Low Dissolved Oxygen Tension Research Articles

Pseudomonas putida KT2440 endures temporary oxygen limitations.

The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids. At those conditions, the growth and production of P. putida KT2440 were decelerated compared to well-aerated reference cultivations, but remarkably, final biomass and rhamnolipid titers were similar. The robust growth behavior was confirmed across different cultivation systems, media compositions, and laboratories, even when P. putida KT2440 was repeatedly exposed to dual carbon and oxygen starvation. Quantification of the nucleotides ATP, ADP, and AMP revealed a decrease of intracellular ATP concentrations with increasing duration of oxygen starvation, which can, however, be restored when re-supplied with oxygen. Only small changes in the proteome were detected when cells encountered oscillations in dissolved oxygen tensions. Concluding, P. putida KT2440 appears to be able to cope with repeated oxygen limitations as they occur in large-scale bioreactors, affirming its outstanding suitability as a whole-cell biocatalyst for industrial-scale bioprocesses.

Methanotrophic Community Detected by DNA-SIP at Bertioga's Mangrove Area, Southeast Brazil.

Methanotrophic bacteria can use methane as sole carbon and energy source. Its importance in the environment is related to the mitigation of methane emissions from soil and water to the atmosphere. Brazilian mangroves are highly productive, have potential to methane production, and it is inferred that methanotrophic community is of great importance for this ecosystem. The scope of this study was to investigate the functional and taxonomic diversity of methanotrophic bacteria present in the anthropogenic impacted sediments from Bertioga´s mangrove (SP, Brazil). Sediment sample was cultivated with methane and the microbiota actively involved in methane oxidation was identified by DNA-based stable isotope probing (DNA-SIP) using methane as a labeled substrate. After 4 days (96 h) of incubation and consumption of 0.7 mmol of methane, the most active microorganisms were related to methanotrophs Methylomonas and Methylobacter as well as to methylotrophic Methylotenera, indicating a possible association of these bacterial groups within a methane-derived food chain in the Bertioga mangrove. The abundance of genera Methylomonas, able to couple methane oxidation to nitrate reduction, may indicate that under low dissolved oxygen tensions, some aerobic methanotrophs could shift to intraerobic methane oxidation to avoid oxygen starvation.

Mixomics analysis of Bacillus subtilis: effect of oxygen availability on riboflavin production

BackgroundRiboflavin, an intermediate of primary metabolism, is one kind of important food additive with high economic value. The microbial cell factory Bacillus subtilis has already been proven to possess significant importance for the food industry and have become one of the most widely used riboflavin-producing strains. In the practical fermentation processes, a sharp decrease in riboflavin production is encountered along with a decrease in the dissolved oxygen (DO) tension. Influence of this oxygen availability on riboflavin biosynthesis through carbon central metabolic pathways in B. subtilis is unknown so far. Therefore the unveiled effective metabolic pathways were still an unaccomplished task till present research work.ResultsIn this paper, the microscopic regulation mechanisms of B. subtilis grown under different dissolved oxygen tensions were studied by integrating 13C metabolic flux analysis, metabolomics and transcriptomics. It was revealed that the glucose metabolic flux through pentose phosphate (PP) pathway was lower as being confirmed by smaller pool sizes of metabolites in PP pathway and lower expression amount of ykgB at transcriptional level. The latter encodes 6-phosphogluconolactonase (6-PGL) under low DO tension. In response to low DO tension in broth, the glucose metabolic flux through Embden–Meyerhof–Parnas (EMP) pathway was higher and the gene, alsS, encoding for acetolactate synthase was significantly activated that may result due to lower ATP concentration and higher NADH/NAD+ ratio. Moreover, ResE, a membrane-anchored protein that is capable of oxygen regulated phosphorylase activity, and ResD, a regulatory protein that can be phosphorylated and dephosphorylated by ResE, were considered as DO tension sensor and transcriptional regulator respectively.ConclusionsThis study shows that integration of transcriptomics, 13C metabolic flux analysis and metabolomics analysis provides a comprehensive understanding of biosynthesized riboflavin’s regulatory mechanisms in B. subtilis grown under different dissolved oxygen tension conditions. The two-component system, ResD–ResE, was considered as the signal receiver of DO tension and gene regulator that led to differences between biomass and riboflavin production after triggering the shifts in gene expression, metabolic flux distributions and metabolite pool sizes.

