Showing papers on "Chemostat published in 2008"

Glycerol fermentation by (open) mixed cultures: a chemostat study.

Abstract: Glycerol is an important byproduct of bioethanol and biodiesel production processes This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose Both reactors operated at a pH of 8 and a dilution rate of 01 h−1 Glycerol was mainly converted into ethanol and formate When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations Glucose fermentation mainly generated acetate, ethanol and butyrate At higher substrate concentrations, acetate and ethanol were the dominant products Co-fermentations of glucose–glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively) The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved Biotechnol Biotechnol Bioeng 2008;100: 1088–1098 © 2008 Wiley Periodicals, Inc

Bacterial physiology, regulation and mutational adaptation in a chemostat environment.

Abstract: The chemostat was devised over 50 years ago and rapidly adopted for studies of bacterial physiology and mutation Despite the long history and earlier analyses, the complexity of events in continuous cultures is only now beginning to be resolved The application of techniques for following regulatory and mutational changes and the identification of mutated genes in chemostat populations has provided new insights into bacterial behaviour Inoculation of bacteria into a chemostat culture results in a population competing for a limiting amount of a particular resource Any utilizable carbon source or ion can be a limiting nutrient and bacteria respond to limitation through a regulated nutrient-specific hunger response In addition to transcriptional responses to nutrient limitation, a second regulatory influence in a chemostat culture is the reduced growth rate fixed by the dilution rate in individual experiments Sub-maximal growth rates and hunger result in regulation involving sigma factors and alarmones like cAMP and ppGpp Reduced growth rate also results in increased mutation frequencies The combination of a strongly selective environment (where mutants able to compete for limiting nutrient have a major fitness advantage) and elevated mutation rates (both endogenous and through the secondary enrichment of mutators) results in a population that changes rapidly and persistently over many generations Contrary to common belief, the chemostat environment is never in "steady state" with fixed bacterial characteristics usable for clean comparisons of physiological or regulatory states Adding to the complexity, chemostat populations do not simply exhibit a succession of mutational sweeps leading to a dominant winner clone Instead, within 100 generations large populations become heterogeneous and evolving bacteria adopt alternative, parallel fitness strategies Transport physiology, metabolism and respiration, as well as growth yields, are highly diverse in chemostat-evolved bacteria The rich assortment of changes in an evolving chemostat provides an excellent experimental system for understanding bacterial evolution The adaptive radiation or divergence of populations into a collection of individuals with alternative solutions to the challenge of chemostat existence provides an ideal model system for testing evolutionary and ecological theories on adaptive radiations and the generation of bacterial diversity

Physiological and transcriptional responses to high concentrations of lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae.

Abstract: Based on the high acid tolerance and the simple nutritional requirements of Saccharomyces cerevisiae, engineered strains of this yeast are considered biocatalysts for industrial production of high-purity undissociated lactic acid. However, high concentrations of lactic acid are toxic to S. cerevisiae, thus limiting its growth and product formation. Physiological and transcriptional responses to high concentrations of lactic acid were studied in anaerobic, glucose-limited chemostat cultures grown at different pH values and lactic acid concentrations, resulting in a 50% decrease in the biomass yield. At pH 5, the yield decrease was caused mostly by osmotically induced glycerol production and not by the classic weak-acid action, as was observed at pH 3. Cultures grown at pH 5 with 900 mM lactic acid revealed an upregulation of many genes involved in iron homeostasis, indicating that iron chelation occurred at high concentrations of dissociated lactic acid. Chemostat cultivation at pH 3 with 500 mM lactate, resulting in lower anion concentrations, showed an alleviation of this iron homeostasis response. Six of the 10 known targets of the transcriptional regulator Haa1p were strongly upregulated in lactate-challenged cultures at pH 3 but showed only moderate induction by high lactate concentrations at pH 5. Moreover, the haa1Δ mutant exhibited a growth defect at high lactic acid concentrations at pH 3. These results indicate that iron homeostasis plays a major role in the response of S. cerevisiae to high lactate concentrations, whereas the Haa1p regulon is involved primarily in the response to high concentrations of undissociated lactic acid.

