Kinetic modeling of kefiran production in mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae

Growth and kefiran production rates of Lactobacillus kefiranofaciens were significantly enhanced in a mixed culture with Saccharomyces cerevisiae as compared with those in a pure culture. Because a positive effect on growth and kefiran production of L. kefiranofaciens in a mixed culture was observed, the elucidation of interaction between L. kefiranofaciens and S. cerevisiae may lead to higher productivity. Hence, microbial performance of each strain was investigated and analyzed by a mathematical model. The mathematical model for kefiran fermentation in a mixed culture of L. kefiranofaciens and S. cerevisiae was established, and the impact of S. cerevisiae on cell growth, kefiran formation, and substrate assimilation of L. kefiranofaciens were considered. The behavior of L. kefiranofaciens in a mixed culture was predicted using a developed mathematical model in this work, and the predictions were compared with the results from mixed culture experiments. The overall mathematical model is capable of describing the behavior of S. cerevisiae in a mixed culture as a lactic acid consumer, nitrogen source competitor and protective function inducer for L. kefiranofaciens. Furthermore, the constructed model described the phenomena in mixed cultures under aerobic and anaerobic conditions. Finally, the optimal inoculation ratios of S. cerevisiae to L. kefiranofaciens at 7-fold and 10-fold under aerobic and anaerobic conditions were obtained by applying the mixed culture model, respectively.     

Comparison of Cultures from Rectoanal-Junction Mucosal Swabs and Feces for Detection of Escherichia coli O157 in Dairy Heifers

Fecal culture for Escherichia coli O157:H7 was compared to rectoanal mucosal swab (RAMS) culture in dairy heifers over a 1-year period. RAMS enrichment culture was as sensitive as fecal culture using immunomagnetic separation (IMS) (P = 0.98, as determined by a chi-square test). RAMS culture is less costly than fecal IMS culture and can yield quantitative data.     

Culture conditions of Aspergillus oryzae for production of enzyme preparation

Submerged culture was better than solid culture in the production of proteinase and peptidases from Aspergillus oryzae 460. On the contrary, solid culture was better than submerged culture in the production of α-amylase, carboxymethyl cellulase, and pectinlyase from the same fungus.The soy souce mash (moromi) made with the enzyme preparation from submerged culture was highly viscous and the soy sauce produced was characteristic in low contents of alcohol and reducing sugar, low pH value, and less aroma. Soy sauce made with the enzyme preparation from solid culture was superior on these points to that from submerged culture.Wheat bran was best as the raw material for the enzyme preparation in easy koji making, large amount produced, and low cost.In enzyme production from a solid culture, addition of urea (0.8% to wheat bran) nearly doubled the leucine aminopeptidase for Leu-Gly-Gly. The incubation period was reduced to 30 to 40 h from 50 to 60 h using germinated spores and moisture-controlled culture with forced aeration.     

Production of raw cassava starch-digestive glucoamylase by Rhizopus sp. in liquid culture

Among about 200 Rhizopus strains isolated in Thailand, Rhizopus sp. MB46 was selected as a producer of raw cassava starch-digestive glucoamylase. Rice bran was effective for the enzyme production in a solid culture as well as wheat bran. Addition of turpentine oil into the rice bran solid culture increased the productivity. Rhizopus sp. MB46 was found to produce glucoamylase in a liquid culture containing 1% rice bran but not in one consisting of 10% raw cassava starch of 2% glucose. The productivity per 1 g solids in the medium in liquid culture was finally improved 6-times by utilization of n-hexane-treated rice bran, supplement of 0.1% meat extract and addition of gauze as a support. The activity was superior to that in turpentine oil-supplemented solid culture.     