Toward efficient microaerobic processes using engineered Escherichia coli W3110 strains

Operational and economic constraints in large‐scale bioreactors often result in local or global microaerobic conditions, which lead to less efficient bioprocesses. Escherichia coli adapts to microaerobicity by activating fermentation pathways that accumulate acidic by‐products, in detriment of growth rate (μ) and biomass yield on glucose (YX/S). In this study, the metabolism of E. coli was modified to better cope with microaerobicity. For that purpose, genes coding for global regulators like carbon source responsive B protein and aerobic respiratory control A protein, or for fermentative pathways were inactivated. The performance of a wild‐type (W3110) and engineered E. coli strains was evaluated in batch cultures at constant low dissolved oxygen tension (3% air sat.). By combining the partial elimination of fermentation pathways and the expression of the Vitreoscilla hemoglobin (VHb), a 32% decrease on carbon waste as by‐products, 24 % increase on YX/S and 13% increase of μ were obtained. Flux balance analysis of the best strain estimated major differences in the fluxes through the pentose phosphate pathway and tricarboxylic acid cycle as consequence of VHb presence. Overall, our results show that E. coli can be genetically modified to overcome some of the disadvantages of microaerobic growth, which is potentially useful for better bioreactor scale‐up and operation.

Poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) Production in Recombinant Aeromonas hydrophila 4AK4 Harboring phbA, phbB and vgb Genes

AbstractPoly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) (PHBHHx), a copolyester consisting of 3‐hydroxybutyrate (3HB) and 3‐hydroxyhexanoate (3HHx), can be synthesized by Aeromonas hydrophila strain 4AK4 using long chain fatty acids as the carbon source. The wild type A. hydrophila 4AK4 accumulated PHBHHx consisting of 12‐15 mol% 3HHx regardless of growth conditions. When phbA, phbB and vgb genes encoding β‐ketothiolase, acetoacetyl‐CoA reductase and vitreoscilla hemoglobin, respectively, were introduced together into A. hydrophila 4AK4, the recombinant strain grew to over 20 g/L cell dry weight (CDW) after 48 h of shake flask cultivation in co‐substrates of dodecanoate and gluconate (weight ratio 1:1), and the CDW contained 50% PHBHHx consisting of 9 mol% 3HHx. Under similar conditions, the wild type strain produced only 12 g/L CDW containing 32% PHBHHx with 15 mol% 3HHx. In comparison, recombinant harboring phbA and phbB produced 35% PHBHHx with 9 mol% 3HHx in 15 g/L CDW under the same conditions. The obvious differences in terms of the cell growth and PHBHHx production were attributed to the expression of vgb in A. hydrophila 4AK4, which was clearly observed in carbon monoxide difference spectra. The expression of vgb in the recombinant not only improved cell growth and PHBHHx accumulation, but also increased the plasmid stability during cell growth, especially under low dissolved oxygen tension in fermentors. PHBHHx production could be further increased to over 60% of the CDW by the over expression of phaC and phaJ from Aeromonas caviae encoding PHBHHx synthase and (R)‐specific enoyl‐CoA hydratase, respectively. Over expression of phaC, phaJ and phaP, alone or in various combinations, also increased the 3HHx content of PHBHHx from 14‐34%. The above results showed that A. hydrophila was amenable to genetic manipulation, and that these modifications could be exploited to produce compounds with different properties for commercial and research applications.

Toxicity and chemistry of aspen wood leachate to aquatic life: field study.

