Oxygen-limited Continuous Cultures Research Articles

Metabolism of sucrose in a non-fermentative Escherichia coli under oxygen limitation

Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap carbon sources, like sucrose. However, oxygen-limiting conditions often lead to by-product formation and reduced ATP yields. While by-product formation is typically decreased by gene deletion, the breakdown of oligosaccharides with inorganic phosphate instead of water could increment the ATP yield. To observe the effect of oxygen limitation during sucrose consumption, a non-fermentative Escherichia coli K-12 strain was transformed with genes enabling sucrose assimilation. It was observed that the combined deletion of the genes adhE, adhP, mhpF, ldhA, and pta abolished the anaerobic growth using sucrose. Therefore, the biomass-specific conversion rates were obtained using oxygen-limited continuous cultures. Strains performing the breakdown of the sucrose by hydrolysis (SUC-HYD) or phosphorolysis (SUC-PHOSP) were studied in such conditions. An experimentally validated in silico model, modified to account for plasmid and protein burdens, was employed to calculate carbon and electron consistent conversion rates. In both strains, the biomass yields were lower than expected and, strikingly, SUC-PHOSP showed a yield lower than SUC-HYD. Flux balance analyses indicated a significant increase in the non-growth-associated ATP expenses by comparison with the growth on glucose. The observed fructose-1,6-biphosphatase and phosphoglucomutase activities, as well as the concentrations of glycogen, suggest the operation of ATP futile cycles triggered by a combination of the oxygen limitation and the metabolites released during the sucrose breakdown.

Model‐based derivation, analysis and control of unstable microaerobic steady‐states—Considering Rhodospirillum rubrum as an example

Microaerobic (oxygen-limited) conditions are critical for inducing many important microbial processes in industrial or environmental applications. At very low oxygen concentrations, however, the process performance often suffers from technical limitations. Available dissolved oxygen measurement techniques are not sensitive enough and thus control techniques, that can reliable handle these conditions, are lacking. Recently, we proposed a microaerobic process control strategy, which overcomes these restrictions and allows to assess different degrees of oxygen limitation in bioreactor batch cultivations. Here, we focus on the design of a control strategy for the automation of oxygen-limited continuous cultures using the microaerobic formation of photosynthetic membranes (PM) in Rhodospirillum rubrum as model phenomenon. We draw upon R. rubrum since the considered phenomenon depends on the optimal availability of mixed-carbon sources, hence on boundary conditions which make the process performance challenging. Empirically assessing these specific microaerobic conditions is scarcely practicable as such a process reacts highly sensitive to changes in the substrate composition and the oxygen availability in the culture broth. Therefore, we propose a model-based process control strategy which allows to stabilize steady-states of cultures grown under these conditions. As designing the appropriate strategy requires a detailed knowledge of the system behavior, we begin by deriving and validating an unstructured process model. This model is used to optimize the experimental conditions, and identify properties of the system which are critical for process performance. The derived model facilitates the good process performance via the proposed optimal control strategy. In summary the presented model-based control strategy allows to access and maintain microaerobic steady-states of interest and to precisely and efficiently transfer the culture from one stable microaerobic steady-state into another. Therefore, the presented approach is a valuable tool to study regulatory mechanisms of microaerobic phenomena in response to oxygen limitation alone. Biotechnol. Bioeng. 2014;111: 734-747. © 2013 Wiley Periodicals, Inc.