Growth of Thiobacillus ferrooxidans on ferrous iron in chemostat culture: Influence of product and substrate inhibition

Abstract: Thiobacillus ferrooxidans was grown at pH 1.6 in continuous flow chemostat culture on ferrous sulphate as growth limiting substrate at dilution rates between 0.02–1.33 h−1. Iron oxidation and growth were subject to product inhibition by ferric iron and under some conditions substrate inhibition by ferrous iron. Product inhibition could be predominantly competitive or non-competitive, and the mode observed depended partly on previous steady state conditions. The inhibition phenomena resulted in unique anomalous washout curves and complex relationships between steady-state substrate, product and biomass concentrations, for which mathematical models are developed. For the growth states subject to non-competitive inhibition by Fe3+ at D 0.073–0.99 h−1, the growth yield coefficient corrected for maintenance (YG) was 1.33 g dry wt (g atom Fe2+ oxidized)−1 and the maintenance coefficient (m) was 0.43 g atom Fe2+ oxidized (g dry wt)−1 h−1). For predominantly competitive states (D, 0.05–0.268 h−1) with 2–70 mM Fe3+ in steady states, YG was 0.36–0.38 and m was 0–0.04. A consequence of product inhibition and substrate inhibition was the possibility of more than one steady state product value and yield for a single steady state substate concentration. This was demonstrated experimentally. Substrate saturation coefficient, Ks (giving half maximum specific growth rate) for Fe2+, was 0.7–2.4 mM and maximum specific growth rate (μm) 1.25–1.78 h−1. The results presented reveal unusual and novel properties of T. ferrooxidans relevant to describing its activities in natural environments or in mineral leaching systems.

Chemostat-Based Micro-Array Analysis in Baker's Yeast

Abstract: Chemostat cultivation of micro-organisms offers unique opportunities for experimental manipulation of individual environmental parameters at a fixed, controllable specific growth rate. Chemostat cultivation was originally developed as a tool to study quantitative aspects of microbial growth and metabolism. Renewed interest in this cultivation method is stimulated by the availability of high-information-density techniques for systemic analysis of microbial cultures, which require high reproducibility and careful experimental design. Genome-wide analysis of transcript levels with DNA micro-arrays is currently the most commonly applied of these high-information-density analysis tools for microbial gene expression. Based on published studies on the yeast Saccharomyces cerevisiae, a critical overview is presented of the possibilities and pitfalls associated with the combination of chemostat cultivation and transcriptome analysis with DNA micro-arrays. After a brief introduction to chemostat cultivation and micro-array analysis, key aspects of experimental design of chemostat-based micro-array experiments are discussed. The main focus of this review is on key biological concepts that can be accessed by chemostat-based micro-array analysis. These include effects of specific growth rate on transcriptional regulation, context-dependency of transcriptional responses, correlations between transcript profiles and contribution of the corresponding proteins to cellular function and fitness, and the analysis and application of evolutionary adaptation during prolonged chemostat cultivation. It is concluded that, notwithstanding the incompatibility of chemostat cultivation with high-throughput analysis, integration of chemostat cultivation with micro-array analysis and other high-information-density analytical approaches (e.g. proteomics and metabolomics techniques) offers unique advantages in terms of reproducibility and experimental design in comparison with standard batch cultivation systems. Therefore, chemostat cultivation and derived methods for controlled cultivation of micro-organisms are anticipated to become increasingly important in microbial physiology and systems biology.

Growth rate regulated genes and their wide involvement in the Lactococcus lactis stress responses

Abstract: Background The development of transcriptomic tools has allowed exhaustive description of stress responses. These responses always superimpose a general response associated to growth rate decrease and a specific one corresponding to the stress. The exclusive growth rate response can be achieved through chemostat cultivation, enabling all parameters to remain constant except the growth rate.

Physiological State, Growth Mode, and Oxidative Stress Play a Role in Cd(II)-Mediated Inhibition of Nitrosomonas europaea 19718

