Shift from growth to nutrient-limited maintenance kinetics during biofilter acclimation

During long-term operation of a biofilter, the mandatory absence of net cell growth forces the cells into maintenance metabolism, which is of relatively low rate compared to substrate consumption during the active growth of the acclimation phase. A model based on this shift in metabolism can explain the postacclimation decrease in activity sometimes reported for biofilters. The cessation of growth can be caused by nutrient depletion in the bed. Postacclimation nutrient addition increases activity primarily by allowing a return to the high substrate consumption rate of active growth, and only secondarily helps raise bed activity because of the ultimately higher amount of biomass in the bed. Simulations incorporating the acclimation period and the role of maintenance metabolism predict about 4 logarithms of growth during acclimation of a hexane biofilter, which was confirmed experimentally. Changes in a biofilter's biomass during the acclimation phase can be estimated from substrate conversion data using two approximate methods. The first follows the cumulative amount of substrate converted and uses the estimated yield of cells from substrate during active growth to estimate the total biomass created. The second method follows a rate constant for conversion of substrate in the bed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 330-339, 1997.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.     

Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence

The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E. coli strain. Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor). The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion). Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells. These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain. These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures.     

Inactivation of the Pta-AckA Pathway Causes Cell Death in Staphylococcus aureus

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD⁺, and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism.     

Constrained Allocation Flux Balance Analysis

New experimental results on bacterial growth inspire a novel top-down approach to study cell metabolism, combining mass balance and proteomic constraints to extend and complement Flux Balance Analysis. We introduce here Constrained Allocation Flux Balance Analysis, CAFBA, in which the biosynthetic costs associated to growth are accounted for in an effective way through a single additional genome-wide constraint. Its roots lie in the experimentally observed pattern of proteome allocation for metabolic functions, allowing to bridge regulation and metabolism in a transparent way under the principle of growth-rate maximization. We provide a simple method to solve CAFBA efficiently and propose an “ensemble averaging” procedure to account for unknown protein costs. Applying this approach to modeling E. coli metabolism, we find that, as the growth rate increases, CAFBA solutions cross over from respiratory, growth-yield maximizing states (preferred at slow growth) to fermentative states with carbon overflow (preferred at fast growth). In addition, CAFBA allows for quantitatively accurate predictions on the rate of acetate excretion and growth yield based on only 3 parameters determined by empirical growth laws.     

Metabolic Footprint Analysis Uncovers Strain Specific Overflow Metabolism and D-Isoleucine Production of Staphylococcus Aureus COL and HG001

During infection processes, Staphylococcus aureus is able to survive within the host and to invade tissues and cells. For studying the interaction between the pathogenic bacterium and the host cell, the bacterial growth behaviour and its metabolic adaptation to the host cell environment provides first basic information. In the present study, we therefore cultivated S. aureus COL and HG001 in the eukaryotic cell culture medium RPMI 1640 and analyzed the extracellular metabolic uptake and secretion patterns of both commonly used laboratory strains. Extracellular accumulation of D-isoleucine was detected starting during exponential growth of COL and HG001 in RPMI medium. This non-canonical D-amino acid is known to play a regulatory role in adaptation processes. Moreover, individual uptake of glucose, accumulation of acetate, further overflow metabolites, and intermediates of the branched-chain amino acid metabolism constitute unique metabolic footprints. Altogether these time-resolved footprint analyses give first metabolic insights into staphylococcal growth behaviour in a culture medium used for infection related studies.     

Influence of the Crc global regulator on substrate uptake rates and the distribution of metabolic fluxes in Pseudomonas putida KT2440 growing in a complete medium

When the soil bacterium Pseudomonas putida grows in a complete medium, it prioritizes the assimilation of preferred carbon sources, optimizing its metabolism and growth. This regulatory process is orchestrated by the Crc and Hfq proteins. The present work examines the changes that occur in metabolic fluxes when the crc gene is inactivated and cells grow exponentially in LB complete medium. Analyses were performed at three different moments during exponential growth, examining the assimilation rates for the compounds present in LB, changes in the proteome, and the changes in metabolic fluxes predicted by the iJN1411 metabolic model for P. putida KT2440. During the early exponential phase, consumption rates for sugars, many organic acids and most amino acids were higher in a Crc-null strain than in the wild type, leading to an overflow of the metabolic pathways and the leakage of pyruvate and acetate. These accelerated consumption rates decreased during the mid-exponential phase, when cells mostly used sugars and alanine. At later times, pyruvate was recovered from the medium and utilized. The higher consumption rates of the Crc-null strain reduced the growth rate. The lack of the Crc/Hfq regulatory system thus led to unbalanced metabolism with poorly optimized metabolic fluxes.     

