of high levels of acetate in the aerated cultures. Paradoxically, despite an almost eight fold increase in acetate accumulation to levels widely reported to be highly detrimental to protein production, when induction wet cell weight (WCW) rose from 100% to 137.5%, MAO specific activity in these aerated processes showed a 3 fold increase. By contrast, for oxygenated cultures induced at WCW''s 100% and 137.5% specific activity levels were broadly similar, but fell rapidly after the maxima were reached. Induction at high biomass levels (WCW 175%) led to very low levels of specific MAO activity relative to induction at lower WCW''s in both aerated and oxygenated cultures. Oxygen enrichment of these cultures was a useful strategy for boosting specific growth rates, but did not have positive effects upon specific enzyme activity. Based upon our findings, consideration of the amino acid composition of MAO and previous studies on related enzymes, we propose that this effect is due to oxidative damage to the MAO enzyme itself during these highly aerobic processes. Thus, the optimal process for MAO production is aerated, not oxygenated, and induced at moderate cell density, and clearly represents a compromise between oxygen supply effects on specific growth rate/induction cell density, acetate accumulation, and high specific MAO activity. This work shows that the negative effects of oxygen previously reported in free enzyme preparations, are not limited to these acellular environments but are also discernible in the sheltered environment of the cytosol of E. coli cells. [Copyright &y& Elsevier]

Objectives: To assess the perioperative outcomes in patients >75 years old to better define the role of laparoscopic renal surgery in this expanding segment of the U.S. population. The outcomes of elderly patients undergoing laparoscopic renal procedures are less well defined than those of younger patients. Methods: We retrospectively reviewed the records of 115 patients >75 years old who had undergone laparoscopic renal surgery. The clinical and pathologic features and complication rates were analyzed. The patients were substratified according to the type of laparoscopic procedure performed and extreme age (>80 years). Results: The mean age of our 115 patients was 79.9 years (range 75-91). The median estimated blood loss was 200 mL (interquartile range 100-387). The transfusion rate was 9.6%. The median length of stay was 4 days (interquartile range 3-5). The overall complication rate for the entire cohort was 22.6% and was 13.7% in those >80 years. The number of patients who experienced a complication was 22 (19.1%). Patients with a Charlson comorbidity index >2 were more likely to experience a perioperative complication than were patients with a Charlson comorbidity index <1 (35% vs 16%; P = .034). Conclusions: Our data support the safety and feasibility of laparoscopic renal procedures in elderly patients. Furthermore, this safety extends to patients of advanced age (>80 years). The overall complication rates and other perioperative parameters appear to be comparable to those in published studies; however, patients with a Charlson comorbidity index >2 are at a greater risk of complications in the perioperative period. [Copyright &y& Elsevier]

Abstract: Oxidative stress events have been shown to be associated with reduced consumption of nutrients in yeasts, but there are very few studies in filamentous fungi. In the present study we investigated the impact of oxidative stress on glucose and ammonia utilization in batch cultures of Aspergillus niger B1-D. The addition of 1mm H2O2 significantly reduced both glucose and ammonia uptake rates in these cultures. Associated with the decreased nutrient uptake, the activity of glyceraldehyde-3-phosphate dehydrogenase was greatly reduced; conversely, the activity of glucose-6-phosphate dehydrogenase remained unchanged. During the period of reduced nutrient uptake, the intracellular ATP and NADPH levels decreased while the amount of trehalose increased. The activities of glutamine synthetase and glutamate dehydrogenase, two key enzymes of ammonia assimilation, remained unchanged in response to H2O2 up to 1mm, suggesting the decreased ammonia uptake rate noted under such conditions is not due to enzyme inactivation caused by oxidative stress, but may be due to an insufficient supply of ATP and NADPH, which are required for ammonia assimilation. [Copyright &y& Elsevier]

Abstract: The use of near infrared spectroscopy (NIRS) was investigated in the context of an efficient high cell density fed-batch industrial Pichia pastoris bioprocess for the production of a therapeutic mammalian protein. This process represented a considerable challenge from the viewpoint of using NIRS to model key analytes because it involved two carbon sources (glycerol and methanol) added at differing rates and times, used a chemically complex medium, and showed a change in liquid phase behaviour due to cell growth. Models for biomass, glycerol, methanol and product were constructed. Different methods of spectral collection and mathematical procedures were used relative to which analyte in the fermentation matrix was being modelled and the rationale behind the model building is clearly described. Regardless of the mode of spectral collection it was essential to consider the changes in modelled analyte concentration relative to changes in other spectral contributors (analytes). The study considerably extends the use of NIRS in fermentation processes to high cell density complex industrial production processes, and comments on how this further developments the technology towards routine in situ NIRS monitoring of bioprocesses. [Copyright &y& Elsevier]

The effects of addition of menadione (MD) to induce elevated intracellular superoxide levels (superoxide stress) and of elevated oxygen availability (oxygen stress) was investigated in batch and chemostat cultures of a recombinant Aspergillus niger (B1-D). Culture responses to the two stressors were clearly distinct in terms of the changes in recombinant protein production, overall protein synthesis, and morphology. MD addition resulted in a significant increase in intracellular O2⋅− levels, but gassing with O2-enriched air (∼25%) led to decreased intracellular O2⋅− levels. Under elevated O2 availability, the activity of the alternative respiratory pathway was enhanced, reducing endogenous O2⋅− generation, but the consequent decreased energy availability in such cultures is proposed to be the main reason leading to the decreased intracellular protein content and foreign protein secretion in batch cultures and chemostat cultures at low dilution rates (D<0.1 h−1). The negative effects of O2 enrichment on protein synthesis were less pronounced at high D due to the relative dominance of the main respiratory pathway at high growth rates. Under stressed cultivation conditions, the mean number of tips per clump correlated well with the secretion of recombinant protein. Morphological change with O2 enrichment was not directly caused by the increased O2⋅− generation as proposed in some previous studies, but possibly by the significantly altered culture metabolism. [Copyright &y& Elsevier]