Physiological effects of the addition of n-dodecane as an oxygen vector during steady-state Bacillus licheniformis thermophillic fermentations perturbed by a starvation period or a glucose pulse

The effect of the presence of n-dodecane as a potential oxygen vector during oxygen-limited continuous cultures of a Bacillus strain was studied, under extreme nutrient supply conditions: glucose excess, limitation and starvation. The addition of n-dodecane to the aqueous phase of a mechanically agitated and aerated fermentation increased the k L a by up to 35%. The n-dodecane additions to Bacillus licheniformis cells during starvation (oxygen limitation with concomitant glucose starvation) caused a severe detrimental progressive change in cell physiological state with respect to cytoplasmic membrane polarisation and permeability which was mitigated against by alleviating either the oxygen limitation (by increasing the mean energy dissipation rate or by the addition of n-dodecane as an oxygen vector) or by alleviating the carbon limitation (by resuming the carbon feed or by the addition of a glucose pulse). Further that during periods of excess glucose (glucose pulse) a much higher k L a was required to prevent the onset of anaerobic mixed acid fermentation than could be provided by the addition of n-dodecane alone. n-Dodecane can be used to increase the k L a when added in sufficient quantities to the aqueous phase of a mechanically agitated and aerated bioreactor but the magnitude of this increase is process and vessel geometry specific.

Phase-Specific Protein Expression in the Dimorphic YeastSaccharomyces cerevisiae

In oxygen-limited continuous culture,Saccharomyces cerevisiaeformed pseudohyphae by unipolar budding. We developed a continuous cultivation sequence to discriminate phase-specific from metabolically regulated proteins during dimorphism. Computer-aided substractive analysis of 2D–PAGE protein patterns allowed the detection of proteins specifically expressed during yeast and pseudohyphal phases. Image analysis resolved 3 spots that were specific to the pseudohyphal phase and 2 spots that were specific to yeast phase. In addition to phase-specific proteins, important regulation of protein expression took place. A group of 9 proteins was highly over-expressed during the yeast phase when another group of 12 was under-expressed. This phenomenon was reversed during the pseudohyphal phase. These experiments showed that dimorphism inS. cerevisiaeis associated with the expression of specific proteins and suggest that yeast phase-specific proteins maintain the yeast form or repress pseudohyphae formation.

Production of poly(3-hydroxyalkanoates) by Pseudomonas putida KT2442 in continuous cultures

The synthesis of poly(3-hydroxyalkanoates) (PHA) by Pseudomonas putida KT2442 growing on long-chain fatty acids was studied in continuous cultures. The effects of the growth rate on the biomass and polymer concentration were determined and it was found that the PHA concentrations decreased with increasing growth rates. The highest volumetric productivity was 0.13 g PHA l-1 h-1 at a specific growth rate (μ) of 0.1 h-1. The molecular mass of the polymer remained constant at all growth rates but changes in the monomeric composition of the PHA synthesized were observed. Variation of the carbon to nitrogen (C/N) ratio of the substrate feed at μ=0.1 h-1 revealed optimal PHA formation at C/N=20 mol/mol. In order to optimize PHA production P. putida KT2442 was cultivated to high cell densities in oxygen-limited continuous cultures. In this way a maximum biomass concentration of 30 g/l containing approximately 23% PHA was achieved. This corresponds to a volumetric productivity of 0.69 g l-1 h-1.

Oxygen and/or glucose limitation in a chemostat culture of Candida utilis mathematical model identification

The glucose and/or dissolved oxygen limited continuous culture of yeast Candida utilis was studied. Six different mathematical models were designed to describe and analyze the experiments. The model considering the production of surface active compounds at autoanaerobic conditions and dissolved oxygen consumption for nongrowth associated exogeneous respiration yields the best fit. The results may be applied for aerobic waste water treatment plants, process analysis and simulation.

A flocculating strain of Pichia stipitis for the conversion of glucose/xylose mixtures