Aerosols as a Source of Widespread Mycoplasma Contamination of Tissue Cultures

Mycoplasma isolates were cultured from 15 antibiotic-free cell cultures obtained from a single laboratory. Complement-fixation tests showed that these isolates were antigenically related to each other but were unrelated to M. hominis type 1, M. hominis type 2, M. arthritidis, M. laidlawii type B, Mycoplasma sp. H.Ep. #2 (Barile), or M. salivarium. Examination of serum used to feed the infected cell lines revealed no Mycoplasma. Infection resulting from cross-contamination by a single Mycoplasma strain from one cell culture to another was investigated. Although the organisms were not found in the air over the work area, aerosols containing these contaminants were produced in tissue culture bottles during the trypsinization of cell monolayers. The minimal infectious dose of Mycoplasma for tissue cultures was measured, and it was determined that one organism was capable of initiating an infection in a tissue culture. The pattern of contamination and the small dose required for infection indicated that Mycoplasma contamination was spread from one tissue culture to another via aerosols. It was demonstrated that Mycoplasma can be transferred from one cell culture to another through the use of a common burette for dispensing medium.     

Influence of algae species,substrata and culture conditions on attached microalgal culture

The objective of this study was to understand and optimize the formation of microalgae biofilms in specific culture conditions. Firstly, the adhesion of six freshwater algae species was compared. Chlorococcum sp. was selected because of the high adhesion biomass productivity (ABP) and adhesion rate achieved. Secondly, the adhesion of Chlorococcum sp. was compared with nine commonly used supporting materials, and glass fiber-reinforced plastic proved to be the optimal substrata. Thirdly, based on response surface methodology experiments, a second-order polynomial model was developed to examine the effect of culture period, initial total nitrogen concentration (ITNC) in manure wastewater, pH and culture volume of the growth chamber on the adhesion of Chlorococcum sp. using glass fiber-reinforced plastic. The experimental and modeling results showed that ITNC, pH and culture volume as well as the interactions between culture period and ITNC, culture period and culture volume were significant on ABP. Optimum culture conditions were predicted at a culture period of 11 days, ITNC of 70 mg L^?1, pH of 8 and culture volume of 340 mL, under which the predicted maximum ABP was 4.26 g m^?2 day^?1. The prediction was close to validation experimental results, indicating that the model could be used to guide and optimize the attached culture of Chlorococcum sp. using glass fiber-reinforced plastic.     

Detection of Brucella canis from inguinal lymph nodes of naturally infected dogs by PCR

The aim of this study was to standardize and evaluate a PCR assay for the detection of Brucella canis (B. canis) in lymph node samples of naturally infected dogs. The performance of the PCR was compared with the results of bacteriological culture as reference method. Forty-eight inguinal lymph node samples were collected from 48 dogs (18 males and 30 females) that died in the city's pound in the years 2007-2008 and were examined by microbiological culture and the PCR assay. B. canis was isolated from 4 (8.3%) of 48 lymph node samples. Forty-four (91.7%) of the samples were bacteriological culture negative. B. canis DNA was directly detected from all culture positive lymph node samples (n = 4) by PCR. All of the culture negative samples were confirmed as negative by PCR. When the culture method was used as a gold standard, sensitivity and specificity of the PCR assay were found to be 100%. The limit of PCR detection of B. canis DNA was 1.4 × 10¹ CFU/g at least. In conclusion, the PCR assay has been shown to have a diagnostic performance equal to bacteriological culture for detection of B. canis. By a non-hazardous protocol for laboratory workers, the assay can be performed in one day.     

Continuous culture of CHO-K1 cells producing thrombomodulin and estimation of culture conditions

Recombinant Chinese hamster ovary (CHO-K1) cells expressing human soluble thrombomodulin (rsTM) were cultured in a continuous culture system with a fluidized-bed reactor. Cells were grown in a medium containing 1% serum for 10 d, and then cultured in a serum-free medium. The protein production rate increased remarkably in the serum-free culture, with a decrease in the lactate production rate. This suggests that CHO-K1 cells exhibit different physiological characteristics in response to serum removal from the medium, which resulted in a higher rsTM concentration (about 60 mg/l). A procedure for estimating protein productivity was developed using experimental glucose and lactate measurements. In this procedure, cell density was estimated from the glucose consumption rate, and the specific protein (rsTM) production rate was obtained from the ratio of lactate production/glucose consumption (ΔL/ΔG). Since the cell density and protein productivity in repeated batch culture were well estimated, the procedure was applied to continuous culture in a fluidized-bed bioreactor culture. The estimation procedure was also found to be effective in this continuous culture using the models derived from the repeated batch culture.     