Overflow metabolism at the thermodynamic limit of life: How carboxydotrophic acetogens mitigate carbon monoxide toxicity

Carboxydotrophic metabolism is gaining interest due to its applications in gas fermentation technology, enabling the conversion of carbon monoxide to fuels and commodities. Acetogenic carboxydotrophs play a central role in current gas fermentation processes. In contrast to other energy-rich microbial substrates, CO is highly toxic, which makes it a challenging substrate to utilize. Instantaneous scavenging of CO upon entering the cell is required to mitigate its toxicity. Experiments conducted with Clostridium autoethanogenum at different biomass-specific growth rates show that elevated ethanol production occurs at increasing growth rates. The increased allocation of electrons towards ethanol at higher growth rates strongly suggests that C. autoethanogenum employs a form of overflow metabolism to cope with high dissolved CO concentrations. We argue that this overflow branch enables acetogens to efficiently use CO at highly variable substrate influxes by increasing the conversion rate almost instantaneously when required to remove toxic substrate and promote growth. In this perspective, we will address the case study of C. autoethanogenum grown solely on CO and syngas mixtures to assess how it employs acetate reduction to ethanol as a form of overflow metabolism.     

Physiological response of central metabolism in Escherichia coli to deletion of pyruvate oxidase and introduction of heterologous pyruvate carboxylase

We studied the physiological response of Escherichia coli central metabolism to the expression of heterologous pyruvate carboxylase (PYC) in the presence and absence of pyruvate oxidase (POX). These studies were complemented with expression analysis of central and intermediary metabolic genes and conventional in vitro enzyme assays to evaluate glucose metabolism at steady-state growth conditions (chemostats). The absence of POX activity reduced nongrowth-related energy metabolism (maintenance coefficient) and increased the maximum specific rate of oxygen consumption. The presence of PYC activity (i.e., with POX activity) increased the biomass yield coefficient and reduced the maximum specific oxygen consumption rate compared to the wildtype. The presence of PYC in a poxB mutant resulted in a 42% lower maintenance coefficient and a 42% greater biomass yield compared to the wildtype. Providing E. coli with PYC or removing POX increased the threshold specific growth rate at which acetate accumulation began, with an 80% reduction in acetate accumulation observed at a specific growth rate of 0.4 h-1 in the poxB-pyc+ strain. Gene expression analysis suggests utilization of energetically less favorable glucose metabolism via glucokinase and the Entner-Doudoroff pathway in the absence of functional POX, while the upregulation of the phosphotransferase glucose uptake system and several amino acid biosynthetic pathways occurs in the presence of PYC. The physiological and expression changes resulting from these genetic perturbations demonstrate the importance of the pyruvate node in respiration and its impact on acetate overflow during aerobic growth.     

An in vivo analysis of the energetics of aldose oxidation by Acinetobacter calcoaceticus

Summary A continuous culture study was made of the energetics of oxidation of various aldose sugars by Acinetobacter calcoaceticus LMD 79.41. The consumption of aldoses during carbon- and energy-limited growth of the organism on mixtures of acetate and an aldose was independent of the pH of the culture. Acid production, however, was strongly dependent on this parameter. It is shown that aldose consumption without concurrent acid production is due to formation of the corresponding lactone, the hydrolysis of which is pH-dependent. The cell yield of A. calcoaceticus on mixtures of acetate and glucose or xylose was much higher than during growth on acetate alone. This increase in cell yield was, however, dependent on the pH of the culture. Only at pH values which permitted a high rate of lactone hydrolysis an enhancement of the cell yield was observed. These results suggest that lactone hydrolysis has an important bearing on the efficiency of periplasmic oxidation of aldoses in bacteria.     

