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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 © 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Fungi are amongst the most industrially important microorganisms in current use within the biotechnology industry. Most such fungal cultures are highly aerobic in nature, a character that has been frequently referred to in both reactor design and fungal physiology. The most fundamentally significant outcome of the highly aerobic growth environment in fermenter vessels is the need for the fungal culture to effectively combat in the intracellular environment the negative consequences of high oxygen transfer rates. The use of oxygen as the respiratory substrate is frequently reported to lead to the development of oxidative stress, mainly due to oxygen-derived free radicals, which are collectively termed as reactive oxygen species (ROS). Recently, there has been extensive research on the occurrence, extent, and consequences of oxidative stress in microorganisms, and the underlying mechanisms through which cells prevent and repair the damage caused by ROS. In the present study, we critically review the current understanding of oxidative stress events in industrially relevant fungi. The review first describes the current state of knowledge of ROS concisely, and then the various antioxidant strategies employed by fungal cells to counteract the deleterious effects, together with their implications in fungal bioprocessing are also discussed. Finally, some recommendations for further research are made.

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 H(2)O(2) 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 H(2)O(2) 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.

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 H(2)O(2) completely prevents killing by this oxidant at high concentrations. It shows that A. niger adapts to exposure to H(2)O(2) 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 H(2)O(2) detoxification, coincides with recommencement of growth. Results from monitoring the extracellular H(2)O(2) concentration clearly indicate a very rapid detoxification rate for H(2)O(2) in adapted A. niger cultures. A mathematical model predicts only very low concentrations of intracellular H(2)O(2) accumulating in such cultures. Our results also show that glutathione plays a role in the oxidative defence against H(2)O(2) in A. niger. On addition of H(2)O(2), the intracellular pool of glutathione increases while the redox state of glutathione becomes more oxidized.

Inoculation of bioreactors with shake-flask cultures present the organism with an immediate shift from an environment with little O2 to one in which O2 is typically at 100% saturation. The inoculation of such shake-flasks cultures into bioreactors sparged with 1 vvm air or 1 vvm air/O2 mix i.e. 50% O2 enrichment is an oxidatively stressful event, as judged by immediate increases in the intracellular concentrations of superoxide anion radical (O2*-) (from 4,600 to 11,600 RLU mg DCW(-1) and 5,500 to 23,000 RLU mg DCW(-1) respectively) and changes in the activities of the major antioxidant enzymes superoxide dismutase and catalase in all cultures. There are further effects on metabolic indices, particularly decreased nutrient consumption in oxygenated cultures (from 0.16 to 0.12 g starch g DCW h(-1)) and decreased protein production, indicating that inoculation of the bioreactor exerts a global burden on the cellular metabolic networks.

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 [O(2) (.-)]) and activities of cellular defensive enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxide [GPx]) were quantified at varying temperature (25, 30, 35, 40 degrees C) to more fully characterize culture response in different growth phases. Elevated culture temperature led to increased O(2) (.-) levels in various culture phases. In the exponential phase this was due to an enhanced generation of O(2) (.-), 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 H(2)O(2) in this A. niger. The combination of elevated temperature (35 degrees C) and increased O(2) supply (50% enrichment) led to decreased levels of O(2) (.-) compared to the cultivation at 35 degrees 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., (Copyright 2003 Wiley Periodicals.)

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., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 691-701, 2003.)