Utilization of Lactose, Glucose, and Galactose by a Mixed Culture of Streptococcus thermophilus and Lactobacillus bulgaricus in Milk Treated with Lactase Enzyme

The mechanism responsible for an increased rate of acid production when yogurt starter cultures are grown in milk treated with lactase enzyme was investigated by studying carbohydrate utilization and acid development by a pure culture of Streptococcus thermophilus and a mixed yogurt starter culture consisting of S. thermophilus and Lactobacillus bulgaricus. In milk containing glucose, galactose, and lactose, glucose and lactose (but not free galactose) were fermented. Fermentation of lactose in control milk was accompanied by the release of free galactose, with the result that carbohydrate utilization was less efficient than in treated milk. This phenomenon also occurred when lactose was fermented by S. thermophilus in broth culture. Carbohydrate utilization by the mixed yogurt culture was more rapid when the lactose in milk was partially prehydrolyzed. Our results suggest that the more rapid acid development that took place when a mixed yogurt starter culture was grown in milk containing prehydrolyzed lactose was the result of a more rapid and efficient utilization of carbohydrate by S. thermophilus when free glucose in addition to lactose was available for fermentation. The evidence presented also suggests that uptake and utilization of glucose and lactose by S. thermophilus are different in broth and milk cultures.     

Microbial Control of the Culture of Artemia Juveniles through Preemptive Colonization by Selected Bacterial Strains

The use of juvenile Artemia as feed in aquaculture and in the pet shop industry has been getting more attention during the last decade. In this study, the use of selected bacterial strains to improve the nutritional value of dry food for Artemia juveniles and to obtain control of the associated microbial community was examined. Nine bacterial strains were selected based on their positive effects on survival and/or growth of Artemia juveniles under monoxenic culture conditions, while other strains caused no significant effect, significantly lower rates of survival and/or growth, or even total mortality of the Artemia. The nine selected strains were used to preemptively colonize the culture water of Artemia juveniles. Xenic culture of Artemia under suboptimal conditions yielded better survival and/or growth rates when they were grown in the preemptively colonized culture medium than when grown in autoclaved seawater. The preemptive colonization of the culture water had a drastic influence on the microbial communities that developed in the culture water or that were associated with the Artemia, as determined with Biolog GN community-level physiological profiles. Chemotaxonomical characterization based on fatty acid methyl ester analysis of bacterial isolates recovered from the culture tanks was performed, and a comparison with the initially introduced strains was made. Finally, several modes of action for the beneficial effect of the bacterial strains are proposed.     

Immature embryo and anther culture of chromosome addition lines of rye in chinese spring wheat

Significant variation among Chinese Spring wheat (Triticum aestivum L.) and a set of seven addition lines in which chromosomes from rye (Secale cereale L.) were incorporated into the Chinese Spring background was observed for callus formation and plant regeneration from anther cultures and for plant regeneration from immature embryo cultures. Callus initiation from immature embryo cultures was uniformly high. Rye chromosome 4 contains factors which significantly increase both anther culture responses relative to Chinese Spring. Rye chromosomes 6 and 7 both contain positive factors for regeneration from immature embryo culture. While no correlation was found between anther culture and embryo culture responses, a positive correlation was observed between the two anther culture response variables.     

Calcium-dependent mechanism of somatic embryogenesis in Panax ginseng cell cultures expressing the rolC oncogene

The Panax ginseng 2c3 embryogenic cell culture was earlier obtained by callus cell transformation with Agrobacterium rhizogenes rolC. Calcium channel blockers (LaCl₃, verapamil, and niflumic acid) reduced the production of somatic embryos in the 2c3 culture, implicating the Ca²⁺ signaling system in plant somatic embryogenesis. The protein kinase inhibitors W7 and H7 also decreased the yield of somatic embryos in the 2c3 culture. The total CDPK expression in the 2c3 culture was 1.2-to 1.5-fold lower than in a control callus culture as a result of a silencing of the genes belonging to the PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a) subfamilies. Expression of the PgCDPK2 subfamily genes (PgCDPK2b and PgCDPK2d) was increased. It was assumed that some genes of the PgCDPK1, PgCDPK2, and PgCDPK3 subfamilies were responsible for generation of embryogenic cells in the 2c3 culture. For the first time, rolC expression and embryogenesis were associated with changes in the expression of certain CDPK genes.     