Abstract: The goal of this study was to determine the impact of physiological growth states (batch exponential and batch stationary growth) and growth modes (substrate-limited chemostat, substrate-sufficient exponential batch, and substrate-depleted stationary batch growth) on several measures of growth and responses to Cd(II)-mediated inhibition of Nitrosomonas europaea strain 19718. The specific oxygen uptake rate (sOUR) was the most sensitive indicator of inhibition among the different responses analyzed, including total cell abundance, membrane integrity, intracellular 16S rRNA/DNA ratio, and amoA expression. This observation remained true irrespective of the physiological state, the growth mode, or the mode of Cd(II) exposure. Based on the sOUR, a strong time-dependent exacerbation of inhibition (in terms of an inhibition coefficient [Ki]) in exponential batch cultures was observed. Long-term inhibition levels (based on Ki estimates) in metabolically active chemostat and exponential batch cultures were also especially severe and comparable. In contrast, the inhibition level in stationary-phase cultures was 10-fold lower and invariable with exposure time. Different strategies for surviving substrate limitation (a 10-fold increase in amoA expression) and starvation (the retention of 16S rRNA levels) in N. europaea cultures were observed. amoA expression was most negatively impacted by Cd(II) exposure in the chemostat cultures, was less impacted in exponential batch cultures, and was least impacted in stationary batch cultures. Although the amoA response was consistent with that of the sOUR, the amoA response was not as strong. The intracellular 16S rRNA/DNA ratio, as determined by fluorescence in situ hybridization, also did not uniformly correlate with the sOUR under conditions of inhibition or no inhibition. Finally, Cd(II)-mediated inhibition of N. europaea was attributed partially to oxidative stress.

Transcription factor control of growth rate dependent genes in Saccharomyces cerevisiae: A three factor design

Abstract: Characterization of cellular growth is central to understanding living systems. Here, we applied a three-factor design to study the relationship between specific growth rate and genome-wide gene expression in 36 steady-state chemostat cultures of Saccharomyces cerevisiae. The three factors we considered were specific growth rate, nutrient limitation, and oxygen availability. We identified 268 growth rate dependent genes, independent of nutrient limitation and oxygen availability. The transcriptional response was used to identify key areas in metabolism around which mRNA expression changes are significantly associated. Among key metabolic pathways, this analysis revealed de novo synthesis of pyrimidine ribonucleotides and ATP producing and consuming reactions at fast cellular growth. By scoring the significance of overlap between growth rate dependent genes and known transcription factor target sets, transcription factors that coordinate balanced growth were also identified. Our analysis shows that Fhl1, Rap1, and Sfp1, regulating protein biosynthesis, have significantly enriched target sets for genes up-regulated with increasing growth rate. Cell cycle regulators, such as Ace2 and Swi6, and stress response regulators, such as Yap1, were also shown to have significantly enriched target sets. Our work, which is the first genome-wide gene expression study to investigate specific growth rate and consider the impact of oxygen availability, provides a more conservative estimate of growth rate dependent genes than previously reported. We also provide a global view of how a small set of transcription factors, 13 in total, contribute to control of cellular growth rate. We anticipate that multi-factorial designs will play an increasing role in elucidating cellular regulation.

Continuous fermentation of undetoxified dilute acid lignocellulose hydrolysate by Saccharomyces cerevisiae ATCC 96581 using cell recirculation.

Abstract: Saccharomyces cerevisiae ATCC 96581 was cultivated in a chemostat reactor with undetoxified dilute acid softwood hydrolysate as the only carbon and energy source. The effects of nutrient addition, dilution rate, cell recirculation, and microaerobicity were investigated. Fermentation of unsupplemented dilute acid lignocellulose hydrolysate at D = 0.10 h(-1) in an anaerobic continuous reactor led to washout. Addition of ammonium sulfate or yeast extract was insufficient for obtaining steady state. In contrast, dilute acid lignocellulose hydrolysate supplemented with complete mineral medium, except for the carbon and energy source, was fermentable under anaerobic steady-state conditions at dilution rates up to 0.14 h(-1). Under these conditions, washout occurred at D = 0.15 h(-1). This was preceded by a drop in fermentative capacity and a very high specific ethanol production rate. Growth at all different dilution rates tested resulted in residual sugar in the chemostat. Cell recirculation (90%), achieved by cross-flow filtration, increased the sugar conversion rate from 92% to 99% at D = 0.10 h(-1). Nutrient addition clearly improved the long-term ethanol productivity in the recirculation cultures. Application of microaerobic conditions on the nutrient-supplemented recirculation cultures resulted in a higher production of biomass, a higher cellular protein content, and improved fermentative capacity, which further improves the robustness of fermentation of undetoxified lignocellulose hydrolysate.

