Anaerobic Continuous Culture Research Articles

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: II. Analysis of metabolic rates and pathways under oscillation and steady-state conditions

The oscillation phenomena reported in the preceding article for the anaerobic continuous fermentation of glycerol by Klebsiella pneumoniae are analyzed in terms of metabolic fluxes (metabolic rates and yields) and stoichiometry of pathways. Significant oscillations in the fluxes of CO(2), H(2), formic acid, ethanol, and reducing equivalents are observed which show obvious relationships to each other. Changes in the consumption or production rates of glycerol, acetic acid, 1,3-propanediol, and ATP are irregular and have relatively small amplitudes compared with their absolute values. By comparing the metabolic fluxes under oscillation and steady state that have nearly the same environmental conditions it could be shown that pyruvate metabolism is the main step affected under oscillation conditions. The specific formation rates of all the products originating from pyruvate metabolism (CO(2), H(2), formic acid, ethanol, acetic acid, lactic acid, and 2,3-butanediol) show significant differences under conditions of oscillation and steady state. In contrast, the specific rates of substrate uptake, ATP generation, and formation of products deriving either directly from glycerol (1,3-propanediol) or from the upstream of pyruvate metabolism (e.g., succinic acid) are not, or at least not significantly, affected during oscillation. Stoichiometric analysis of metabolic pathways confirms that other enzyme systems, in addition to pyruvate: formate-lyase, must be simultaneously involved in the pyruvate decarboxylation under both oscillation and steady-state conditions. The results strongly suggest oscillations of activities of these enzymes under oscillation conditions. It appears that the reason for the occurrence of oscillation and hysteresis lies in an unstable regulation of pyruvate metabolism of different enzymes triggered by substrate excess and drastic change(s) of environmental conditions.

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: I. The phenomena and characterization of oscillation and hysteresis

Oscillation and hysteresis phenomena are observed in the anaerobic continuous fermentation of glycerol by Klebsiella pneumoniae in long-term cultivations under a variety of conditions. In this work, the conditions for the occurrence of these phenomena are reported and the patterns of cell growth and metabolism under oscillation are characterized. During an oscillation period, the formation rates of CO(2), H(2), and formate and the consumption rate of alkali periodically pass values of maxima and minima, the latter being close to zero. The formation of biomass and fermentation products such as 1,3-propanediol, acetate, and ethanol also undergo periodic changes which shift maxima and minima. Sustained oscillation occurs only under conditions of substrate excess within a distinct regime. At pH 7.0, it is only found at dilution rates above 0.15 h(-1) under the experimental conditions. At lower pH values, oscillations are more likely to happen, even at a relatively low dilution rate and low substrate excess. Whereas the amplitude of oscillations at pH 7.0 depends on both the dilution rate and the residual glycerol concentration (C(Glyc)) the interval of oscillations appears to be only a function of C(Glyc). An increase of C(Glyc) in culture damps the oscillation and leads to its disappearance at C(Glyc) = 1100 to 1200 mmol/L (pH 7.0). The operation mode was also found to be an important parameter in determining the stability and actual state of the culture, resulting in hysteresis under certain conditions, particularly at low pH values. Generally, a large perturbation of cultivation conditions tends to cause oscillation and hysteresis. The results unambiguously demonstrate that the oscillation and hysteresis phenomena shown in this work are bound to genuine metabolic fluctuations of the microorganism. They reveal several differences and new features compared with those reported in the literature and cannot be readily explained by the mechanisms known so far.

Modeling the survivability ofSalmonella typhimuriumin the chicken cecae using an anaerobic continuous-culture of chicken cecal bacteria

An anaerobic continuous-culture containing 29-different bacterial isolates of normal avian microflora obtained from the cecae of adult chickens was used to model the survivability of Salmonella typhimurium in the avian cecae. When the continuous-culture was challenged with S. typhimurium at concentrations between 10 1 -10 5 log 10 CFU the pathogen was unable to survive within the culture. When challenged with 10 6 CFU S. typhimurium , the number of Salmonella CFU decreased to 10 2 within 5-days, however S. typhimurium was not cleared from the culture and remained at a concentration of 10 2 for 65-days at which time the fermentation was terminated. When chicks were provided the continuous-culture on day-of-hatch, then challenged with S. typhimurium two days later with up to 10 4 CFU Salmonella, no cecal colonization was observed 5-days after challenge. However chicks challenged with 10 6 CFU Salmonella averaged 10 2 S. typhimurium in cecal contents five days later. Chicks provided the continuous-culture on day-of-hatch and challenged with 10 2 or 10 4 CFU Salmonella had significantly (P<0.05) fewer cecal Salmonella compared to untreated chicks. Salmonella challenge did not effect chick cecal or continuous-culture fermentation parameters and fermentation parameters within the continuous-culture and chick cecae were similar. Based upon similar fermentation parameters and Salmonella colonization levels it appears that this anaerobic continuousculture of cecal bacteria is an excellent model of the cecae of chickens, and may serve as a valuable tool that can be used to better understand the interactions between Salmonella and normal avian flora in the cecae, which is the primary reservoir of Salmonella in commercial poultry. Keywords: Salmonella, continuous-culture, chicks, model.

