Continuous-flow Culture System Research Articles

Effect of feeding frequency on growth performance, feed transit and digestive enzyme activity of Acipenser dabryanus juveniles

Dabry's sturgeon (Acipenser dabryanus) is critically endangered in China. However, the lack of research on the feeding strategy of this species is detrimental to its conservation management. The present study evaluated the effects of feeding frequency (1, 2, 3, 4 and 5 times per day) on the growth performance, feed transit and digestive enzyme activity of juvenile Dabry's sturgeons (initial weight 0.42 ± 0.09 g) in a continuous flow culture system at a water temperature of 18.2–18.5°C. Single-meal and normal-feeding (1, 2, 3, 4 and 5 times per day) experimental methods were adopted to assess the effect of different feeding frequencies on feed transit. The fish in each treatment of the normal-feeding experiment were fed normally according to the original feeding frequency (1, 2, 3, 4 and 5 times per day). The optimum feeding frequency for juvenile Dabry's sturgeons was determined to be 4 times per day based on the growth performance, feed conversion rate and digestive enzyme activity. The results of the single-meal experiment demonstrated that changes in daily feeding frequency modified residence time in the stomach (S1–S5: 27.2–10.4 h) and intestine (S2–S5: range from 14 to 29.85 h). However, the obtained gastric emptying time varied significantly depending on the experimental approach. The single-meal experiment approach resulted in a longer gastric emptying time of fish fed 1 and 2 times per day (S1: >35 h; S2: 35 h) than those in the normal-feeding experiment (N1: 16 h; N2: 24 h), demonstrating that the single-meal method might change the gastrointestinal emptying strategy of fish. Furthermore, the digestive enzyme activity had daily rhythmic changes and the average digestive enzyme activity was affected by feeding frequency. The average digestive enzyme activity of the fish in N3 or N4 was higher than those of fish in other groups (p < 0.05). In summary, this study provides much useful information for the healthy farming of juvenile Dabry's sturgeons and towards the understanding of digestive physiology, which is of great significance for the protection of Dabry's sturgeon.

Enhanced Growth of Acidovorax sp. Strain 2AN during Nitrate-Dependent Fe(II) Oxidation in Batch and Continuous-Flow Systems

Microbial nitrate-dependent, Fe(II) oxidation (NDFO) is a ubiquitous biogeochemical process in anoxic sediments. Since most microorganisms that can oxidize Fe(II) with nitrate require an additional organic substrate for growth or sustained Fe(II) oxidation, the energetic benefits of NDFO are unclear. The process may also be self-limiting in batch cultures due to formation of Fe-oxide cell encrustations. We hypothesized that NDFO provides energetic benefits via a mixotrophic physiology in environments where cells encounter very low substrate concentrations, thereby minimizing cell encrustations. Acidovorax sp. strain 2AN was incubated in anoxic batch reactors in a defined medium containing 5 to 6 mM NO₃⁻, 8 to 9 mM Fe²⁺, and 1.5 mM acetate. Almost 90% of the Fe(II) was oxidized within 7 days with concomitant reduction of nitrate and complete consumption of acetate. Batch-grown cells became heavily encrusted with Fe(III) oxyhydroxides, lost motility, and formed aggregates. Encrusted cells could neither oxidize more Fe(II) nor utilize further acetate additions. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced, and further additions of acetate and Fe(II)-EDTA could be oxidized. Experiments using a novel, continuous-flow culture system with low concentrations of substrate, e.g., 100 μM NO₃⁻, 20 μM acetate, and 50 to 250 μM Fe²⁺, showed that the growth yield of Acidovorax sp. strain 2AN was always greater in the presence of Fe(II) than in its absence, and electron microscopy showed that encrustation was minimized. Our results provide evidence that, under environmentally relevant concentrations of substrates, NDFO can enhance growth without the formation of growth-limiting cell encrustations.

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration

The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla(TEM-52) -carrying plasmid (lactose-negative mutant of B1-54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. Fresh faeces from a healthy volunteer, negative for cephalosporin-resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed-field gel electrophoresis profiles (PFGE). The avian extended-spectrum β-lactamase-positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1-54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla(TEM-52) -carrying plasmid. These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.