Short Communication Optimization of chemically defined medium for recombinant Pichia pastoris for biomass production

Abstract: A chemically defined medium was optimized for the maximum biomass production of recombinant Pichia pastoris in the fermentor cultures using glycerol as the sole carbon source. Optimization was done using the statistical methods for getting the optimal level of salts, trace metals and vitamins for the growth of recombinant P. pastoris. The response surface methodology was effective in optimizing nutritional requirements using the limited number of experiments. The optimum medium composition was found to be 20 g/L glycerol, 7.5 g/L (NH4)2SO4, 1 g/L MgSO4� 7H2O, 8.5 g/L KH2PO4, 1.5 mL/L vitamin solution and 20 mL/L trace metal solution. Using the optimized medium 11.25 g DCW/L biomass was produced giving a yield coefficient of 0.55 g biomass/g of glycerol in a batch culture. Chemostat cultivation of recombinant P. pastoris was done in the optimized medium at different dilution rates to determine the kinetic parameters for growth on glycerol. Maximum specific growth rate of 0.23 h � 1 and Monod saturation constant of 0.178 g/L were determined by applying Monod model on the steady state data. Products of fermentation pathway, ethanol and acetate, were not detected by HPLC even at higher dilution rates. This supports the notion that P. pastoris cells grow on glycerol by a respiratory route and are therefore an efficient biomass and protein producers. 2008 Elsevier Ltd. All rights reserved.

Further Results on Stabilization of Periodic Trajectories for a Chemostat With Two Species

Abstract: We discuss an important class of problems involving the tracking of prescribed trajectories in the chemostat model. We provide new tracking results for chemostats with two species and one limiting substrate, based on Lyapunov function methods. In particular, we use a linear feedback control of the dilution rate and an appropriate time-varying substrate input concentration to produce a locally exponentially stable oscillatory behavior. This means that all trajectories for the nutrient and corresponding species concentrations in the closed loop chemostat that stay near the oscillatory reference trajectory are attracted to the reference trajectory exponentially fast. We also obtain a globally stable oscillatory reference trajectory for the species concentrations, using a nonlinear feedback control depending on the dilution rate and the substrate input concentration. This guarantees that all trajectories for the closed loop chemostat dynamics are attracted to the reference trajectory. Finally, we construct an explicit Lyapunov function for the corresponding global error dynamics. We demonstrate the efficacy of our method in a simulation.

Element content of Pseudomonas fluorescens varies with growth rate and temperature: A replicated chemostat study addressing ecological stoichiometry

Abstract: Ecological stoichiometry is emerging as a central organizing framework upon which our perceptions of aquatic trophic dynamics are being reshaped. The microbial component of aquatic systems is crucial to overall nutrient dynamics, yet little data are available addressing the ecological stoichiometry of microorganisms. Pseudomonas fluorescens, a commonly encountered bacterium, was used as a model organism to investigate the relationships among temperature, growth rate, and element stoichiometry. P. fluorescens was grown in chemostats at low dilution rates (ranging between 0.03 and 0.13 h21) and realistic environmental temperatures (ranging between 14uC and 28uC). Cells accumulated elements as an interactive function of temperature and growth rate. The highest element concentrations corresponded to cells growing slowly under low temperatures and to cells growing rapidly under warm conditions. Additionally, small cells had higher concentrations of elements than did large cells. Element ratios (C : N, C : P, and N : P) varied more as a function of growth rate than of temperature. The same dissolved resource pool could conceivably yield bacteria of differing element content simply as an interactive function of growth rate and temperature.

Transcription of hexose transporters of Saccharomyces cerevisiae is affected by change in oxygen provision.

Abstract: The gene family of hexose transporters in Saccharomyces cerevisiae consists of 20 members; 18 genes encoding transporters (HXT1-HXT17, GAL2) and two genes encoding sensors (SNF3, RGT2). The effect of oxygen provision on the expression of these genes was studied in glucose-limited chemostat cultivations (D = 0.10 h-1, pH 5, 30°C). Transcript levels were measured from cells grown in five steady state oxygen levels (0, 0.5, 1, 2.8 and 20.9% O2), and from cells under conditions in which oxygen was introduced to anaerobic cultures or removed from cultures receiving oxygen. The expression pattern of the HXT gene family was distinct in cells grown under aerobic, hypoxic and anaerobic conditions. The transcription of HXT2, HXT4 and HXT5 was low when the oxygen concentration in the cultures was low, both under steady state and non-steady state conditions, whereas the expression of HXT6, HXT13 and HXT15/16 was higher in hypoxic than in fully aerobic or anaerobic conditions. None of the HXT genes showed higher transcript levels in strictly anaerobic conditions. Expression of HXT9, HXT14 and GAL2 was not detected under the culture conditions studied. When oxygen becomes limiting in a glucose-limited chemostat cultivation, the glucose uptake rate per cell increases. However, the expression of none of the hexose transporter encoding genes was increased in anaerobic conditions. It thus seems that the decrease in the moderately low affinity uptake and consequently the relative increase of high affinity uptake may itself allow the higher specific glucose consumption rate to occur in anaerobic compared to aerobic conditions.