A Novel Bifidogenic Growth Stimulator Produced by Propionibacterium freudenreichii

A novel bifidogenic growth stimulator (BGS) was present in the cell-free filtrate and in methanol extract fraction of the starter (Propionibacterium freudenreichii) cells for the manufacture of Swiss-type cheese. After purification of the BGS isolated from the lyophilized cells, the mass (217.037) was determined by high-resolution electron impact mass spectrometry (MS) and liquid chromatography-MS spectra. Various experimental analyses indicated that the chemical structure of the BGS was 2-amino-3-carboxy-1, 4-naphthoquinone (ACNQ). We examined the activity of the P. freudenreichii ET culture containing the BGS on a gut bacterial composition using an anaerobic continuous culture system and found that the BGS seems to enhance the selective utilization of oligosaccharides by bifidobacteria. Furthermore, a placebo-controlled study of the effects of BGS on fecal flora and stool frequency was carried out in healthy human subjects. A drink with the sterilized ET-3 culture was administered once a day for 7 days. Bifidobacterium percentage in the fecal flora and stool frequency were significantly increased by administration of the P. freudenreichii culture. The ACNQ exhibited growth stimulation of bifidobacteria at an extremely low concentration and enhanced the activities of NADH oxidase and NADH peroxidase in bifidobacteria. It was revealed that ACNQ works as a good electron acceptor of NAD (P)H diaphorase. The ACNQred was easily auto-oxidized and also acted as a better electron donor of NAD (P) H peroxidase. These ACNQ-mediated reactions seem to play roles in NAD (P) +-regeneration processes and seem to be responsible for the growth stimulation of bifidobacteria.

Kinetic, dynamic, and pathway studies of glycerol metabolism byKlebsiella pneumoniae in anaerobic continuous culture: IV. Enzymes and fluxes of pyruvate metabolism

The activities of pyruvate kinase (PK), pyruvate: formate-lyase (PFL), pyruvate dehydrogenase (PDH), and citrate synthase (CS) involved in the anaerobic glycerol conversion by Klebsiella pneumoniae were studied in continuous culture under conditions of steady states and sustained oscillations. Both the in vitro and in vivo activities of PK, PFL, and PDH are strongly affected by the substrate concentration and its uptake rate, as is the in vitro activity of CS. The flux from phosphoenolpyruvate to pyruvate is found to be mainly regulated on a genetic level by the synthesis rate of PK, particularly at low substrate concentration and low growth rate. In contrast, the conversion of pyruvate to acetyl-CoA is mainly regulated on a metabolic level by the in vivo activities of PFL and PDH. The ratio of in vitro to in vivo activities is in the range of 1 to 1.5 for PK, 5 to 17 for PFL and 5 to 80 for PDH under the experimental conditions. The regulation of in vivo activity and synthesis of these enzymes is sensitive to fluctuations of culture conditions, leading to oscillations of both the in vitro and in vivo activities. In particular, PFL is strongly affected during oscillations; its average in vitro activity is only about half of its corresponding steady-state value under similar environmental conditions. The average in vitro activities of PDH and PK under oscillations are close to their corresponding steady-state values. In contrast to all other enzymes measured for the glycerol metabolism by K. pneumoniae PFL and PDH are more effectively in vivo utilized under oscillations than under steady state, underlining the peculiar role of pyruvate metabolism in the dynamic responses of the culture.

