Single-stage Continuous Culture Research Articles

MiniBioReactor Array (MBRA) in vitro gut model: a reliable system to study microbiota-dependent response to antibiotic treatment.

BackgroundAntimicrobial drugs are mostly studied for their impact on emergence of bacterial antibiotic resistance, but their impact on the gut microbiota is also of tremendous interest. In vitro gut models are important tools to study such complex drug–microbiota interactions in humans.MethodsThe MiniBioReactor Array (MBRA) in vitro microbiota system; a single-stage continuous flow culture model, hosted in an anaerobic chamber; was used to evaluate the impact of three concentrations of a third-generation cephalosporin (ceftriaxone) on faecal microbiota from two healthy donors (treatment versus control: three replicates per condition). We conducted 16S microbiome profiling and analysed microbial richness, diversity and taxonomic changes. β-Lactamase activities were evaluated and correlated with the effects observed in the MBRA in vitro system.ResultsThe MBRA preserved each donor’s specificities, and differences between the donors were maintained through time. Before treatment, all faecal cultures belonging to the same donor were comparable in composition, richness, and diversity. Treatment with ceftriaxone was associated with a decrease in α-diversity, and an increase in β-diversity index, in a concentration-dependent manner. The maximum effect on diversity was observed after 72 h of treatment. Importantly, one donor had a stronger microbiota β-lactamase activity that was associated with a reduced impact of ceftriaxone on microbiota composition.ConclusionsMBRA can reliably mimic the intestinal microbiota and its modifications under antibiotic selective pressure. The impact of the treatment was donor- and concentration-dependent. We hypothesize these results could be explained, at least in part, by the differences in β-lactamase activity of the microbiota itself. Our results support the relevance and promise of the MBRA system to study drug–microbiota interactions.

Xylose\u2013glucose co-fermentation to ethanol by Escherichia coli strain MS04 using single- and two-stage continuous cultures under micro-aerated conditions

BackgroundSimultaneous co-fermentation of mixed sugars is an important feature to consider in the production of ethanol from lignocellulosic biomass hydrolysates because it enhances the overall ethanol yield and volumetric productivity during fermentation. Continuous cultures can be used during ethanol production from lignocellulosic hydrolysates to prevent catabolite repression by glucose on other sugars, such as xylose, and thus promote the simultaneous and total consumption of sugars and reduce fermentation time. The use of single- and two-stage continuous cultures under micro-aerated conditions for simultaneous consumption of xylose and glucose, and fermentation to ethanol by ethanologenic Escherichia coli strain MS04 was studied. Mineral medium supplemented with glucose, xylose and sodium acetate, was used to compare continuous cultures performance to batch cultures.ResultsSingle-stage continuous cultures under micro-aerated conditions allowed the total co-consumption of a mixture of glucose and xylose (7.5 and 42.5 g/L, respectively) in mineral medium, with steady state ethanol production of 18 g/L, and a volumetric ethanol productivity of 0.9 g/L h, when low dilution rates (0.05 h−1) were used. However, the volumetric productivity was lower than the batch process under similar conditions (1.3 g/L h). Conversely, micro-aerated two-stage continuous culture enhanced the volumetric productivity up to 1.6 g/L h at a dilution rate of 0.15 h−1, with a total consumption of sugars and a slight reduction of the overall ethanol yield.ConclusionsThe total and simultaneous consumption of glucose and xylose by the ethanologenic E. coli strain MS04 was accomplished by using two-stage continuous culture under micro-aerated conditions with an increase in the volumetric ethanol productivity of 23% and 78% when compared to batch and single-stage continuous cultures, respectively. Multi-stage continuous cultivation can be used to promote the simultaneous consumption of all sugars contained in biomass hydrolysates, and thus increase the volumetric ethanol productivity of the fermentation process.

Enhanced microbial lipid production by Cryptococcus albidus in thehigh-cell-density continuous cultivation with membrane cell recycling and two-stage nutrient limitation.