Revisiting the role of yeast Sfp1 in ribosome biogenesis and cell size control: a chemostat study.

Abstract: Saccharomyces cerevisiae SFP1 promotes transcription of a large cluster of genes involved in ribosome biogenesis. During growth in shake flasks, a mutant deleted for SFP1 shows a small size phenotype and a reduced growth rate. We characterized the behaviour of an sfp1Δ mutant compared to an isogenic reference strain growing in chemostat cultures at the same specific growth rate. By studying glucose (anaerobic)- and ethanol (aerobic)-limited cultures we focused specifically on nutrient-dependent effects. Major differences in the genome-wide transcriptional profiles were observed during glucose-limited growth. In particular, Sfp1 appeared to be involved in the control of ribosome biogenesis but not of ribosomal protein gene expression. Flow cytometric analyses revealed size defects for the mutant under both growth conditions. Our results suggest that Sfp1 plays a role in transcriptional and cell size control, operating at two different levels of the regulatory network linking growth, metabolism and cell size.

Simpler noninstrumented batch and semicontinuous cultures provide mammalian cell kinetic data comparable to continuous and perfusion cultures.

Abstract: Perfusion culture optimization in multiple noninstrumented small-scale flasks allows reduced expense and time associated with process development. These cultures normally use a different process mode because at small scales it is not practical to retain the cells for medium perfusion. In this work, the kinetics of growth, nutrient consumption, metabolite, and product formation were compared in spinner cultures operated in batch, semicontinuous, chemostat, and perfusion modes. Fed-batch was also included to provide an added comparison. Using logistic fitting for more reliable specific rate estimates in transient conditions, the growth phase of batch cultures predicted similar kinetics to fed-batch and continuous processes. For daily medium exchange rates up to 50%, the semicontinuous mode also predicted the perfusion process kinetics. Differences between the chemostat and semicontinuous culture results were only observed at higher exchange rates with the greatest daily culture perturbation. Overall, the batch or semicontinuous cultures were shown to readily provide results similar to the far more complex to operate chemostat or perfusion cultures.

How flocculation can explain coexistence in the chemostat.

Abstract: We study a chemostat model in which two microbial species grow on a single resource. We show that species coexistence is possible when the species which would normally win the exclusive competition aggregates in flocs. Our mathematical analysis exploits the fact that flocculation is fast compared to biological growth, a common hypothesis in floc models. A numerical study shows the validity of this approach in a large parameter range. We indicate how our model yields a mechanistic justification for the so-called density-dependent growth.

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

Abstract: High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanellaoneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O2 deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.

Biological control of the chemostat with nonmonotonic response and different removal rates.

Abstract: We show the global stabilization of the chemostat with nonmonotonic growth, adding a new species as a "biological" control, in presence of different removal rates for each species. This result is obtained by an extension of the Competitive Exclusion Principle in the chemostat, for the case of two species with different removal rates and at least one nonmonotonic response.

Modeling of ferrous iron oxidation by a Leptospirillum ferrooxidans-dominated chemostat culture.

Abstract: The objective of this study was to evaluate a direct classical bioengineering approach to model data generated from continuous bio-oxidation of Fe(2+) by a Leptospirillum ferrooxidans-dominated culture fed with either 9 g or 18 g Fe(2+) L(-1) under chemostat conditions (dilution rates were between 0.051 and 0.094 h(-1)). The basic Monod and Pirt equations have successfully been integrated in an overall mass balance procedure, which has not been previously presented in this detail for Fe(2+) oxidation. To ensure chemostat conditions, it was found that the range of the dilution rates had to be limited. A too long retention time might cause starvation or non-negligible death rate whereas, a too short retention time may cause a significant alteration in solution chemistry and culture composition. Modeling of the experimental data suggested that the kinetic- and yield parameters changed with the overall solution composition. However, for respective feed solutions only minor changes of ionic strength and chemical speciation can be expected within the studied range of dilution rates, which was confirmed by thermodynamic calculations and conductivity measurements. The presented model also suggests that the apparent Fe(3+) inhibition on specific Fe(2+) utilization rate was a direct consequence of the declining biomass yield on Fe(2+) due to growth uncoupled Fe(2+) oxidation when the dilution rate was decreased. The model suggested that the maintenance activities contributed up to 90% of the maximum specific Fe(2+) utilization rate, which appears close to the critical dilution rate. Biotechnol. Bioeng. 2008;99: 378-389. (c) 2007 Wiley Periodicals, Inc.