Impact of different benthic animals on phosphorus dynamics across the sediment-water interface

As a diagenetic progress, bioturbation influences solute exchange across the sediment-water interface (SWI). Different benthic animals have various mechanical activities in sediment, thereby they may have different effects on solute exchange across the SWI. This laboratory study examined the impacts of different benthic animals on phosphorus dynamics across the SWI. Tubificid worms and Chironomidae larvae were introduced as model organisms which, based on their mechanical activities, belong to upward-conveyors and gallery-diffusers, respectively. The microcosm simulation study was carried out with a continuous flow culture system, and all sediment, water, and worms and larvae specimens were sampled from Taihu Lake, China. To compare their bioturbation effects, the same biomass (17.1 g wet weight (ww)/m 2) was adopted for worms and larvae. Worms altered no oxygen penetration depth in sediment, while larvae increased the O 2 penetration depth, compared to the control treatment. Their emergence also enhanced sediment O 2 uptake. The oxidation of ferrous iron in pore water produced ferric iron oxyhydroxides that adsorbed soluble reactive phosphorus (SRP) from the overlying water and pore water. Larvae built obviously oxidized tubes with about 2 mm diameter and the maximum length of 6 cm in sediment, and significantly decreased ferrous iron and SRP in the pore water compared to the control and worms treatments. Worms constructed no visually-oxidized galleries in the sediment in contrast to larvae, and they did not significantly alter SRP in the pore water relative to the control treatment. The adsorption of ferric iron oxyhydroxides to SRP caused by worms and larvae inhibited SRP release from sediment. Comparatively, worms inhibited more SRP release than larvae based on the same biomass, as they successively renewed the ferric iron oxyhydroxides rich oxidation layer through their deposition.

Planktonic and biofilm communities from 7-day-old chicken cecal microflora cultures: characterization and resistance to Salmonella colonization.

Information implicating bacterial biofilms as contributory factors in the development of environmental bacterial resistance has been increasing. There is a lack of information regarding the role of biofilms within the microbial ecology of the gastrointestinal tract of food animals. This work used a continuous-flow chemostat model derived from the ceca of 7-day-old chicks to characterize these communities and their ability to neutralize invasion by Salmonella enterica serovar Typhimurium. We characterized and compared the biofilm and planktonic communities within these microcosms using automated ribotyping and the Analytical Profile Index biotyping system. Eleven species from eight different genera were identified from six culture systems. Klebsiella pneumoniae was isolated from all planktonic communities and four of the biofilm communities. Three of the communities resisted colonization by Salmonella enterica serovar Typhimurium, two communities suppressed growth, and one community succumbed to colonization. In cultures that resisted colonization, no Salmonella could be isolated from the biofilm; in cultures that succumbed to colonization, Salmonella was consistently found within the biofilms. This study was one of a series that provided a molecular-based characterization of both the biofilm and planktonic communities from continuous-flow culture systems derived from the cecal microflora of chicks, ranging in age from day-of-hatch to 14 days old. The one common factor relating to successful colonization of the culture was the presence of Salmonella within the biofilm. The capacity to sequester the introduced Salmonella into the biofilm appears to be a contributing factor to the inability of these cultures to withstand colonization by the Salmonella.

The role of disulfide bond isomerase A (DsbA) ofEscherichia coliO157:H7 in biofilm formation and virulence

The role of periplasmic disulfide oxidoreductase DsbA in Shiga toxin-producing Escherichia coli O157:H7 (STEC) was investigated. Deletion of dsbA (DeltadsbA) significantly decreased cell motility and alkaline phosphatase activity in STEC. STEC DeltadsbA also showed greater sensitivity to menadione and under low pH conditions. Significant reductions in surface attachment to both biotic (HT-29 epithelial cells) and abiotic (polystyrene and polyvinyl chloride) surfaces were observed in STEC DeltadsbA. In addition, no biofilm formation was detected in STEC DeltadsbA compared to wild-type cells in glass capillary tubes under continuous flow-culture system conditions. In the nematode model Caenorhabditis elegans-killing assay, the deletion of dsbA in STEC resulted in attenuated virulence compared to wild-type cells. STEC DeltadsbA was also found to have a reduced ability to colonize the nematode gut. These results suggest that DsbA plays important roles in biofilm formation and virulence in STEC cells.