As a potential feedstock for biofuel production, a high-cell-density continuous culture for the lipid production by Cryptococcus albidus was investigated in this study. The influences of dilution rates in the single-stage continuous cultures were explored first. To reach a high-cell-density culture, a single-stage continuous culture coupled with a membrane cell recycling system was carried out at a constant dilution rate of 0.36/h with varied bleeding ratios. The maximum lipid productivity of 0.69g/L/h was achieved with the highest bleeding ratio of 0.4. To reach a better lipid yield and content, a two-stage continuous cultivation was performed by adjusting the C/N ratio in two different stages. Finally, a lipid yield of 0.32g/g and lipid content of 56.4% were obtained. This two-stage continuous cultivation, which provided a higher lipid production performance, shows a great potential for an industrial-scale biotechnological production of microbial lipids and biofuel production.

Continuous production of isopropanol and butanol using Clostridium beijerinckii DSM 6423

Clostridium beijerinckii DSM 6423 was studied using different continuous production methods to give maximum and stable production of isopropanol and n-butanol. In a single-stage continuous culture, when wood pulp was added as a cell holding material, we could increase the solvent productivity from 0.47 to 5.52 g L⁻¹ h⁻¹ with the yield of 54% from glucose. The overall solvent concentration of 7.51 g L⁻¹ (39.4% isopropanol and 60.6% n-butanol) with the maximum solvent productivity of 0.84 g L⁻¹ h⁻¹ was obtained with two-stage continuous culture. We were able to run the process for more than 48 overall retention times without losing the ability to produce solvents.

Optimization of ammonium nitrate concentration in single-stage continuous cultures of Aspergillus niger with biomass retention

AbstractThe effect of ammonium nitrate concentration in the citric acid biosynthesis by Aspergillus niger NC-12 in single-stage continuous cultures with biomass retention was investigated. Experiments were carried out in a BIOMER laboratory fermenter with 5 dm3 working volume. At the initial stage of each cultivation, the substrate in the bioreactor contained 1.5 g NH4NO3 dm−3. After 120 h onwards, the bioreactor was fed continuously at a constant dilution rate of 0.009 h−1. NH4NO3 concentration in the feed was varied from one culture to another, ranging between 0.5 g dm−3 and 2.5 g dm−3. Promising results were obtained when NH4NO3 concentration of 1.5 g dm−3 was used. The observed concentration of citric acid (c P) and yield of citric acid with respect to the introduced sucrose (Y P/S) were 117.88 g dm−3 and 78.59 %, respectively. The efficiency coefficient of citric acid biosynthesis (K ef) was very high, amounting to 83.38.

Production of Solvent (acetone-butanol-ethanol) in Continuous Fermentation by Clostridium saccharobutylicum DSM 13864 Using Gelatinised Sago Starch as a Carbon Source

Solvent production by Clostridium saccharobutylicum DSM 13864 was carried out in a single stage continuous culture using 2 L stirred tank fermenter with gelatinised sago starch as a carbon source. From the study it was found that the condition could be adjusted to suit for acids production (high dilution rate and high pH) or solvent production (low dilution rate and low pH) by manipulating the dilution rate and culture pH of single stage continuous fermentation. The highest solvent concentration in outflow (9.10 g/L) was obtained at pH 4.5 and dilution rate of 0.05 h 1 , which corresponds to overall productivity of 0.46 g/L.h. However, the highest total solvent productivity (0.85 g/L.h) was obtained at dilution rate of 0.11 h -1 and pH 4.5, which gave a total solvent yield of 0.29 g solvent/g sago starch. Although the total solvent productivity was greatly increased in continuous culture, the final solvent concentration attained in outflow was decreased by about 53% as compared to batch culture.

The use of a model ileum to investigate the effects of novel and existing antimicrobials on indigenous porcine gastrointestinal microflora: using vancomycin as an example

