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The use of in-situ near infrared spectroscopy (NIRS) as a tool for monitoring four key analytes in a CHO-K1 animal cell culture was investigated. Previous work using on-line NIRS to monitor bioprocesses has involved its application ex-situ where the analyzer is physically outside the fermentor, or to microbial bioprocesses. This novel application of NIRS to monitor analytes within an animal cell culture using a steam sterilizable in-situ fiber optic probe is very important for furthering the use of NIRS within the bioprocessing industry. The method of calibration used to develop the models involved the use of large data sets so that all likely variation in stoichiometry was incorporated within the models. Successful models for glucose, lactate, glutamine, and ammonia were built with Standard Error of Predictions (SEP's) of 0.072 (g/L), 0.0144 (g/L), 0.308 (mM), and 0.036 (mM), respectively of the total concentration range. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 13–19, 2003. [ABSTRACT FROM AUTHOR]

In the present study the relationship between oxidative stress and elevated culture temperature was examined in an industrially relevant fungal culture, Aspergillus niger B1-D. For the first time, both the intracellular levels of the main stressor species (superoxide radical [O2.-]) and activities of cellular defensive enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxide [GPx]) were quantified at varying temperature (25, 30, 35, 40°C) to more fully characterize culture response in different growth phases. Elevated culture temperature led to increased O2.- levels in various culture phases. In the exponential phase this was due to an enhanced generation of O2.-, whereas in stationary phase a decreased dismutation rate may also have contributed. CAT activities generally increased with culture temperature, whereas GPx activity changed little as temperature rose, indicating that GPx played only a minor role in destroying H2O2 in this A. niger. The combination of elevated temperature (35°C) and increased O2 supply (50% enrichment) led to decreased levels of O2.- compared to the cultivation at 35°C gassed with air, probably due to enhanced activity of the alternative fungal respiratory pathway. Our findings indicate that while elevated cultivation temperature does clearly induce oxidative stress events, mechanistically, it does so by a rather more complex route than previous studies indicate. Elevated temperature caused a marked disparity in the activities of SOD and CAT, very distinct from the integrated increase in activity of these enzymes in response to oxidative stress. © 2003 Wiley Periodicals. Biotechnol Bioeng 83: 772–779, 2003. [ABSTRACT FROM AUTHOR]

Bioprocesses that employ mycelial microorganisms are commercially important. The application of optical techniques for the measurement of biomass in such processes is limited by the morphological heterogeneity exhibited by the mycelial microorganism employed. We investigated the influence of morphology on the near-infrared (NIR) spectra of the biomass of Streptomyces fradiae, a filamentous microorganism, by studying the spectra of mycelial suspensions that were manipulated to generate a range of morphological forms. Computerized image analysis was used to characterize the morphological forms. Principal component analysis was used to assess the spectral variations and study correlations to the manipulated mycelial morphology. Although morphology was found to influence the near infrared transmittance spectra of biomass, the influence was less pronounced than in the visible region, the spectral information at longer wavelengths (1600–2350 nm) showing greater stability to morphological variations. Long-wave NIR spectral information is therefore likely to be more useful in estimating biomass in mycelial bioprocesses. Furthermore, the NIR reflectance spectra of dried biomass were found to show correlations to the morphological variations introduced, suggesting that NIR spectra may be useful in obtaining morphology related information. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 715–724, 2003. [ABSTRACT FROM AUTHOR]

The physiological responses of chemostat cultures of the filamentous fungus, Aspergillus niger (B1-D) to simulated and actual oxidative stress, imposed respectively by addition of exogenous menadione (MD; a superoxide radical generating reagent) and gassing the culture with oxygen enriched air (25%, 50%, 75%, and 100% [v/v]), were examined. Changes in the levels of intracellular superoxide anions and defensive enzyme activities, such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), were monitored, together with glutathione and respiratory activity in both the dynamic phase and when a new steady state was established. Culture response to MD addition was distinct from that upon exposure to enriched oxygen conditions, in that MD caused elevated levels of intracellular protein, whereas oxygen enrichment caused reduced protein content, especially at low dilution rates. An unexpectedly low level of superoxide radical was found in oxygen-enriched steady-state cultures (50%) at a range of dilution rates, which was not caused by elevated SOD activity. Under these conditions, it was noted that the ratio of rotenone-insensitive/total respiration increased, suggesting increased activity of the alternative respiratory pathway. This may have had the effect of reducing the endogenous generation of superoxide radicals under oxygen rich conditions, but also may have reduced the ATP yield due to the non–proton-pumping nature of the alternative respiratory pathway. Thus, the negative culture effects noted in many studies at high oxygen levels may not simply be due to elevated endogenous superoxide generation, but could be in part due to the consequences of metabolic changes in the culture that seek to minimize superoxide generation. The dynamic culture response was characterized by rapid elevation of intracellular superoxide anions and associated protective enzymes, especially SOD, and was clearly distinct from the adaptive response just described. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 691–701, 2003. [ABSTRACT FROM AUTHOR]