Survival and function of rat hepatocytes cocultured with nonparenchymal cells or sinusoidal endothelial cells on biodegradable polymers under flow conditions

Background/Purpose: The authors have investigated hepatocyte transplantation using biodegradable polymer scaffolds as a possible treatment of end-stage liver disease. The purpose of this study was to investigate the survival rate and function of hepatocytes alone or cocultured with other cell types on 3-dimensional biodegradable polymers for 7 days under continuous flow conditions in vitro. Methods: Hepatocytes (group 1, n = 8), hepatocytes with nonparenchymal cells (group 2, n = 7), or hepatocytes with sinusoidal endothelial cells (group 3, n = 6) were isolated from Lewis rats and seeded onto the polymer scaffolds. The polymer devices subsequently were placed under continuous flow conditions for 7 days. Albumin production from the constructs was measured each day, and urea nitrogen synthesis was examined on day 7. The devices also were examined by histology at day 7. Results: Histology results showed the presence of numerous viable hepatocytes on polymer devices, with no differences in hepatocyte viability between the 3 groups. Albumin secretion in the culture medium gradually decreased by day 7. There also were no significant differences in albumin production or urea nitrogen synthesis between the 3 groups at day 7. Conclusions: Hepatocytes could survive on the 3-dimensional polymer scaffolds under flow conditions for 7 days, and albumin secretion and urea synthesis of hepatocytes were seen at day 7. Nonparenchymal cells and sinusoidal endothelial cells had no measurable effect on hepatocyte function in our continuous flow culture system. J Pediatr Surg 35:1287-1290. Copyright © 2000 by W.B. Saunders Company.

Optimization and stability of glucoamylase production by recombinant strains ofAspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L−1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L−1 in batch culture and 373 ± 9 mg GAM L−1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h−1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]−1 h−1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h−1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h−1, the addition of 5 g mycopeptone L−1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 ± 12 to 496 ± 10 mg GAM L−1) and a 37% increase in specific production rate (from 12.0 ± 0.4 to 16.4 ± 1.6 mg GAM [g biomass]−1 h−1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:407–418, 1998.

The amelioration of aluminium toxicity by silicon in wheat ( Triticum aestivum L.): malate exudation as evidence for an in planta mechanism

Two wheat (Triticum aestivum L.) cultivars, one aluminium tolerant (Atlas 66) and one sensitive (Scout 66), were grown in a continuous-flow culture system (≤pH 5.0) containing aluminium (0–100 μM) and silicon (0–2000 μM) in factorial combination. Treatment with silicon resulted in a highly significant amelioration of aluminium toxicity as assessed by root growth in both cultivars. Amelioration was influenced by wheat cultivar and silicon concentration, as 2000 μM silicon significantly ameliorated the toxic effects of 100 μM aluminium in Atlas 66, and only 5 μM silicon alleviated the effect of 1.5 μM aluminium on Scout 66. Nutrient medium pH was critical, as an amelioration by silicon was apparent only at pH > 4.2 for Atlas 66, and at pH > 4.6 for Scout 66. Silicon neither reduced levels of toxic aluminium species in the growth solutions, nor the amount of aluminium taken up by roots. In experiments to assess exudation of malate by Atlas 66 roots treated with 100 μM aluminium, the presence of 2000 μM silicon (pH 4.6) was found to have a negligible effect on exudation. In contrast, citrate, a known aluminium chelator, reduced aluminium-induced exudation of malate at 5–40 μM and completely inhibited it at 100 μM citrate. The results indicate that silicon does not reduce aluminium phytotoxicity as a result of aluminium/silicon interactions in the external media, and that the mechanism of amelioration has an in planta component.

A Modified Continuous Flow Culture System for Studying Microbial Interactions Related to the Gastro-Intestinal Tract: Application of the Probiotic Organism Enterococcus faecium SF68

A Modified Continuous Flow Culture System for Studying Microbial Interactions Related to the Gastro-Intestinal Tract: Application of the Probiotic Organism Enterococcus faecium SF68

Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system.

A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-µm pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal.

