Single-stage Chemostat Research Articles

Effects of different carbohydrates on the simulation of human intestinal bacterial flora with in vitro culture

To investigate the optimal growth condition of human fecal bacterial flora in vitro by comparing the effect of different carbohydrates as cultural media. Three fecal samples (1, 2, 3) were collected and inoculated into a single-stage chemostat system, in which starch medium (VI) and starch polysaccharide medium(XP) were used. Samples were collected for bacterial genomic DNA extraction and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Bacterial composition and short chain fatty acid (SCFA) were then analyzed. The single stage chemostat system reached steady after operating 8 days, when evaluated by the PCR-DGGE. Bacterial 16s rRNA high-throughput sequencing showed that the intestinal bacteria of these three volunteers was mainly composed of four bacterial phyla, namely, Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria. When the influence of bacterial abundance was considered, the similarity of bacterial composition between the original fecal samples to the harvested flora after culture was 0.847, 0.825, 0.968 in VI medium and 0.927, 0.926, 0.836 in XP medium, respectively. The similarity was decreased to 0.553, 0.580, 0.623 with VI medium and 0.617, 0.520, 0.574 with XP medium, when the number of bacterial species was considered. The variation of host individual also influenced the simulation. VI medium favored fecal sample 3, while XP medium more benefited sample 1 and 2. Bacteroides and Lachnospiraceae_incertae_sedis grew in both VI and XP medium. However, some species were only detected in VI medium and some were specifically found in the XP medium. The SCFA concentration in fermenters was 15-35 mmol/L, mainly propionate and butyrate. The chemostat system works for stimulating human gut bacterial flora in vitro. The bacterial composition is affected by different carbohydrate in the culture medium yet with close simulation higher than 80%.

Evaluation of microbial community reproducibility, stability and composition in a human distal gut chemostat model

In vitro gut models provide several advantages over in vivo models for the study of the human gut microbiota. However, because communities developed in these models are inevitably simplified simulations of the in vivo environment, it is necessary to broadly define the differences between in vitro consortia and the communities from which they are derived. In this study we characterized microbial community development in a twin-vessel single-stage chemostat model of the human distal gut ecosystem using both gel (Denaturing Gradient Gel Electrophoresis) and phylogenetic microarray (Human Intestinal Tract Chip) based techniques. Five different sets of twin-vessels were inoculated with feces from three different healthy adult donors and allowed to reach steady state compositions. We found that twin-vessel single-stage chemostats could develop and maintain stable, diverse, and reproducible communities that reach steady state compositions in all five runs by at most 36days post-inoculation. As noted in other in vitro studies, steady state communities were enriched in Bacteroidetes but not Clostridium cluster XIVa, Bacilli or other Firmicutes relative to the fecal inocula. Communities developed within this model had higher within-run reproducibility than between-run repeatability when using consecutive fecal donations. Both fecal inocula and steady state chemostat communities seeded with feces from different donors had distinct compositions. We conclude that twin-vessel single-stage chemostat models represent a valid simulation of the human distal gut environment and can support complex, representative microbial communities ideal for experimental manipulation.

The two stage immobilized column reactor with an integrated solvent recovery module for enhanced ABE production

The production of acetone, butanol, and ethanol (ABE) by fermentation is a process that had been used by industries for decades. Two stage immobilized column reactor system integrated with liquid-liquid extraction was used with immobilized Clostridium acetobutylicum DSM 792, to enhance the ABE productivity and yield. The sugar mixture (glucose, mannose, galactose, arabinose, and xylose) representative to the lignocellulose hydrolysates was used as a substrate for continuous ABE production. Maximum total ABE solvent concentration of 20.30 g L(-1) was achieved at a dilution rate (D) of 0.2h(-1), with the sugar mixture as a substrate. The maximum solvent productivity (10.85 g L(-1)h(-1)) and the solvent yield (0.38 g g(-1)) were obtained at a dilution rate of 1.0 h(-1). The maximum sugar mixture utilization rate was achieved with the present set up which is difficult to reach in a single stage chemostat. The system was operated for 48 days without any technical problems.