A dark, toxic leachate has been observed around woodpiles of trembling aspen (Populus tremuloides Michx.) cut in winter for pulp or structural lumber. We measured production of leachate from 18 m3 of harvestable aspen logs stacked in an open field near Dawson Creek, British Columbia, Canada. The logpile began producing leachate during the first winter thaw and continued to do so for the duration of the two-year study (mean, 250 L/collection). Aspen leachate was characterized by dark color, acidic pH (5.0-6.5), elevated conductivity (200-500 microS/cm), high to very high biochemical oxygen demand (500-5,000 mg/L) and total organic carbon concentrations (500-2,000 mg/L), variable levels of phenolic compounds (2-27 mg/L), and low dissolved oxygen tensions (<2 mg/L). In tests with rainbow trout (Oncorhynchus mykiss), Daphnia magna, and luminescent bacteria, the leachate varied from weakly toxic (median lethal concentration, >10%) to very toxic (median lethal concentration, <1%). The volume of leachate generated by the logpile was correlated with total precipitation (rain or snow) since the last collection. Loads of chemical constituents or toxicity (lethal concentration x volume) in the leachate did not decline over the duration of the study. Less than 10% of the total mass of leachable material in the aspen logs was removed during two years of exposure.

Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor

Media and growth conditions were optimized for the microaerobic cultivation of Magnetospirillum gryphiswaldense in flasks and in a fermentor, resulting in significantly increased cell and magnetosome yields, compared with earlier studies. A reliable method was established for the automatic control of low dissolved oxygen tensions (pO(2)) in the fermentor (oxystat). Growth and magnetosome formation by M. gryphiswaldense, M. magnetotacticum and Magnetospirillum sp. AMB-1 were studied at various oxygen concentrations. Despite differences in their growth responses with respect to oxygen, we found a clear correlation between pO(2) and magnetosome formation in all three Magnetospirillum strains. Magnetite biomineralization was induced only below a threshold value of 20 mbar O(2) and optimum conditions for magnetosome formation were found at a pO(2) of 0.25 mbar (1 bar = 10(5) Pa). A maximum yield of 6.3 mg magnetite l(-1) day(-1) was obtained with M. gryphiswaldense grown under oxystat conditions, which is the highest magnetosome productivity reported so far for a magnetotactic bacterium. In conclusion, the presented results provide the basis for large-scale cultivation of magnetospirilla under defined conditions.

The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli.

Escherichia coli MC4100 was grown in anaerobic glucose-limited chemostat cultures, either in the presence of an electron acceptor (fumarate, nitrate, or oxygen) or fully fermentatively. The steady-state NADH/NAD ratio depended on the nature of the electron acceptor. Anaerobically, the ratio was highest, and it decreased progressively with increasing midpoint potential of the electron acceptor. Similarly, decreasing the dissolved oxygen tension resulted in an increased NADH/NAD ratio. As pyruvate catabolism is a major switch point between fermentative and respiratory behavior, the fluxes through the different pyruvate-consuming enzymes were calculated. Although pyruvate formate lyase (PFL) is inactivated by oxygen, it was inferred that the in vivo activity of the enzyme occurred at low dissolved oxygen tensions (DOT </= 1%). A simultaneous flux from pyruvate through both PFL and the pyruvate dehydrogenase complex (PDHc) was observed. In anaerobic cultures with fumarate or nitrate as an electron acceptor, a significant flux through the PDHc was calculated on the basis of the redox balance, the measured products, and the known biochemistry. This result calls into question the common assumption that the complex cannot be active under these conditions. In vitro activity measurements of PDHc showed that the cellular content of the enzyme varied with the internal redox state and revealed an activity for dissolved oxygen tension of below 1%. Whereas Western blots showed that the E3 subunit of PDHc (dihydrolipoamide dehydrogenase) did not vary to a large extent under the conditions tested, the E2 subunit (dihydrolipoamide acetyltransferase) amount followed the trend that was found for the in vitro PDHc activity. From this it is concluded that regulation of the PDHc is exerted at the E1/E2 operon (aceEF). We propose that the external redox state (measured as the midpoint potentials of those terminal acceptors with which the cell has sufficient capacity to react) is reflected by the internal redox state. The latter may subsequently govern both the expression and the activity of the two pyruvate-catabolizing enzymes.

Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation

The effects of dissolved oxygen tension and mechanical forces on fungal morphology were both studied in the submerged fermentation of Aspergillus awamori. Pellet size, the hairy length of pellets, and the free filamentous mycelial fraction in the total biomass were found to be a function of the mechanical force intensity and to be independent of the dissolved oxygen tension provided that the dissolved oxygen tension was neither too low (5%) nor too high (330%). When the dissolved oxygen concentration was close to the saturation concentration corresponding to pure oxygen gas, A. awamori formed denser pellets and the free filamentous mycelial fraction was almost zero for a power input of about 1 W/kg. In the case of very low dissolved oxygen tension, the pellets were rather weak and fluffy so that they showed a very different appearance. The amount of biomass per pellet surface area appeared to be affected only by the dissolved oxygen tension and was proportional to the average dissolved oxygen tension to the power of 0.33. From this it was concluded that molecular diffusion was the dominant mechanism for oxygen transfer in the pellets and that convection and turbulent flow in the pellets were negligible in submerged fermentations. The biomass per wet pellet volume increased with the dissolved oxygen tension and decreased with the size of the pellets. This means that the smaller pellets formed under a higher dissolved oxygen tension had a higher intrinsic strength. Correspondingly, the porosity of the pellets was a function of the dissolved oxygen tension and the size of pellets. Within the studied range, the void fraction in the pellets was high and always much more than 50%. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 409-419, 1998.

Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation

The effects of dissolved oxygen tension and mechanical forces on fungal morphology were both studied in the submerged fermentation of Aspergillus awamori. Pellet size, the hairy length of pellets, and the free filamentous mycelial fraction in the total biomass were found to be a function of the mechanical force intensity and to be independent of the dissolved oxygen tension provided that the dissolved oxygen tension was neither too low (5%) nor too high (330%). When the dissolved oxygen concentration was close to the saturation concentration corresponding to pure oxygen gas, A. awamori formed denser pellets and the free filamentous mycelial fraction was almost zero for a power input of about 1 W/kg. In the case of very low dissolved oxygen tension, the pellets were rather weak and fluffy so that they showed a very different appearance. The amount of biomass per pellet surface area appeared to be affected only by the dissolved oxygen tension and was proportional to the average dissolved oxygen tension to the power of 0.33. From this it was concluded that molecular diffusion was the dominant mechanism for oxygen transfer in the pellets and that convection and turbulent flow in the pellets were negligible in submerged fermentations. The biomass per wet pellet volume increased with the dissolved oxygen tension and decreased with the size of the pellets. This means that the smaller pellets formed under a higher dissolved oxygen tension had a higher intrinsic strength. Correspondingly, the porosity of the pellets was a function of the dissolved oxygen tension and the size of pellets. Within the studied range, the void fraction in the pellets was high and always much more than 50%.

Kinetic analysis of alkaline protease activity, recombinant protein production and metabolites for infected insect (Sf9) cells under different DO levels

An analysis of protease activity, recombinant protein yield ( β-galactosidase and epoxide hydrolase) and metabolite concentration (glucose, glutamine, ammonia, and lactate) for Sf9 cells under varied dissolved oxygen tension and before and after viral infection are reported. Different levels of bulk proteolytic activity were found under three different dissolved oxygen (DO) levels (50%, 25%, and 15% of air saturation). The culture with lowest protease activity had the highest specific recombinant protein yield, suggesting a negative correlation and an appropriate timing for cell harvest. At low dissolved oxygen tension (15% of air saturation), epoxide hydrolase production was lowest and the lactate/glucose yield, Y lac/glu, was highest, marking low glucose utilization efficiency. Ammonia and lactate were consumed by cells at 25% DO, but not at low and high DO. Thus, the possibility that ammonia and lactate exert an inhibitory effect on protein production is very low given an appropriate DO setpoint. Our results facilitate systematic development of optimized media formulations and reactor operating strategies for maximizing protein yield in insect cell systems.

A substrate feeding strategy--using an oxystat for L-phenylalanine fermentation by Corynebacterium glutamicum

A substrate feeding strategy using an oxystat was first successfully applied to a fed-batch phenylalanine fermentation. The control method allowed the fermentation to be under low dissolved oxygen tension, which was favourable phenylalanine formation, and to be from substrate inhibition during the course of fed-batch operation. The final product concentration was 3 times higher than in a batch culture.