In the production of ethanol from glucose/xylose mixtures by Pichia stipitis, the sugar uptake rate is the rate limiting step in the process. Therefore, a high biomass concentration is needed for a complete conversion of a mixed substrate in a single reactor. A flocculating strain of P. stipitis can offer the advantage of a high biomass concentration in the reactor that is almost independent of the dilution rate. The sugar concentrations in an oxygen-limited continuous culture were varied and CaCl 2 was added to promote flocculation. Flocculation occurred under these conditions, and a flocculating type of P. stipitis was isolated. The flocculating strain of P. stipitis, which formed pseudomycelium, is deposited at the CBS as P. stipitis CBS 7505. To study the possibilities of P. stipitis CBS 7505, continuous culture experiments were carried out in an airlift-loop reactor. A biomass concentration of 14.1 g l −1 was possible at a dilution rate of 0.05 h -1, leading to a simultaneous conversion of glucose and xylose. Computer simulations showed the conversion of glucose is probably oxygen limited. The results of flocculation are comparable with those of coimmobilization of P. stipitis and Saccharomyces cerevisiae. The specific and volumetric ethanol production rates of the two processes are almost identical. However, a higher ethanol yield was found in the flocculation experiments, but more xylose is converted in the case of coimmobilization. The conversion of waste streams that contain hexose as well as pentose sugars with flocculating P. stipitis is possible. Due to the low yield of ethanol, the extra profit made in comparison to that obtained from the conversion of the same waste stream with S. cerevisiae is minimal.

Growth, death, and oxygen uptake kinetics of Pichia stipitis on xylose

Pichia stipitis NRRL Y-7124 has potential application in the fermentation of xylose-rich waste streams, produced by wood hydrolysis. Kinetic models of cell growth, death, and oxygen uptake were investigated in batch and oxygen-limited continuous cultures fed a rich synthetic medium. Variables included rates of dilution (D) and oxygen transfer (K(1)a) and concentrations of xylose (X), ethanol (E), and dissolved oxygen (C(ox)). Sustained cell growth required the presence of oxygen. Given excess xylose, specific growth rate (micro) was a Monod function of C(ox). Specific oxygen uptake rate was proportional to mu by a yield coefficient relating biomass production to oxygen consumption; but oxygen uptake for maintenance was negligible. Thus steady-state C(OX) depended only on D, while steady-state biomass concentration was controlled by both D and K(1)a. Given excess oxygen, cells grew subject to Monod limitation by xylose, which became inhibitory above 40 g/L. Ethanol inhibition was consistent with Luong's model, and 64. 3 g/L was the maximum ethanol concentration allowing growth. Actively growing cells died at a rate that was 20% of micro. The dying portion increased with E and X.

Growth, Respiration, and Polypeptide Patterns of Bradyrhizobium sp. ( Arachis ) Strain 3G4b20 from Succinate- or Oxygen-Limited Continuous Cultures

Succinate- or oxygen-limited continuous cultures were used to study the influences of different concentrations of dissolved oxygen and ammonia on the growth, respiration, and polypeptide patterns of Bradyrhizobium sp. (Arachis) strain 3G4b20. During succinate-limited growth, molar growth yields on succinate (Y(succ)) ranged from 38.9 to 44.4 g (dry weight) of cells mol of succinate and were not greatly influenced by changes in dilution rates or changes in the oxygen concentrations that we tested. Succinate, malate, and fumarate induced the highest rates of oxygen uptake in all of the steady states in which the supply rates of (NH(4))(2)SO(4) ranged between 322 and 976 mumol h. However, the amino acids aspartate, asparagine, and glutamate could also be used as respiratory substrates, especially when the (NH(4))(2)SO(4) supply rate was decreased to 29 mumol h. Glutamine-dependent respiration was seen only when the (NH(4))(2)SO(4) supply rate was 29 mumol h and thus appears to be under tight ammonia control. Nitrogenase activity was detected only when the culture was switched from a succinate-limited steady state to an oxygen-limited steady state. Comparison of major silver-stained proteins from three steady states by two-dimensional gel electrophoresis revealed that nearly 60% were affected by oxygen and 24% were affected by ammonia. These data are consistent with reports that oxygen has a major regulatory role over developmental processes in Rhizobium sp. and Bradyrhizobium sp.

Physiology and virulence determinants of Neisseria gonorrhoeae grown in glucose-, oxygen- or cystine-limited continuous culture.