The response of the yeast Saccharomyces cerevisiae to sudden vs. gradual changes in environmental stress monitored by expression of the stress response protein Hsp12p.

Abstract: The response of the yeast Saccharomyces cerevisiae to sudden vs. gradual changes in different environmental stress conditions during both respiratory growth and aerobic fermentative growth in the presence of excess glucose was investigated by monitoring the level and rate of expression of the stress response protein Hsp12p using the fluorescent fusion construct Hsp12p-Gfp2p. The initial expression level and the rate of Hsp12p synthesis was significantly greater under glucose-limited conditions in the chemostat (D<0.14 h(-1)) compared with when excess glucose was present in the auxostat. Decreasing the dilution rate and the glucose concentration further in the A-stat resulted in increased Hsp12p expression, which was more marked when a rapid rather than a gradual change was affected. Common stress factors such as NaCl, ethanol and elevated temperature caused stress responses in both D-stat and auxo-accelerostat culture. The magnitude of the stress response depended on the stress factor, cultivation conditions as well as the rate of change of the stress factor. The rate of Hsp12p synthesis increased due to all applied stresses, with the observed increase between 2 and 20 times lower when the stress was applied gradually rather than rapidly. The results suggested that the Hsp12p expression rate is a good indicator of applied stress in S. cerevisiae.

Response of Pseudomonas putida F1 cultures to fluctuating toluene loads and operational failures in suspended growth bioreactors

Abstract: The response of Pseudomonas putida F1 to process fluctuations and operational failures during toluene biodegradation was evaluated in a chemostat suspended growth bioreactor. The ability of P. putida F1 to rapidly increase its specific toluene degradation capacity resulted in no significant variation in process removal efficiency when toluene load was increased from 188 to 341 g m−3 h−1. Likewise, bacterial activity rapidly reached steady state performance (in less than 1.5 h after the restoration of steady state operational conditions) following an 8 h process shutdown, or after episodes of toluene or mineral nutrients deprivation. Process performance was however highly sensitive to pH, as pH levels below 4.5 dramatically inhibited bacterial activity, decreasing severely process robustness and inducing a cycle of periodic process collapses and recoveries. This pH mediated deterioration of bacterial activity was confirmed by further respirometric tests, which revealed a 50–60% reduction in the O2 consumption rate during the degradation of both toluene and 3-methyl catechol when pH decreased from 5.05 to 4.55. Finally, process robustness was quantified according to methods previously described in literature.

Engineering and analysis of a Saccharomyces cerevisiae strain that uses formaldehyde as an auxiliary substrate.

Abstract: We demonstrated that formaldehyde can be efficiently coutilized by an engineered Saccharomyces cerevisiae strain that expresses Hansenula polymorpha genes encoding formaldehyde dehydrogenase (FLD1) and formate dehydrogenase (FMD), in contrast to wild-type strains. Initial chemostat experiments showed that the engineered strain coutilized formaldehyde with glucose, but these mixed-substrate cultures failed to reach steady-state conditions and did not exhibit an increased biomass yield on glucose. Subsequent transcriptome analyses of chemostat cultures of the engineered strain, grown on glucose-formaldehyde mixtures, indicated that the presence of formaldehyde in the feed caused biotin limitations. Further transcriptome analysis demonstrated that this biotin inactivation was prevented by using separate formaldehyde and vitamin feeds. Using this approach, steady-state glucose-limited chemostat cultures were obtained that coutilized glucose and formaldehyde. Coutilization of formaldehyde under these conditions resulted in an enhanced biomass yield of the glucose-limited cultures. The biomass yield was quantitatively consistent with the use of formaldehyde as an auxiliary substrate that generates NADH and subsequently, via oxidative phosphorylation, ATP. On an electron pair basis, the biomass yield increase observed with formaldehyde was larger than that observed previously for formate, which is tentatively explained by different modes of formate and formaldehyde transport in S. cerevisiae.