Stability of plasmid vector plJ303 in Streptomyces lividans TK24 during laboratory‐scale fermentations

Plasmid plJ303 stability in Streptomyces lividans cultures has been studied by measuring plasmid copy number under various growth conditions. An increase in mean plasmid copy number was normally seen during early rapid growth in both shaken culture and stirred vessel fermentations at 28 degrees C. Maximum copy numbers were consistently attained in early stationary phase followed by a decline (of variable amount) upon further incubation. The imposition of environmental stress (high growth temperature, i.e., 37 degrees C, and low dissolved oxygen tension, i.e., <5% air saturation) led to a plasmid copy number of zero and a 50% reduction, respectively. Interestingly, the relative proportions of plasmid topoisomers changed with time since progressively more supercoiled forms were observed throughout the stationary phase. Plasmid dimers were also observed in some cultures, and no evidence of structural plasmid instability was found. In general, this host-vector system seemed remarkably stable under normal growth conditions. However, copious organic acid production by the host was observed and was thought to be undesirable for good heterologous gene expression of a secreted protein.

Influence of dissolved oxygen tension on the synthesis of catechol 1,2-dioxygenase by Pseudomonas putida

Both basal noninduced and induced levels of catechol 1,2-dioxygenase in strains of Pseudomonas putida during glutamate-limited continuous culture have been shown to increase with dissolved oxygen tension over the range investigated (4–80% air saturation). Fluctuating rates of enzyme synthesis were observed following induction by addition of catechol with an initial lag which was greater at low dissolved oxygen tensions. The results indicate that the rates of metabolism of aromatic cmopounds via the ortho pathway may be enhanced if the dissolved oxygen tension is maintained at a high value.

Electron transport system associated with direct glucose oxidation in Gluconobacter oxydans

The mode of electron transport associated with the dehydrogenase enzymes located on the cytoplasmic membrane inGluconobacter oxydans (ATCC 9937) has been postulated. High turnover of dehydrogenases under oxygen enrichment conditions is explained on the basis of a simplistic electron transport chain comprising cytochrome c553 (MW 23000) as a subunit of dehydrogenase and a cytochrome b562. The electron transport chain under low dissolved oxygen tension (DOT) is shown to comprise a number of cytochrome c species with very low midpoint potential difference.

11β-Hydroxylation of coxtexolone by Curvularia lunata

The 11β-hydroxylase of Curvularia lunata (NRRL 2380) was demonstrated to be nonconstitutive and intracellularly inducible by cortexolone. The pH change and the color development associated with the mycelial growth can serve as convenient criteria for 11β-hydroxylation process. The feeding time of steroid substrate affects the hydroxylation rate significantly. Feeding of substrate was found to be best at around 8 h of incubation. Hydroxylation could be enhanced when induction was performed at low dissolved oxygen tension (DOT), followed later by DOT elevation in the culture. The effect of DOT on enzyme expression was investigated showing an optimum DOT value of 5.5 ppm for maximum hydroxylation rate.

Ethanol tolerance of Pichia stipitis and Candida shehatae strains in fed-batch cultures at controlled low dissolved oxygen levels

Fed-batch cultivations of Pichia stipitis and strains of Candida shehatae with d-xylose or d-glucose were conducted at controlled low dissolved oxygen tension (DOT) levels. There were some marked differences between the strains. In general growth was inhibited at lower ethanol concentrations than fermentation, and ethanol levels of up to 47 g·l-1 were produced at 30°C. Ethanol production was mainly growth associated. The yeast strains formed small amounts of monocarboxylic acids and higher alcohols, which apparently did not enhance the ethanol toxicity. The maximum ethanol concentration obtained on d-xylose could not be increased by using a high cell density culture, nor by using d-glucose as substrate. The latter observation suggested that the low ethanol tolerance of these xylose-fermenting yeast strains was not a consequence of the metabolic pathway used during pentose fermentation. In contrast with the C. shehatae strains, it was apparent with P. stipitis CSIR-Y633 that when the ethanol concentration reached about 28 g·l-1, ethanol assimilation exceeded ethanol production, despite cultivation at a low DOT of 0.2% of air saturation. Discontinuing the aeration enabled ethanol accumulation to proceed, but with concomitant xylitol production and cessation of growth.