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: IV. Enzymes and fluxes of pyruvate metabolism

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: IV. Enzymes and fluxes of pyruvate metabolism

Kinetic, dynamic, and pathway studies of glycerol metabolism byKlebsiella pneumoniae in anaerobic continuous culture: III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation

The initial steps of glycerol dissimilation and 1,3-propanediol (1, 3-PD) formation by Klebsiella pneumoniae anaerobically grown on glycerol were studied by quantifying the in vitro and in vivo activities of enzymes in continuous culture under conditions of steady state and oscillation and during transient phases. The enzymes studied included glycerol dehydrogenase (GDH), glycerol dehydratase (GDHt), and 1,3-propanediol oxidoreductase (PDOR). Three conclusions can be drawn from the steady-state results. First, glycerol concentration in the culture is a key parameter that inversely affects the in vitro activities (concentrations) of all three enzymes, but has a positive effect on their in vivo activities. Growth rate significantly affects the ratio of in vitro and in vivo enzyme activities under low glycerol concentrations, but not under glycerol excess. Second, whereas the flux through the oxidative pathway of glycerol dissimilation is governed mainly by the regulation of in vivo enzyme activity on a metabolic level, the flux through the reductive pathway is largely controlled by the synthesis of enzymes. Third, GDHt is a major rate-liming enzyme for the consumption of glycerol and the formation of 1,3-PD in K. pneumoniae at high glycerol concentrations. Results from oscillating cultures revealed that both in vitro and in vivo activities of the enzymes oscillated. The average values of the in vitro activities during an oscillation cycle agreed well with their corresponding values for nonoscillating cultures under similar environmental conditions. Experiments with step changes in the feed concentration of glycerol demonstrated that growth and product formation are very sensitive to changes of substrate concentration in the culture. This sensitivity is due to the dynamic responses of the genetic and metabolic networks. They should be considered when modeling the dynamics of the culture and attempting to improve the formation of 1,3-PD.

The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems

The potential of the gfp (green fluorescent protein) gene from the jellyfish Aequoria victoria as a visible marker for tracking lactic acid bacteria introduced into complex anaerobic ecosystems was examined. A red-shifted mutant gfp gene was placed downstream of the constitutive Lactococcus lactis P32 promoter and fluorescence resulting from green fluorescent protein (GFP) expression was detected in Escherichia coli and L. lactis. The construct (pKPSPsgfp) was also introduced into Enterococcus faecalis, Streptococcus gordonii and Streptococcus bovis by mobilisation with pVA797. In all cases fluorescence was detectable at the level of individual cells, and isolated colonies. Although oxygen is required for GFP to fluoresce, colonies or cells grown anaerobically were able to fluoresce upon subsequent exposure to air. A combination of selective plating and colony fluorescence allowed a gfp marked strain of E. faecalis to be enumerated after addition to an anaerobic continuous culture of human faecal flora.

The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems

The potential of the gfp (green fluorescent protein) gene from the jellyfish Aequoria victoria as a visible marker for tracking lactic acid bacteria introduced into complex anaerobic ecosystems was examined. A red-shifted mutant gfp gene was placed downstream of the constitutive Lactococcus lactis P32 promoter and fluorescence resulting from green fluorescent protein (GFP) expression was detected in Escherichia coli and L. lactis. The construct (pKPSPsgfp) was also introduced into Enterococcus faecalis, Streptococcus gordonii and Streptococcus bovis by mobilisation with pVA797. In all cases fluorescence was detectable at the level of individual cells, and isolated colonies. Although oxygen is required for GFP to fluoresce, colonies or cells grown anaerobically were able to fluoresce upon subsequent exposure to air. A combination of selective plating and colony fluorescence allowed a gfp marked strain of E. faecalis to be enumerated after addition to an anaerobic continuous culture of human faecal flora.

Image analysis for the study of the development of anaerobic biofilms on materials characteristic of landfilled refuse

The techniques of anaerobic continuous culture, microscopy and image analysis were combined for the study of attachment, colonisation and biofilm development on surfaces, by strictly anaerobic microbial populations. The Anaerobic Continuous Culture Microscopy Unit (ACCMU) is a miniature continuous culture chamber, which is detachable, self-sealing, oxygen-impermeable, transparent and fits onto a microscope stage. The ACCMU system was used to record colonisation processes on different materials, by naturally occurring, mixed species, functioning microbial associations, isolated from the anaerobic layers of a landfill site. The same fields were sampled through time, allowing a colonisation rate (in μm2 d−1) to be calculated. Paper, wood, cotton and polyester fabric remained poorly or not colonised over a three week study period. Cellophane and selected non-biodegradable materials (glass, plastic and polythene) were rapidly colonised in 2–3 weeks.