Characterization of changes in PER.C6 cellular metabolism during growth and propagation of a replication-deficient adenovirus vector

PER.C6 cells were cultivated for propagation of a replication-defective adenovirus vector in serum-free suspension bioreactors. Cellular metabolism during cell growth and adenovirus propagation was fully characterized using on-line and off-line methods. The energy metabolism was found to accelerate transiently after adenovirus infection with increases in glucose and oxygen consumption rates. Similar to other mammalian cells, glucose utilization was highly inefficient and a high lactate:glucose yield was observed, both before and after virus infection. A higher consumption of most of the essential amino acids was observed transiently after the infection, likely due to increased protein synthesis requirements for virus propagation. To improve virus propagation, a medium exchange strategy was implemented to increase PER.C6 cell concentration for infection. During cell growth, a 50% increase in glucose consumption and lactate production rates was observed after initiation of the medium exchange in comparison to the batch phase. This decrease in medium capacity only affected the central carbon metabolism and no increase in amino acid consumption was observed. In addition, even though cell concentrations of up to 10 x 10(6) cells/mL were reproducibly obtained by medium exchange, infections at cell concentrations higher than 1 x 10(6) cells/mL did not proportionally improve volumetric adenovirus productivities. No measured nutrient limitation was observed at those high cell concentrations, indicating that adenovirus cell-specific productivity at higher cell concentrations is highly dependent on cell physiology. These results provide a better understanding of PER.C6 cellular metabolism and a basis for intensifying PER.C6 growth and adenovirus propagation.     

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.     

From population ecology to metabolism: growth of Trypanosoma evansi,and implications of glucose depletion,in a live host

Little attention has been paid to the potential top-down effects of population ecology on metabolism. Because it is capable of a fast growth that dramatically modifies its blood environment, the parasitic protozoan, Trypanosoma evansi, is a promising model for the study of density-dependent metabolic processes. We assess the in vivo growth rate, doubling time, biomass yield, glycolytic flux, and enzyme expression of T. evansi. Then, we explore the metabolic changes likely occurring during its growth. At low T. evansi densities, most host glucose is used to produce energy, which results in the release of pyruvate. Part of glucose is used for NADPH production through the pentose phosphate pathway. At high T. evansi densities, fructose, mannose, and glycerol become additional energy sources. Part of host glucose is used for biosynthesis and for NADPH production through alternative metabolic pathways, which results in the release of succinate, alanine, and acetate. The ability of organisms to adjust to resource changes is crucial to their survival. Irrespective if the triggering mechanism is direct (nutrient limitation) or indirect (a pheromone-like factor), nutrient availability is necessarily the main evolutionary factor responsible for the density-dependent metabolic properties of trypanosome populations.     

Acetate accumulation and regulation by process parameters control in Chinese hamster ovary cell culture

Chinese hamster ovary (CHO) cells represent a group of predominantly used mammalian hosts for producing recombinant therapeutic proteins. Known for their rapid proliferation rates, CHO cells undergo aerobic glycolysis that is characterized by fast glucose consumption, that ultimately gives rise to a group of small-molecule organic acids. However, only the function of lactate has been extensively studied in CHO cell culture. In this study, we observed the accumulation of acetate from the late exponential phase to harvest day, potentially contributing to the pH decline in late culture stage regardless of lactate consumption. In addition, we evaluated the acidification of the fresh media and the cell culture suspension, and the data revealed that acetate presented a lower acidification capacity compared to lactate and exhibited limited inhibitory effect on cells with less than 20 mM supplemented in the media. This study also explored the ways to control acetate accumulation in CHO cell culture by manipulating the process parameters such as temperature, glucose, and pH control. The positive correlation between the specific glucose consumption rate and acetate generation rate provides evidence of the endogenous acetate generation from overflow metabolism. Reducing these parameters (temperature, glucose consumption) and HCl-controlled low pH ultimately suppress acetate build-up. In addition, the specific acetate generation rate and relevant glucose consumption rate are found to be a metabolic trait associated with specific cell lines. Taken together, the results presented in these experiments provide a means to advance industrial CHO cell culture process control and development.     