Oxygen consumption and responses to hypoxia of ammocoetes of the southern hemisphere lampreyGeotria australis

The standard rate of oxygen consumption of ammocoetes (larvae) ofGeotria australis with a mean weight of c. 0.5 g was 9.6, 31.4 and 59.4μl g(-1) h(-1) at 4.5, 15.5 and 25.0°C respectively, which gives an overall Q10 of 2.4. The regression coefficient for the logarithmic relationship between oxygen consumption and body weight at 15.5°C was 0.704. The ammocoetes ofG. australis have a much lower rate of oxygen consumption at 15.5 and 25.0°C than those of holarctic lampreys. This presumably reflects the lower oxygen delivery pressure to their tissues and helps account for their slow growth rate. At 15.5°C, ammocoetes ofG. australis emerged from the substrate at 21-25 mm Hg and, unlike those of the Northern HemisphereIchthyomyzon greeleyi, died at 14-17 mm Hg. Thus, despite having a thinner water/blood barrier in the gills and blood with a higher oxygen affinity and capacity than holarctic ammocoetes, the larvae ofG. australis cannot survive very low dissolved oxygen tensions. This is apparently related to an inability of larvalG. australis to meet the high oxygen requirements of the respiratory pump at these oxygen tensions. During metamorphosis, oxygen consumption at 15.5°C rose from approximately 27μl g(-1) h(-1) at the beginning of transformation to 33.2μl g(-1) h(-1) by Stage 3 and then rapidly to 66μl g(-1) h(-1) at Stage 6. It remained near this level in Stage 7 and the downstream migrant.

The effects of low dissolved oxygen tension during the aerobic treatment of piggery slurry in completely mixed reactors.

The energy requirement for aerobic treatment of animal excreta is minimal when the dissolved oxygen (DO) level is as low as possible. The efficiency of continuous aerobic treatment at 15°C in terms of the removal of carbonaceous materials was found to be the same at DO levels as low as 1% of saturation as at high DO levels. However, up to 45% of the total input nitrogen and 90% of the soluble nitrogen were lost from the slurry during treatment at low DO. This loss was the result of the simultaneous occurrence of nitrification and denitrification within the completely mixed reactor. It was also observed that low DO levels favoured nitrification during aerobic treatment. This resulted from the activity of denitrifying bacteria which raised the pH and removed the inhibitory effect of acidic conditions upon the nitrifiers. Control of the pH value at pH 7.1 during aerobic treatment resulted in the complete oxidation of the ammonia content of piggery slurry.

Oxygen consumption by Campylobacter sputorum subspecies Bubulus with formate as substrate.

The kinetics of oxygen utilization by the microaerophile Campylobacter sputorium subspecies bubulus was studied. With formate as substrate two enzyme systems were found to be responsible for electron transfer between formate and oxygen. In the case of lactate oxidation one enzyme system could account for the activity measured. One of the formate-oxidizing systems possessed a high affinity for oxygen [Km(O2) = approx. 4 microM O2]. From inhibitor studies it was concluded that a respiratory chain was involved in its activity. Respiration by this system must be responsible for proton translocation and electron transport-linked phosphorylation at formate oxidation. The other enzyme system had an extremely low affinity for oxygen [Km (O2) = approx. 1 mM O2]. It was tentatively identified as the H2O2-producing formate oxidase previously found in C. sputorum. The H2O2 production by this enzyme is implicated in an explantation of the microaerophilic nature of C. sputorum. Sensitivity of formate dehydrogenase to H2O2 was demonstrated. The influence of the formate concentration on aerobic formate oxidation was determined. The pH- and temperature dependencies of oxygen uptake with formate as substrate were examined at air-saturation and at a low dissolved oxygen tension.