In Vitro Lactose Fermentation by Human Colonic Bacteria Is Modified by Lactobacillus acidophilus Supplementation , ,

Adaptation of the colonic flora to lactose may contribute to lactose digestion in lactose maldigesters, and supplementation with Lactobacillus acidophilus may modify colonic fermentation of lactose and short-chain fatty acid production. We evaluated the capability of colonic bacteria to ferment lactose and the ability of L. acidophilus to modify lactose fermentation by the colonic microflora in vitro. An anaerobic continuous culture was established and inoculated with fresh samples of human feces. Lactose infusion was maintained at 25 g/d and pH at 6.7. L. acidophilus strain LA-1 (1.5 × 1010 cells) was introduced into the fermenter on d 0 or added daily on d 0 through 4. The control was the continuous culture without the addition of lactobacilli. Rapid adaptation of colonic bacteria to lactose occurred within 1–2 d, with a significant decrease in lactose concentration and increase in β-galactosidase activity, and lactose concentrations fell below 3 mmol/L by d 7. Supplementation with strain LA-1 resulted in a significantly greater decrease in lactose concentration and greater increase in acetate and propionate production within the first day compared with the control group. However, there was no significant difference between the fermentation treated with L. acidophilus daily and the control after the first day. These data suggest that the colonic bacteria adapt quickly to lactose, causing efficient utilization of lactose. L. acidophilus supplementation may enhance lactose fermentation during early periods when the adaptation is not established in this model.

Image analysis for the study of the development of anaerobic biofilms on materials characteristic of landfilled refuse

The techniques of anaerobic continuous culture, microscopy and image analysis were combined for the study of attachment, colonisation and biofilm development on surfaces, by strictly anaerobic microbial populations. The Anaerobic Continuous Culture Microscopy Unit (ACCMU) is a miniature continuous culture chamber, which is detachable, self-sealing, oxygen-impermeable, transparent and fits onto a microscope stage. The ACCMU system was used to record colonisation processes on different materials, by naturally occurring, mixed species, functioning microbial associations, isolated from the anaerobic layers of a landfill site. The same fields were sampled through time, allowing a colonisation rate (in μm2 d−1) to be calculated. Paper, wood, cotton and polyester fabric remained poorly or not colonised over a three week study period. Cellophane and selected non-biodegradable materials (glass, plastic and polythene) were rapidly colonised in 2–3 weeks.

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: II. Analysis of metabolic rates and pathways under oscillation and steady‐state conditions

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: II. Analysis of metabolic rates and pathways under oscillation and steady‐state conditions

Effect of CO2 absorption on the measurement of CO2 evolution rate in aerobic and anaerobic continuous cultures

A general and simple equation is presented to account for the effect of CO2 absorption and the dissociation of carbonic acid in liquid on the measurement of the CO2 evolution rate (QCO2) of both anaerobic and aerobic continuous cultures. For aerobic cultures the same equation applies to the measurement of the respiratory quotient (RQ). The deviation of QCO2 and RQ, calculated from gas-phase measurements, from their true values can be assessed with two parameters: one accounts for the influence of pH, resulting from dissociation of carbonic acid, the other for the influence of operating conditions. Plots are given to show the influences of culture and operating conditions; they may be used as a guideline for choosing proper operating conditions for a reliable measurement of CO2 evolution rate and RQ value.

Effect of CO 2 absorption on the measurement of CO 2 evolution rate in aerobic and anaerobic continuous cultures

Effect of CO 2 absorption on the measurement of CO 2 evolution rate in aerobic and anaerobic continuous cultures

Occurrence of glycopeptide resistance among Enterococcus faecium isolates from conventional and ecological poultry farms.

Occurrence of glycopeptide resistance among Enterococcus faecium isolates from conventional and ecological poultry farms.

Metabolic and energetic aspects of the growth response of Streptococcus rattus to environmental acidification in anaerobic continuous culture.

Streptococcus rattus, a serotype b strain of mutans streptococci, was grown in an anaerobic glucose-limited chemostat. The molar growth yield of glucose [Yglucose, g dry wt (mol glucose)-1] together with the maximum growth yields (Ymax) and maintenance coefficients for glucose utilization and calculated ATP generation were estimated as a function of pH. When the pH was lowered from 7.0 to 5.0, Yglucose decreased, with a concomitant gradual change in the composition of the end product from a mixture of formate, acetate and ethanol to one mostly of lactate. Whereas the Ymax for glucose decreased without any change in the Ymax for ATP on acidification, both of the maintenance coefficients markedly increased. Kinetic and immunochemical examinations indicated the presence of an F1F0-type proton-translocating ATPase in the membrane fraction prepared from bacterial cells grown under acidic conditions; no detectable level of the enzyme was found in cells grown at neutral pH. However, when incubated with glucose under non-growing conditions, these acid-adapted and unadapted cells showed an insignificant difference in the ability to maintain the intracellular pH alkaline relative to the acidic environments. These results suggest that the organism responds and adapts to environmental acidification by sacrificing some energy cost in terms of both the efficiency of glucose utilization to generate ATP and the extra maintenance required to continue biomass production as efficiently as under neutral pH.