The modification of an existing in vitro model of the porcine ileum is described, together with its initial validation in terms of specific bacterial content, resistance to transient pathogen colonisation and test antimicrobial exposure. With the primary aim of producing a standardised test system requiring a low animal cost, a single-stage continuous culture fermenter inoculated with porcine faecal material was shown to be capable of establishing and maintaining a microflora comparable to that of the porcine ileum. Such comparison was based upon total aerobic and anaerobic bacterial population magnitude, total Bacteroides, Bifidobacterium and Enterococcus spp., Escherichia coli and lactic acid bacteria numbers and E. coli strain complexity. The robustness of the model was demonstrated through resistance to transient pathogen ( Salmonella poona) exposure. Test glycopeptide exposure (vancomycin) served both to verify the simulated microflora response as comparable with that reported elsewhere in vivo and to demonstrate the model’s value in the assessment of dietary antimicrobial activity. The trends observed under antibiotic exposure illustrate the influence of vancomycin treatment on the enteric commensal microflora, inhibiting potentially probiotic bacteria that may be vital to the competitive exclusion of the gut and favouring the selection of opportunistic pathogens. Despite the limitations of such a model system, the in vitro porcine ileum produced highly repeatable data at a low cost, with complete control and containment. Given a comparable seeding inoculum the model proved capable of yielding data reproducible over time, describing the test antibiotics influence on the ileal microflora in a form suitable for comparison elsewhere.

Lipid production by Yarrowia lipolytica growing on industrial glycerol in a single-stage continuous culture

Yarrowia lipolytica LGAM S(7)1 presented remarkable growth on industrial glycerol used as sole carbon substrate. Nitrogen-limited flask cultures were accompanied by restricted synthesis of reserve lipid, whilst amounts of citric acid were produced extracellularly. On the contrary, high amounts of reserve lipid (up to 3.5 g/l, 43% w/w of lipids in dry biomass) were produced in highly aerated continuous cultures. Lipid production was favoured at low specific dilution rates whilst fat-free material yield increased over the whole range of D ( h −1) . The maximum volumetric productivity obtained was 0.12 g lipid/l h. Storage lipid composition did not present remarkable changes in the specific dilution rates tested. Oleate and linoleate were the dominant cellular fatty acids.

Glycerol production by Candida krusei employing NaCl as an osmoregulator in batch and continuous fermentations

Addition of 40 g NaCl l−1 to a chemically defined medium containing 140 g glucose l−1 in shake-flask culture improved glycerol production by Candida krusei from 16.5 g l−1 to 47.7 g l−1. With 40 g NaCl l−1 at a dilution rate of 0.065 h−1, glycerol concentration, glycerol yield (based on glucose consumed), and productivity in a four-stage cascade bioreactor were higher by 240%, 27% and 28%, respectively, than in a single-stage continuous culture system.

High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain

Batch and continuous cultures of a newly isolated Clostridium butyricum strain were carried out on industrial glycerol, the major by-product of the bio-diesel production process. For both types of cultures, the conversion yield obtained was around 0.55 g of 1,3-propanediol formed per 1 g of glycerol consumed whereas the highest 1,3-propanediol concentration, achieved during the single-stage continuous cultures was 35–48 g l −1. Moreover, the strain presented a strong tolerance at the inhibitory effect of the 1,3-propanediol, even at high concentrations of this substance at the chemostat (e.g. 80 g l −1). 1,3-Propanediol was associated with cell growth whereas acetate and butyrate seemed non growth-associated products. At low and medium dilution rates (until 0.1 h −1), butyrate production was favoured, whereas at higher rates acetate production increased. The maximum 1,3-propanediol volumetric productivity obtained was 5.5 g l −1 h −1. A two-stage continuous fermentation was also carried out. The first stage presented high 1,3-propanediol volumetric productivity, whereas the second stage (with a lower dilution rate) served to further increase the final product concentration. High 1,3-propanediol concentrations were achieved (41–46 g l −1), with a maximum volumetric productivity of 3.4 g l −1 h −1. A cell concentration decrease was reported between the second and the first fermentor.

Biofilms in drinking water systems: a possible reservoir for Helicobacter pylori

A laboratory model system was utilised to investigate the persistence of Helicobacter pylori in mixed-species heterotrophic biofilms. A single-stage continuous culture vessel was linked to a modified-Robbins device (mRD) incorporating removable stainless steel coupons. The system was innoculated with H. pylori (NCTC 11637) and the fate of the organism monitored by polymerase chain reaction (PCR) analysis. Helicobacter pylori was detected in biofilm material for a period of up to 192 h. Theoretical washout would have occurred at around 48 h thus detection of H. pylori for a prolonged period after theoretical washout suggested that the organism possessed the ability to persist in the mixed-species heterotrophic biofilm. Preliminary studies using heat-inactivated H. pylori showed that the organism was not detected in biofilm material at any time post-challenge suggesting that the persistence of H. pylori in such material was a phenomenon requiring the organism to be in a viable state. Further investigations to assess the biological basis for the association of H. pylori with drinking water biofilms and the risk that this may pose to public health are being undertaken.