Piliated Neisseria gonorrhoeae forming small, transparent colonies (P+O-) on clear typing agar have been grown in prolonged continuous culture to ascertain how different growth environments might affect gonococcal physiology and the expression of virulence determinants. Virulence of the penicillin-sensitive P9-2 and the penicillin-resistant KW1 strains was assessed by their ability to survive in polypropylene chambers implanted into the flanks of guinea pigs. Initial continuous culture experiments in the defined medium of Manchee et al. (FEMS Microbiology Letters 7, 115-118, 1980) indicated that growth was actually cystine-limited, rather than the anticipated glucose-limited. Surprisingly, cysteine was not completely metabolized and ammonium salts remained in excess. The molar growth yield on glucose (YGlc) was 65 g dry wt mol-1 and 45% of the glucose carbon metabolized was converted to biomass. Gonococci, whilst retaining the P+O- phenotype for over 100 generations of growth, did not survive in the subcutaneous chambers when inoculated at a variety of doses. When the cystine and glucose concentrations were increased and decreased respectively, growth became glucose-limited, the YGlc increased to 108 g mol-1 for strain KW1 and 75% of the metabolized glucose carbon was converted to biomass. After 17 generations of growth, however, only 2% of the gonococci retained the P+O- phenotype and P-O- bacteria predominated. Nevertheless, these bacteria were virulent in the chamber model, as was strain P9-2, which also retained only 2% of the P+O- phenotype during glucose-limited continuous culture. By contrast, the P+O- phenotype was retained during prolonged cystine- or oxygen-limited growth but only the latter was virulent. SDS-PAGE of membrane extracts confirmed that opaque colonies (O+) selected from the glucose-limited cultures contained a heat-modifiable protein (protein II) whereas transparent colony types lacked such proteins. The initial phenotype of virulent gonococci recovered from the subcutaneous chambers was P+O- but opaque variants dominated after several days. A 40 kDa outer-membrane protein was apparently induced during oxygen-limited continuous culture whereas a 44 kDa protein was absent during cystine-limited growth.

Effects of silicone on oxygen‐limited continuous culture of yeast

Biotechnology and BioengineeringVolume 20, Issue 6 p. 899-901 Communications to the EditorFree Access Effects of silicone on oxygen-limited continuous culture of yeast Marina L. R. Vairo, Marina L. R. Vairo Biochemical Engineering Laboratory, Department of Chemical Engineering, Escola Politécnica, University of São Paulo, C.P. 8174, São Paulo, BrazilSearch for more papers by this authorLeonardo Perego Jr., Leonardo Perego Jr. Biochemical Engineering Laboratory, Department of Chemical Engineering, Escola Politécnica, University of São Paulo, C.P. 8174, São Paulo, BrazilSearch for more papers by this authorLuiz H. Koshimizu, Luiz H. Koshimizu Biochemical Engineering Laboratory, Department of Chemical Engineering, Escola Politécnica, University of São Paulo, C.P. 8174, São Paulo, BrazilSearch for more papers by this authorWalter Borzani, Walter Borzani Biochemical Engineering Laboratory, Department of Chemical Engineering, Escola Politécnica, University of São Paulo, C.P. 8174, São Paulo, BrazilSearch for more papers by this author Marina L. R. Vairo, Marina L. R. Vairo Biochemical Engineering Laboratory, Department of Chemical Engineering, Escola Politécnica, University of São Paulo, C.P. 8174, São Paulo, BrazilSearch for more papers by this authorLeonardo Perego Jr., Leonardo Perego Jr. Biochemical Engineering Laboratory, Department of Chemical Engineering, Escola Politécnica, University of São Paulo, C.P. 8174, São Paulo, BrazilSearch for more papers by this authorLuiz H. Koshimizu, Luiz H. Koshimizu Biochemical Engineering Laboratory, Department of Chemical Engineering, Escola Politécnica, University of São Paulo, C.P. 8174, São Paulo, BrazilSearch for more papers by this authorWalter Borzani, Walter Borzani Biochemical Engineering Laboratory, Department of Chemical Engineering, Escola Politécnica, University of São Paulo, C.P. 8174, São Paulo, BrazilSearch for more papers by this author First published: June 1978 https://doi.org/10.1002/bit.260200609Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume20, Issue6June 1978Pages 899-901 ReferencesRelatedInformation

Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli.