Effects of anaerobic conditions on biomass production and plasmid stability in an immobilized recombinantE. Coli

Cellular aggregation is a natural phenomenon shown by microorganisms which allows the cells to form biofilms. The study of immobilized microorganisms requires the development of microtechniques to evaluate their metabolism and stability. In aerobic conditions, the immobilization of recombinant microorganisms in carrageenan gel beads has been successfully explored to enhance plasmid stability when growth in the support was oxygen‐limited. A series of anaerobic continuous cultures of free and immobilized Escherichia coli B (pTG201) and E. coli HB 101 (pKBF 367–11) have been developed, without selection pressure. In continuous culture of free cells, both exhibited segregational instability. The existence of strong competition between cells containing plasmid (P+ cells) and cells devoid of plasmids (P— cells) was favourable to the colonization of the bioreactor by the faster growing P— cells. Furthermore, pTG 201 was mainly dimeric while the plasmid copy number of pKBF 367–11 increased from 80 to 270, until p...

Cell-bound peptidase activities of Treponema denticola ATCC 33520 in continuous culture.

The oral spirochaete Treponema denticola ATCC 33520 was grown at a mean generation time of 10 h in anaerobic continuous culture in a serum- and carbohydrate-free medium at pH 7.0. The extracellular proteolytic activities of this spirochaete were then investigated by incubating washed cells with 68 2-naphthylamide derivatives of the Extended API System. Chymotrypsin-like, trypsin-like, elastase-like and iminopeptidase activities were demonstrated. The phenylalanine peptidase or chymotrypsin-like activity of T. denticola ATCC 33520, estimated with N-succinyl-L-phenylalanyl-L-leucyl-L-phenylalanine-thiobenzyl ester (SPLP) had a pH optimum at pH 8.5, a specific activity of 36.6 nmol min-1 (mg dry wt)-1 and was inhibited only slightly by HgCl2. The trypsin-like activity, estimated with benzoyl-DL-arginine-7-amido-4-methylcoumarin (BAMC), had a pH optimum at pH9, and a specific activity of 0.3 nmol min-1 (mg dry wt)-1; inhibition by HgCl2 indicated the involvement of active thiol groups. The activity should preferably be termed arginine peptidase activity, according to the carboxy-terminal amino acid of the test substrate. The extracellular proline peptidase activity, estimated with L-proline-7-amido-4-methylcoumarin. HBr (PRAMC), had an activity of 1.5 nmol min-1 (mg dry wt)-1, an optimum at pH 8.5 and the properties of a thiol protease. The main cell-bound and extracellular active peptidase activities of fast-growing cells of T. denticola ATCC 33520 are phenylalanine peptidase, proline peptidase, arginine peptidase and an oligopeptide-dependent alanine peptidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A segmented gas/liquid delivery system for continuous culture of microorganisms on insoluble substrates and its use for growth of Ruminococcus flavefaciens on cellulose

An anaerobic continuous culture device was constructed that permits accurate delivery of media containing insoluble substrates, even at very low volumetric flow rates (<3 ml/h). The system consisted of: (1) a reservoir in which the medium slurry was mixed well by the combined use of stirring and diffusive gas sparging to suspend a cellulose substrate of small (< 45 μm) particle size; (2) a delivery system that segmented the slurry into small (~ 20μl) discrete liquid segments separated by intervening bubbles of CO2 gas; and (3) a stirred, temperature-controlled 2-l fermentation vessel. The device was used to examine substrate consumption, product formation, and cell yield by the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 under steady-state, cellulose-limited conditions at six different dilution rates (D) ranging from 0.017 to 0.101 h−1 (pH 6.4–6.6). Cellulose consumption decreased from 4.00 g/1 (at D=0.017 h−1) to 2.56 g/1 (at D=0.101 h−1). Increases in D resulted in a progressive shift toward production of more acetate and formate, and less succinate. Redox balance calculations revealed a deficiency in reduced products, probably due to the production of H2, which was not directly measured. Reducing sugar values remained low (0.05–0.10 g/1, as glucose) at all D values. The cellulose fermentation was characterized by a low maintenance coefficient (0.07 g cellulose/g cells per hour) and a high true growth yield (YG = 0.24 g cells/g cellulose, corrected for maintenance). Comparison of the data with literature values suggests that the fermentation of cellulose by this organism gives cell yields at least as great as the yields obtained from the fermentation of soluble sugars.