Enrichment of bifidobacteria from human gut contents by oligofructose using continuous culture.

Chemostat cultures of human faecal bacteria were used to determine the bifidogenic effect of oligofructose, a fermentable carbohydrate found in a number of plants. In single stage continuous culture, oligofructose preferentially enriched for bifidobacteria, in comparison to sucrose and inulin. This stimulatory effect was enhanced at a high dilution rate, high substrate concentration and low pH. These parameters are likely to approximate to those that occur in the proximal colon. Studies with a three-stage continuous culture model of the large intestine confirmed the bifidogenic effect of oligofructose. These in vitro data indicate that an increase in the concentration of fructose-based oligosaccharides in the diet may alter the balance of the gut microflora towards bifidobacteria, a purported health-promoting genus.

Frementation of biowaste to H2 by Bacillus licheniformis

Completely damaged wheat grains, unfit for human consumption, were fermented to H2 by Bacillus licheniformis strain JK1. Batch-culture fermentation of wheat slurries [6% (w/v) total solids and 5.8% (w/v) organic solids (OS)] evolved 225, 205 and 203 l of biogas-H, a mixture of H2, CO2 and H2S, per kg OS at pH 6, 7 and 8, respectively. H2 constituted 25% to 41% of the total biogas-H evolved. In single-stage continuous culture, H2 generated/kg OS reduced was 70 l at pH 6 and 74 l at pH 7 and 8.

Substrate inhibition of Chlamydomonas reinhardtii by acetate in heterotrophic culture

The heterotrophic growth of microalgae has been proposed as a potential mode of culture to achieve high cell densities on a large scale. In this paper, the heterotrophic growth of the green microalga, Chlamydomonas reinhardtii,on acetate as sole energy and carbon source in batch and single-stage continuous cultures is reported. No inhibition by acetate was observed at low concentrations (≤ 0·4 g/litre), in which case, cell growth was well described by the Monod model. However, above this acetate concentration, growth was progressively inhibited. Inhibition was observed as a decrease in both the specific growth rate and the observed growth yield. The former was well modelled using the Haldane Model. A model based on constant q m was proposed to describe the latter phenomenon. Whereas it predicted the trend of falling observed cell growth yield with initial acetate concentration in batch cultures, there was significant lack of fit between simulated and experimental data. In continuous cultures, a steady state cell concentration of 0·4 g/litre and maximum cell productivity of 0·02 g/litre/h was obtained for an acetate feed concentration of 0·85 g/litre. This compares well with values achieved by photosynthetic mass culture systems.

Lipid production by an unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichum curvatum grown in single-stage continuous culture

An unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichum curvatum, named UfaM3, blocked in the conversion of stearic to oleic acid was cultivated in single-stage continuous culture. The influence of consumed carbon to nitrogen ratios (C/N ratios, g g−1) obtained at various dilution rates (D) on fatty acid (FA) accumulation and its profiles were studied. In continuous culture in N-limited medium a maximum FA accumulation of 45.6% (g g−1 of dry biomass) was obtained at an optimal D of 0.049 h−1, recording an efficiency of substrate conversion of 0.48 g g−1 and 0.22 g g−1 for biomass and lipids, respectively. The quality of lipid approached cocoa butter at an optimal C/N ratio of between 20 and 30. The C/N ratio in the incoming medium was 38.5 g g−1 with 30 g l−1 of glucose and both C and N sources were completely consumed at a critical D of ≤ 0.07 h−1. The stability of the mutant was demonstrated in the steady-state conditions of the chemostat with regard to the FA composition of its lipids.