Continuous two stage acetone–butanol–ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313

The objective of this study was to optimize continuous acetone–butanol–ethanol (ABE) fermentation using a two stage chemostat system integrated with liquid–liquid extraction of solvents produced in the first stage. This minimized end product inhibition by butanol and subsequently enhanced glucose utilization and solvent production in continuous cultures of Clostridium acetobutylicum B 5313. During continuous two-stage ABE fermentation, sugarcane bagasse was used as the cell holding material for the both stages and liquid–liquid extraction was performed using an oleyl alcohol and decanol mixture. An overall solvent production of 25.32 g/L (acetone 5.93 g/L, butanol 16.90 g/L and ethanol 2.48 g/L) was observed as compared to 15.98 g/L in the single stage chemostat with highest solvent productivity and solvent yield of 2.5 g/L h and of 0.35 g/g, respectively. Maximum glucose utilization (83.21%) at a dilution rate of 0.05 1/h was observed as compared to 54.38% in the single stage chemostat.

Development of a fermentation system to model sessile bacterial populations in the human colon

A fermentation system was designed to model the human colonic microflora in vitro. The system provided a framework of mucin beads to encourage the adhesion of bacteria, which was encased within a dialysis membrane. The void between the beads was inoculated with faeces from human donors. Water and metabolites were removed from the fermentation by osmosis using a solution of polyethylene glycol (PEG). The system was concomitantly inoculated alongside a conventional single-stage chemostat. Three fermentations were carried out using inocula from three healthy human donors.Bacterial populations from the chemostat and biofilm system were enumerated using fluorescence in situ hybridization. The culture fluid was also analysed for its short-chain fatty acid (SCFA) content. A higher cell density was achieved in the biofilm fermentation system (taking into account the contribution made by the bead-associated bacteria) as compared with the chemostat, owing to the removal of water and metabolites. Evaluation of the bacterial populations revealed that the biofilm system was able to support two distinct groups of bacteria: bacteria growing in association with the mucin beads and planktonic bacteria in the culture fluid. Furthermore, distinct differences were observed between populations in the biofilm fermenter system and the chemostat, with the former supporting higher populations of clostridia and Escherichia coli. SCFA levels were lower in the biofilm system than in the chemostat, as in the former they were removed via the osmotic effect of the PEG. These experiments demonstrated the potential usefulness of the biofilm system for investigating the complexity of the human colonic microflora and the contribution made by sessile bacterial populations.

Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates).

Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D(1)) and 0.16 h(-1) in the second fermentor (D(2)) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D(1) and D(2) over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans were found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L(-1) h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermentor. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes.

Continuous, high level production and excretion of a plasmid‐encoded protein by Escherichia coli in a two‐stage chemostat

The stable continuous overproduction of a plasmidencoded protein, beta-lactamase, for at least 50 days by Escherichia coli K-12, RB791(pKN), with release into the culture medium has been demonstrated in two-stage chemostats. The second-stage culture was continuously induced with 0.1 mM IPTG. Continuous expression of beta-lactamase could not be sustained with this strain in a single-stage chemostat because of cell death and selection for lac(-1) cells. Beta-lactamase production in the second stage was sensitive to the second-stage dilution rate and the distribution of the limiting substrate (i.e., glucose) between the first and second stages. The fraction of viable, excreting cells and the average copy number in the induced culture was measurably higher under those conditions of dilution rate and substrate distribution which yielded high beta-lactamase levels. The best operating conditions found at 20 degrees C were a first-stage dilution rate of 0.12 h(-1), a second-stage dilution rate of 0.03 h(-1), and equal glucose feed supplied to each stage. Enzymatically active beta-lactamase was produced at a level of 25% of total cellular protein with 90% excretion yielding 300 mg beta-lactamase/L that was 50% pure at an OD(600) < 6.