Production and Characterization of Monoclonal Antibodies to aVeillonellaspecies from a Continuous-Flow Culture System of Chicken Cecal Bacteria

Administering native intestinal flora to newly hatched chicks protects against cecalSalmonellacolonization, and is known as competitive exclusion. Continuous-flow culture systems have been used to maintain defined competitive exclusion cultures. We have recently demonstrated that such a stable continuous-flow culture, CF3, contains 29 bacterial strains representing ten genera. Broiler chicks treated with CF3 are protected againstSalmonellacolonization of the ceca. Such protection is correlated with elevated concentrations of proprionic acid in the cecal contents of treated chicks. In this study we report on the preparation and characterization of monoclonal antibodies to one of the proprionic acid producing anaerobes contained in CF3, namelyVeillonellaCF3. Five different monoclonal antibodies were characterized with respect to: (1) isotype; (2)Veillonellaspecificity as judged by cross-reactivity profiles with other bacteria; (3) sensitivity as measured by the limit of detection of the number of colony forming units ofVeillonella; and (4) antigen recognition ofVeillonellaby Western Blot analysis. These antibodies have been used to enumerateVeillonellain both the CF3 cultures and in the ceca of young chicks.

連続流動培養装置によって培養されたヒト腸内フローラによる数種のマイコトキシンの分解性

We investigated the effect of cultured human intestinal microflora on several mycotoxins, ochratoxin A (OCTA), patulin, zearalenone, citrinin, aflatoxin B1 and fumonisin B1, B2. Human intestinal microflora derived from a Japanese health adult was cultured using continuous flow culture system we had developed previously. We examined the recoveries of seven mycotoxins after incubation with human intestinal microflora for 8 hr. As a result, zearalenone, citrinin, and aflatoxin B1 were not degraded by human intestinal microflora for 8 hr incubation. It was, however, suggested that OCTA and fumonisin B1, B2 might he partially degraded by human intestinal microflora.

Stability of recombinant protein production by Penicillium chrysogenum in prolonged chemostat culture

A strain (WKW2) of Penicillium chrysogenum transformed with heterologous fungal acetamidase ( amdS) and bacterial β-galactosidase ( lacZ) was grown at a dilution rate of 0.17 h −1 (doubling time of approx. 4.1 h) for 1600 h in a glucose-limited culture. By the end of the experiment the original strain had been almost completely replaced by spontaneous, morphological mutants, but the acetamidase and β-galactosidase activities of the culture were essentially unaltered. Furthermore, when WKW2 and the non-transformed parental strain (NRRL1951) were grown together in glucose- or NH 4 +-limited chemostat cultures, neither strain had a selective advantage over the other. Thus, heterologous gene expression does not result in NRRL1951 having a selective advantage over WKW2. These results suggest that continuous flow culture systems could be used for efficient (and cost effective) production of recombinant proteins.

Stability of recombinant protein production byPenicillium chrysogenumin prolonged chemostat culture

A strain (WKW2) of Penicillium chrysogenum transformed with heterologous fungal acetamidase (amdS) and bacterial β-galactosidase (lacZ) was grown at a dilution rate of 0.17 h−1 (doubling time of approx. 4.1 h) for 1600 h in a glucose-limited culture. By the end of the experiment the original strain had been almost completely replaced by spontaneous, morphological mutants, but the acetamidase and β-galactosidase activities of the culture were essentially unaltered. Furthermore, when WKW2 and the non-transformed parental strain (NRRL1951) were grown together in glucose- or NH4+-limited chemostat cultures, neither strain had a selective advantage over the other. Thus, heterologous gene expression does not result in NRRL1951 having a selective advantage over WKW2. These results suggest that continuous flow culture systems could be used for efficient (and cost effective) production of recombinant proteins.

Control of Salmonella typhimurium Colonization in Broiler Chicks with a Continuous-Flow Characterized Mixed Culture of Cecal Bacteria

A continuous-flow culture system was used to isolate and maintain a mixed culture of cecal bacteria from adult broilers composed of 29 bacterial strains representing 10 genera. Broiler chicks were treated with the mixed culture in the drinking water on the day of hatch and challenged orally with 104Salmonella typhimurium 2 d after treatment. The experiment was repeated in four separate trials using newly hatched chicks. The concentration of propionic acid and total volatile fatty acid (VFA) in the cecal contents was determined 2 d after treatment and at 10 d of age. Compared with controls, the number of treated chicks that were cecal culture-positive for Salmonella decreased (P < .01) in each of the trials. Additionally, the number of Salmonella in the cecal contents of the treated chicks at 10 d of age was decreased (P < .005) compared with controls in each trial. The decreased number of Salmonella in the cecal contents of the 10-d-old treated chicks was correlated with elevated concentrations of propionic acid (P < .05) and total VFA (P < .1) in the cecal contents of the treated chicks 2 d after treatment. The results indicated that VFA-producing bacteria present in the mixed culture became rapidly established in the ceca of the treated chicks and effectively increased resistance to S. typhimurium challenge.