On population dynamics in multi-species cultures of diatoms and dinoflagellates

In order to assess the phenomena possibly underlying population dynamics and species succession in the sea, the following phytoplankton culture experiments were made. In uni-algal and in multi-algal batch cultures, generation times and cell yields gained during logarithmic growth were determined for the diatomsBiddulphia regia andCoscinodiscus concinnus, as well as for the dinoflagellates,Ceratium horridum andProrocentrum micans. In multi-species cultures, none of the tested organisms showed any influence on generation time, compared with uni-algal cultures. In contrast, the cell yield of different species showed considerable changes depending on the species concerned and the species-combination used. The dinoflagellatesC. horridum reached, if cultivated together withB. regia orB. regia andC. concinnus, only 10% of the cell number of uni-algal cultures. In the combinations tested,B. regia produced always more than half of the cell number attained in uni-algal cultures. In multi-species cultures,C. concinnus cell production was not affected. Addition of nitrate and phosphate to stationary-phase multi-species cultures induced further growth. Thus it is concluded that growth is limited by nutrient competition in the multi-species experiments conducted. Possible mechanisms of nutrient competition are discussed.     

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g/L succinic acid with a yield of 0.53 g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.     

Aerobic glucose metabolism of Saccharomyces kluyveri: growth, metabolite production, and quantification of metabolic fluxes.

The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0.29 +/- 0.01 g/g). The glucose metabolism of S. kluyveri was further characterized by the new and powerful techniques of metabolic network analysis. Flux distributions in the central carbon metabolism were estimated for respiro-fermentative growth in aerobic batch cultivation on glucose and respiratory growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate pathway cannot provide the cell with enough NADPH for biomass formation which is why the remaining NADPH will have to be provided by another pathway. During batch cultivation of S. kluyveri the tricarboxylic acid cycle was working as a cycle with a considerable flux, that is in sharp contrast to what has previously been observed in S. cerevisiae at the same growth conditions, where the tricarboxylic acid cycle operates as two branches. This indicates that the respiratory system was not significantly repressed in S. kluyveri during batch cultivation on glucose.     

Comparison of unitrophic and mixotrophic substrate metabolism by acetate-adapted strain of Methanosarcina barkeri

We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed ¹⁴C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH₄ produced, but 14% of the CO₂ generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH₄. ¹⁴CH₄ was also produced from added ¹⁴CO₂, although to a lesser extent than from reduction of the C-1 of acetate. During mixotrophic metabolism, methanol and acetate were catabolized simultaneously. The rates of ¹⁴CH₄ and ¹⁴CO₂ generation from [2-¹⁴C]acetate were logarithmic and higher in mixotrophic than in unitrophic cultures at substrate concentrations of 50 mM. A comparison of the oxidoreductase activities in cell extracts of the acetate-adapted strain grown on acetate and of strain MS grown on methanol or on H₂ plus CO₂ indicated that the pyruvate, α-ketoglutarate, and isocitrate dehydrogenase activities remained constant, whereas the CO dehydrogenase activity was significantly higher (5,000 nmol/min per mg of protein) in the acetate-adapted strain. These results suggested that a significant intramolecular redox pathway is possible for the generation of CH₄ from acetate, that energy metabolism from acetate by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity.     

水浮莲对水稻竞争效应、产量与土壤养分的影响

为了解入侵植物水浮莲在稻田生产中的扩散规律和对水稻农业性状的影响,通过田间水稻田实验,按照de Wit 取代试验方法和添加系列设计方法研究水浮莲与水稻(云稻2 号)混种对植株形态和生物量影响、种间竞争效应、水稻产量以及对土壤养分的影响。结果表明,混种条件下水浮莲母株株高、分蘖数、生物量和开花株数均受到水稻的明显抑制,受到的抑制率显著高于其对水稻的抑制率。水浮莲的种间竞争大于种内竞争(RY 小于1.0)而水稻的种内竞争大于种间竞争(RY 大于1.0),水浮莲混种比例大于和等于1:1 时(RYT大于1.0)与水稻不存在竞争作用,而小于1:1时(RYT 小于1.0)其存在着竞争作用,水浮莲对水稻的竞争力(CB 小于0)小于水稻。混种条件下水稻有效穗数和产量有明显提高,增产比例为3.54%-13.38%。生长过程中水浮莲对土壤钾、磷元素消耗大于水稻,而有机质和氮元素消耗小于水稻;混种条件下水稻明显降低水浮莲对土壤养分消耗,且二者在土壤养分上没有竞争关系。所有这些表明,入侵稻田的水浮莲与水稻生长过程中其形态、生物量等方面都处于劣势,而且一定的水浮莲密度有利于抑制水浮莲对土壤养分的消耗和促进水稻生长繁殖及其产量的提高,因此为满足饲料利用和环境净化,建议在正常耕作稻田中可对水浮莲进行适当的应用。