Escherichia coli B was cultured continuously in succinate-minimal medium under conditions of oxygen limitation in the phauxostat. With decreasing oxygenation and consequent decreasing growth rates, the complement of terminal cytochrome oxidases changed as follows: high growth rates, cytochrome o; intermediate growth rates, cytochromes o and d; lowest growth rates, cytochromes o, d, and a1. Respiratory kinetics exhibited by nongrowing cell suspensions obtained from continuous cultures indicated that terminal oxidase activity was exhibited by cytochrome o (Km for O2 = 0.2 micron; Vmax = 1.1 to 1.5 mumol of O2 per nmol of cytochrome o per min) and cytochrome d (Km for O2 = 0.024 micron; Vmax = 0.7 mumol of O2 per nmol of cytochrome d per min). During oxygen-limited growth, the molar growth yield referred to respiration, and corrected for maintenance respiration [Yo(max)], was 12.6 g (dry weight) per g-atom of oxygen, not significantly different from the succinate-limited value of 12.0 g (dry weight) per g-atom of oxygen. The rate of maintenance respiration of the oxygen-limited culture was only 3.4 mg-atoms of O per g (dry weight) per h, some threefold less than that of the succinate-limited culture. Respiration-driven proton extrusion did not vary with the growth rate or with the complement of terminal oxidases (H+/O = 3.7; standard deviation, 0.07). We conclude that the content of terminal oxidases is without effect on the efficiency of respiratory energy conservation.

Cytochrome Formation, Oxygen-induced Proton Extrusion and Respiratory Activity in Streptococcus faecalis var. zymogenes Grown in the Presence of Haematin

Streptococcus faecalis var. zyrnogenes was grown in oxygen-limited continuous culture in a lactate/tryptone/yeast extract medium containing 7·5 μg haematin ml-l. Low temperature difference spectra of whole organisms and washed membranes showed two peaks in the cytochrome b region, at 558 nm and 562 nm, and a peak at 627 nm indicative of a d-type cytochrome. A CO-binding cytochrome was also present in the haematin-grown bacteria. These cytochromes were not detected in bacteria grown without haematin. The haematin- grown bacteria produced proton pulses in a weakly buffered medium when pulsed with oxygen-saturated buffer. The average →H+/O ratio of such pulses was 1·4 and they were abolished by carbonyl cyanide m-chlorophenylhydrazone (CCCP). The haematin-grown bacteria had a high activity of particulate NADH oxidase, at least 10 times that of bacteria grown without haematin. They also had a high lactate Q 02 which was strongly inhibited by CCCP and gramicidin whereas bacteria grown without haematin had a very low lactate Q 02 largely insensitive to the uncoupling agents. The glucose Q 02 was similar in bacteria grown in the presence or absence of haematin but was stimulated by uncoupling agents in haematin- grown organisms and slightly inhibited by these agents in organisms grown without haematin. These results confirm earlier findings of the ability of S. faecalis to form a functional cytochrome system in the cell membrane when supplied with haematin and show that electron transport to oxygen by such a system is coupled to proton translocation.

The role of O 2-limitation in control of nitrogenase in continuous cultures of Rhizobium sp

Oxygen-limited continuous cultures of the cowpea Rhizobium sp. strain CB756, had high levels of nitrogenase activity, which were not significantly affected by excess ammonium ions or glutamine. When the growth-restricting O 2-limitation was partially relieved, nitrogenase was repressed and this was accompanied by increased adenylylation of glutamine synthetase. It is suggested that the restricted supply of ATP interferes with adenylylation of glutamine synthetase during O 2-limited growth, thus preventing repression of nitrogenase in the presence of excess ammonium ions.