Development of Defined Cultures of Indigenous Cecal Bacteria to Control Salmonellosis in Broiler Chicks

An in vitro continuous-flow (CF) culture system was utilized to isolate and maintain a defined mixed culture of indigenous cecal bacteria from adult broilers. The protective effects of the defined CF culture and dietary lactose on Salmonella typhimurium colonization were evaluated in broiler chicks The CF culture was administered to chicks by crop gavage on the day of hatch. Lactose was provided as 5% (wt/wt) of the feed ration. The chicks were challenged orally with 104S. typhimurium at 3 days of age and evaluated for Salmonella colonization 7 days after challenge. The experiment was repeated in six separate trials using newly hatched chicks and CF culture that was maintained in continuous steady-state conditions from 42 to 190 days. Compared with controls, the mean number of S. typhimurium in the cecal contents of the chicks given CF culture and dietary lactose decreased significantly (P < .01) by 4.2 log10 units. Similarly, the numbers of Salmonella cecal culture-positive chicks was significantly decreased (P < .01) by 55% in the chicks given CF culture and lactose. The results indicated that a defined culture of indigenous cecal bacteria isolated and maintained in CF culture, together with dietary lactose, effectively controlled S. typhimurium cecal colonization in newly hatched broiler chicks.

Myco-protein: A twenty-year overnight success story

Fusarium graminearum for production of myco-protein is currently grown on a glucose-ammonia-biotin-mineral salts medium in a 40 m3 air-lift fermenter. The resulting biomass is RNA reduced, harvested, texturized and sold for human consumption, either directly as a food or as meat or poultry alternatives in pre-prepared meals. Originally intended in the 1960s to combat the world's flagging supply of protein foods, Quorn® myco-protein was marketed in the middle 1980s as a low-calorie, high-fibre, food containing no cholesterol or animal fats. The continuous flow culture system currently used for myco-protein production is described, together with details of the ‘evolution’ of the fungus in prolonged culture.

Control of Escherichia coli populations by a combination of indigenous clostridia and lactobacilli in gnotobiotic mice and continuous-flow cultures.

The function of indigenous lactobacilli in the control of other intestinal microbial species is not clear. Still more controversial is the effect of dietary bacterial supplements containing lactobacilli or other species. This situation is unlikely to change unless the mechanisms that control the colonization of ingested bacteria are better understood, and until more detailed information becomes available on the mechanisms by which certain populations of indigenous bacteria can affect the population sizes of other species. We used gnotobiotic mice and a continuous-flow culture system to study the interactions between Escherichia coli and (i) clostridia (in chloroform-treated cecal suspensions from conventional mice) and (ii) three strains of lactobacilli isolated from conventional mice. In gnotobiotic mice, the lactobacilli suppressed E. coli multiplication in the stomach and the small intestine, but had no demonstrable effect on E. coli multiplication in the large intestine. In contrast, clostridia were most effective in controlling E. coli multiplication in the large intestine. In the presence of both lactobacilli and clostridia, E. coli populations in the various regions of the gastrointestinal tract resembled those found in conventionalized control animals. The control of E. coli populations was not related to changes in pH or intestinal motility. In vitro stimulation of the above-described in vivo interactions required a two-stage continuous-flow culture in which the effluent from the first stage represented the influx to the second. The first stage was inoculated with lactobacilli, and the second stage was inoculated with either a pure culture of E. coli or E. coli and clostridia. In these instances, the E. coli populations in the second stage of the culture resembled in size those found in the large intestine of gnotobiotic mice harboring a similar flora. Although there are some current shortcomings of this in vitro model, we expect that a multistage continuous-flow culture can be developed to satisfactorily model the interactions among bacterial populations along the entire gastrointestinal tract.