Pathways of ethanol production from sucrose by a mutant thermophilic Bacillus in continuous culture

A mutant (strain LLD-15) of Bacillus stearothermophilus strain LLD-R, that lacks l-lactate dehydrogenase activity was studied in anaerobic single-stage continuous culture using a defined minimal medium for its capacity to produce ethanol from sucrose at 70 °C. Cultures were stable at neutral pH, dilution rates below 0·25 h−1 and sucrose concentrations below 15 g l−1, producing mainly ethanol, CO2, formate and acetate plus a little succinate. The data obtained were used to estimate the maintenance energy coefficient at 70 °C and showed that the relative fluxes through the pyruvate-formate lyase (PFL) pathway and an anaerobic pyruvate dehydrogenase (PDH) pathway were about equal, though the later predominates at low dilution rates. At higher dilution rates or at acid pH, l-lactate became the major anaerobic product due to takeover by wild-type revertants. Reversion occurred only above a critical specific sucrose consumption rate of 4·2 g sucrose h−1 (g cells)−1 at pH 7, at which point pyruvate production was also seen. This can be attributed to saturation of the PFL pathway and PDH pathways by increasing glycolytic flux and consequent accumulation of intracellular pyruvate. The growth rate then declines, but a small population of revertant LLD-R cells would always be present in the bioreactor since the mutation in the l-lactate dehydrogenase gene has a significant reversion rate. These revertants have no selective advantage below the critical sugar uptake rate, but at higher rates they divert excess pyruvate to L-lactate, so their growth is unimpaired and they take over in the culture. At lower pH, growth restriction and reversion occur at low sucrose consumption rates due to difficulty in excreting acetic and formic acids against a pH gradient.

Lipid accumulation inRhodotorula glutinis on sugar cane molasses in single-stage continuous culture

Microbial lipids produced byRhodotorula glutinis grown in continuous culture with molasses under nitrogen-limiting conditions were evaluated and the effects of growth rate on fatty acid composition were studied. As the growth rate decreased, cell biomass, lipid content and lipid yield gradually increased. The maximum lipid content recorded was 39% (w/w) of dry cell biomass at a dilution rate of 0.04 h(-1). The growth rate also affected fatty acid composition: oleic acid decreased with decreasing growth rate while stearic acid increased.

Limitation of growth and lactic acid production in batch and continuous cultures of Lactobacillus helveticus

Continuous and batch cultures of Lactobacillus helveticus operated under different conditions were studied with respect to the limitation of growth and lactic acid production by increasing undissociated lactic acid and hydrogen ion concentrations, respectively. In a single-stage continuous culture without pH control a final pH of 3.8 and 65 mm undissociated lactic acid was obtained. In two-stage continuous cultures provided with different growth media and run at different pH values, 65–70 mm free acid was obtained in the second stage. Further batch-culture experiments showed growth limitation at 60–70 mm lactic acid. After growth ceased, production of lactate continued until a lactic acid concentration of about 100 mm was reached; obviously an uncoupling of growth and acid production had occurred. Examining the effect of different concentrations of either lactic acid or hydrochloric acid, added to growing batch cultures of L. helveticus, it was shown that the undissociated lactic acid concentration was responsible for growth limitation and lactic acid production in this organism, whereas the pH value had only an indirect effect.

Ethanol from whey: Continuous fermentation with cell recycle

The production of ethanol from cheese whey lactose has been demonstrated using a single-stage continuous culture fermentation with 100% cell recycle. In a two-step process, an aerobic fed batch operation was used initially to allow biomass buildup in the absence of inhibitory ethanol concentrations. In the anaerobic ethanol-producing second step, a strain of Kluyveromyces fragilis selected on the basis of batch fermentation data had a maximum productivity of 7.1 g ethanol/L/h at a dilution rate of 0.15 h(-1), while achieving the goal of zero residual sugar concentration. The fermentation productivity diminished when the feed sugar concentation exceeded 120 g/L despite the inclusion of a lipid mixture previous shown to enhance batch fermentation productivities.

Kinetics of ethanol production by yeast in continuous culture

The rate of fermentation of glucose by Saccharomyces uvarum in steadystate continuous culture in excess of substrates showed non-competitive inhibition kinetics with respect to ethanol. A model is presented which predicts that growth stops at a finite ethanol concentration, which was calculated to be 95 gl-1 for the system used here. The observed maximum ethanol concentration in a single stage continuous culture was 92 gl-1.