Physiology and polyester formation of Pseudomonas oleovorans in continuous two-liquid-phase cultures

Pseudomonas oleovorans is able to grow on linear aliphatic hydrocarbons of medium chain length as sole energy and carbon source. When nitrogen, sulfur, or magnesium is limiting, P. oleovorans produces an intracellular polyester poly(β-hydroxyalkanoate) (PHA) from the excess alkanoic acid formed from the alkanes supplied in the medium. To study the effect of growth rate and exposure to bulk amounts of n-octane on the physiology and morphology of P. oleovorans, we have established continuous cultures of this organism in two-liquid phase media containing about 15% (v/v) n-octane. P. oleovorans was grown in an ammonium-limited single-stage chemostat at growth rates varying from D = 0.05 to D = 0.46 h −1. In contrast to batch cultures of P. oleovorans grown on n-octane, both rapidly and slowly growing cells remained fully viable during the entire continuous culture experiments, which typically lasted 200–300 h. The cellular morphology of these cells was studied as a function of time by freeze-fracture electron microscopy, which provided information on changes in membrane ultrastructure and revealed large and small PHA granules in slowly and rapidly growing cells, respectively. The cell density, cellular protein content, and PHA content were determined as a function of growth rate. The cell density decreased from 2.25 to 1.32 mg ml −1, while the PHA content of the cells decreased from 46.7% to 8.3% of the total cell dry weight when the dilution rate (=growth rate) increased from 0.09 to 0.46 h −1. The rest biomass concentration, defined as the difference between total biomass and PHA, was almost independent of the cellular growth rate. The cellular protein content relative to the rest biomass increased from 24% to 46% when the growth rate increased from 0.09 to 0.46 h −1, indicating that rapidly growing cells contain more protein than slowly growing cells, which correlates well with the qualitative data of the electron micrographs.

Lipid Turnover in Oleaginous Yeasts

When eight strains of the oleaginous yeasts Candida curvata, Lipomyces starkeyi, Rhodosporidium toruloides and Trichosporon cutaneum were starved of carbon after having accumulated lipid up to 34% of their biomass, the lipid was readily converted to new biomass in all cases except the two strains of L. starkeyi. When C. curvata D was grown in a two-stage chemostat with the second stage as a carbon-starvation vessel (but containing NH+ 4) biosynthesis of new biomass reached 1·9 ± 0·2 g per g lipid utilized. Experiments in a single-stage chemostat undergoing transition from lipid accumulation (nitrogen-limited medium) to carbon-starvation conditions showed that the lipid in C. curvata was rapidly mobilized. When the lipid was pre-labelled with 14C, a transitory pool of rapidly metabolizable non-lipid material appeared within 1.5 h of the initiation of starvation. Rates of lipid loss indicated that initiation of lipid degradation occurred immediately carbon was lost from the external medium.

Simultaneous utilization of glucose and xylose byCandida curvata D in continuous culture

Of ten, mainly oleaginous, yeasts examined for the ability to use glucose and xylose simultaneously, only one,Candida curvata D, was found which could do so. This yeast was examined further in a single-stage chemostat wherein it produced similar biomass yields, lipid contents and fatty acids on glucose plus xylose mixed in varying proportions. This oleaginous yeast would therefore be capable of growing on hydrolysed wood and straw wastes as a potential source of single cell oil.

Continuous production of ?-amylase byBacillus amyloliquefaciens in a two-stage fermentor

The production of a secondary metabolite (α-amylase) by a highly aerobic bacterium (Bacillus amyloliquefaciens) was examined in batch, single-stage chemostat, two-stage stirred tank, and two-stage stirred tank/tubular reactor configurations. The relative performance of these reactor systems as measured by product concentration and volumetric productivity was compared, and the effect of aeration rate on the extent of plug flow in the tubular reactor was examined.

Interspecific interactions in a methane-utilizing mixed culture.

A two-member methane-utilizing mixed culture of bacteria, formed by combining two pure cultures isolated from a naturally occurring methane-utilizing mixed culture, was studied in continuous culture. From the nutritional requirements and substrate ranges of the pure cultures, a mechanism for the interspecific interactions occurring in the mixed culture was proposed. Product formation kinetics were determined in continuous culture for each product involved in the proposed mechanism. From this proposed mechanism a mathematical model was derived based on simple material balance equations around a single-stage chemostat. The steady-state predictions of this model were compared to experimental results obtained from continuous-culture experiments with the two-member methane-utilizing mixed culture. Interspecific interactions occurring in two-member methanol-utilizing and three-member methane-utilizing mixed cultures have also been discussed.

Predation of Escherichia coli by Colpoda steinii.