It has been known for many years that oxygen (O2) may have toxic effects on aerobically growing microorganisms, mainly due to the threat arising from reactive oxygen species (ROS). In submerged culture industrial fermentation processes, maintenance of adequate levels of O2 (usually measured as dissolved oxygen tension (DOT)) can often be critical to the success of the manufacturing process. In viscous cultures of filamentous cultures, actively respiring, supplying adequate levels of O2 to the cultures by conventional air sparging is difficult and various strategies have been adopted to improve or enhance O2 transfer. However, adoption of those strategies to maintain adequate levels of DOT, that is, to avoid O2 limitation, may expose the fungi to potential oxidative damage caused by enhanced flux through the respiratory system. In the past, there have been numerous studies investigating the effects of DOT on fungal bioprocesses. Generally, in these studies moderately enhanced levels of O2 supply resulted in improvement in growth, product formation and acceptable morphological changes, while the negative impact of higher levels of DOT on morphology and product synthesis were generally assumed to be a consequence of "oxidative stress." However, very little research has actually been focused on investigation of this implicit link, and the mechanisms by which such effects might be mediated within industrial fungal processes. To elucidate this neglected topic, this review first surveys the basic knowledge of the chemistry of ROS, defensive systems in fungi and the effects of DOT on fungal growth, metabolism and morphology. The physiological responses of fungal cells to oxidative stress imposed by artificial and endogenous stressors are then critically reviewed. It is clear that fungi have a range of methods available to minimize the negative impacts of elevated ROS, but also that development of the various defensive systems or responses, can itself have profound consequences upon many process-related parameters. It is also clear that many of the practically convenient and widely used experimental methods of simulating oxidative stress, for example, addition of exogenous menadione or hydrogen peroxide, have effects on fungal cultures quite distinct from the effects of elevated levels of O2, and care must thus be exercised in the interpretation of results from such studies. The review critically evaluates our current understanding of the responses of fungal cultures to elevated O2 levels, and highlights key areas requiring further research to remedy gaps in knowledge.

It has been known for many years that oxygen (O2) may have toxic effects on aerobically growing microorganisms, mainly due to the threat arising from reactive oxygen species (ROS). In submerged culture industrial fermentation processes, maintenance of adequate levels of O2 (usually measured as dissolved oxygen tension (DOT)) can often be critical to the success of the manufacturing process. In viscous cultures of filamentous cultures, actively respiring, supplying adequate levels of O2 to the cultures by conventional air sparging is difficult and various strategies have been adopted to improve or enhance O2 transfer. However, adoption of those strategies to maintain adequate levels of DOT, that is, to avoid O2 limitation, may expose the fungi to potential oxidative damage caused by enhanced flux through the respiratory system. In the past, there have been numerous studies investigating the effects of DOT on fungal bioprocesses. Generally, in these studies moderately enhanced levels of O2 supply resulted in improvement in growth, product formation and acceptable morphological changes, while the negative impact of higher levels of DOT on morphology and product synthesis were generally assumed to be a consequence of "oxidative stress." However, very little research has actually been focused on investigation of this implicit link, and the mechanisms by which such effects might be mediated within industrial fungal processes. To elucidate this neglected topic, this review first surveys the basic knowledge of the chemistry of ROS, defensive systems in fungi and the effects of DOT on fungal growth, metabolism and morphology. The physiological responses of fungal cells to oxidative stress imposed by artificial and endogenous stressors are then critically reviewed. It is clear that fungi have a range of methods available to minimize the negative impacts of elevated ROS, but also that development of the... [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]

Continuous chemostat cultures of a recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme, were investigated with regard to their susceptibility to oxidative stress. The culture response to oxidative stress, produced either by addition of exogenous hydrogen peroxide (H2O2) or by high dissolved oxygen tension (DOT), was characterised in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). Since the morphology is so critical in submerged fungal bioprocesses, the key morphological indices were analysed using a semi-automated image analysis system. Both oxidant stressors, H2O2 and elevated DOT, increased both enzyme activities, however, the extent was different: exogenous H2O2 led mainly to increased CAT activity, whereas gassing with O2 enriched air, which resulted in a DOT of 165% of air saturation, increased both enzyme activities more than 2-fold compared with the control steady state culture. Addition of exogenous H2O2 resulted in shorter hyphae compared with control steady state cultures. These findings indicate that it is unsound to use exogenous H2O2 to simulate oxidative stress induced by elevated dissolved oxygen levels since the response to each might be quite different, both in terms of enzymatic (defensive) responses and in terms of culture morphology. [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]

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]

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]