A study of single-stage chemostat cultures of Colpoda steinii. Escherichia coli, and glucose is reported here. Two levels of glucose were fed as the limiting nutrient to the chemostat cultures. The cultures were studied at three holding times. Oscillations developed at short holding time and damped oscillations developed a long-residence times that approached steady-state conditions of populations of C. steinii and E. coli and concentrations of glucose. The experimental data are fitted to and compared with Jost's model.

Daughter cells as an important factor in determining the physiological state of yeast populations.

Cells of Candida utilis grown in a single-stage chemostat at D = 0.05, 0.1, 0.25, and 0.35 hr-1 were separated into a fraction of scar-bearing mother cells and a fraction of scar-free daughter cells. The scar-free cells were transferred into small batch cultures where the length of the maturation phase, changes in length and width of cells, specific growth rate, and specific rate of RNA and protein synthesis were examined for 5 hr. The daughter cells grown at D = 0.05 hr-1 were very small at the moment of separation from the mother cells (about one-third of the mother cell). Their maturation phase (in a batch culture), at the beginning of which they attain the specific growth rate approaching the mumax of the strain used, lasts for 3 hr. On the other hand, daughter cells grown at D = 0.35 hr-1 are almost the same size as the mother cells at the moment of separation. After transfer to a batch culture they begin to bud almost immediately. Similarly, in their other morphological and physiological parameters they differ strikingly from immature daughter cells which are formed at low specific growth rates. The importance of these differences from the point of view of mathematical modeling of growth processes is discussed.

Role of Protozoa in Waste Purification Systems

Pirt and Bazin1 discussed possible adverse effects of bacterial predation by protozoa on the removal of substrate by activated sludge. Their analysis considered not activated sludge but a single-stage chemostat without recycle as used experimentally by Curds2, and mathematically by Curds3 and Canale4, for the study of predator/prey interactions. Pirt and Bazin1 concluded that efficient waste removal could best be achieved with two chemostats in series, eliminating protozoa from the first stage by operating it at a dilution rate in excess of their maximum specific growth rate.

Growth and physiology of a yeast cultivated in batch and continuous culture systems.

Inoculum size has been found to affect significantly the maximum attainable specific growth rate during batch cultivation ofCandida utilis. Lower inoculum size resulted in an increased growth rate and relatively longer lag. The culture is found to be most active in the beginning of the exponential phase as regards its RNA synthesis rate. Batch data were used for predicting the conditions of the yeast population in single-stage continuous culture system. Predicted and the experimental values showed a reasonable agreement. In single-stage chemostat the physiology of the yeast was studied on the basis RNA, DNA and protein synthesis rates at various growth rates. The results indicate that the productivity of cells and the rate of synthesis of macromolecules is highest at the dilution rate values of 0.33 to 0.35 hr−1. In order to attain so-called unrestricted conditions of growth a pluristage pluristream continuous system was employed. It is assumed that under such conditions the specific growth rate and the synthetic activity of yeasts may reach its maximum on a given medium. The results presented do not show such conditions of growth under the experimental conditions employed (D1=0.35 hr−1 andD2=0.2 to 1.7 hr−1) withCandida utilis cultivated on beet molasses medium. Second stage of a two-stage two-stream continuous system is constantly fed with the cells from the foregoing stage; this category of cells on entering the new conditions of the second stage is expected to show some adaptation period. Experiments are reported to this effect.

Performance of a perforated plate column as a multistage continuous fermentor

AbstractMicroorganisms were continuously cultivated in multistage column consisting of ten perforated plate sections to which medium and air were supplied concurrently from the bottom.At steady state the cell concentration in the various stages was gradationally differentiated from the bottom to the top in the direction of medium flow. RNA content per unit cell concentration at each sage was determined. The cells in the lower stages were higher in RNA content than those from the upper stages. Wash out was observed to occur in the column at dilution rates which do not result in wash out in a single stage chemostat system.A study of the flow characteristics revealed that the overall performance of the plate column was equivalent to that of a multistage system, when hole diameter and hole area to column cross sectional area ratio were properly selected. This was true even in highly aerated conditions. These results indicated that the perforated plates in the column hindred intermixing through the plates, and that each stage functioned as an independent stirred vessel. Industrial and research application of this type fermentor was discussed.