Significance of the Cgl1427 gene encoding cytidylate kinase in microaerobic growth of Corynebacterium glutamicum

The Cgl1427 gene was previously found to be relevant to the microaerobic growth of Corynebacterium glutamicum (Ikeda et al. Biosci Biotechnol Biochem 73:2806–2808, 2009). In the present work, Cgl1427 was identified as a cytidylate kinase gene (cmk) by homology analysis of its deduced amino acid sequence with that of other bacterial cytidylate kinases (CMP kinases) and on the basis of findings that deletion of Cgl1427 results in loss of CMP kinase activity. Deletion of the cmk gene significantly impaired the growth of C. glutamicum in oxygen-limiting static culture, and the impaired growth was restored by introducing a plasmid containing the cmk gene, suggesting that this gene plays an important role in the microaerobic growth of C. glutamicum. On the other hand, in the main culture with aerobic shaking, a prolonged lag phase was observed in the cmk disruptant, despite an unchanged growth rate, compared to the behavior of the wild-type strain. The prolongation was observed when using seed culture grown to later growth stages in which oxygen limitation occurred, but it was not observed when using seed culture grown to an earlier growth stage in which oxygen remained relatively plentiful. Since nucleotide biosynthesis in C. glutamicum requires oxygen, we hypothesized that the ability of the cmk disruptant to synthesize nucleotides was influenced by oxygen limitation in the later growth stages of the seed culture, which caused the prolongation of the lag phase in the following shaken culture. To verify this hypothesis, a plasmid containing genes encoding all components of a homologous ribonucleotide reductase, a key enzyme for nucleotide synthesis that requires oxygen for its reaction, was introduced into the cmk disruptant, which significantly ameliorated the lag phase prolongation. Furthermore, this experimental setup almost completely restored the growth of the cmk disruptant in the oxygen-limiting static culture. These results indicate that CMP kinase plays an important role in normal nucleotide biosynthesis under an oxygen-limiting environment.     

Homogenisation of cystic fibrosis sputum by sonication--an essential step for Aspergillus PCR

The importance of Aspergillus as a lung pathogen in cystic fibrosis (CF) is becoming increasingly recognised. However, fungal culture of CF sputum is unreliable and there is no consensus for identifying phenotypes beyond ABPA that may benefit from antifungal therapy. There are no published studies using real-time PCR to detect Aspergillus in CF sputum. The major barrier to sensitive detection of Aspergillus using PCR is sputum homogenisation. This study aimed to optimise sputum homogenisation utilising sonication to improve Aspergillus DNA extraction. Sonication amplitude and duration that enabled sputum homogenisation but ensured preservation of DNA integrity were first determined. 160 sputum samples were collected from CF patients. 49 of the sputum samples were split, one half was used for standard culture and the other half was homogenised with NALC-NaOH before undergoing DNA extraction. The subsequent 111 samples were homogenised with dithiothreitol plus sonication prior to culture and DNA extraction. Real-time PCR targeting a portion of the 18S rDNA of Aspergillus was performed on all DNA extractions. In the 49 samples with no sonication 8 (16%) were culture positive but only 4 of these were PCR positive. However, PCR was positive in 11 culture negative samples. PCR after sonication showed a significant improvement in sensitivity: 33 (30%) were culture and PCR positive, 48 (43%) were culture negative, but PCR positive (p < 0.0001) and 30 (27%) were culture and PCR negative. The combination of dithiothreitol and sonication to homogenise sputum increases PCR yield, with PCR being substantially more sensitive than culture.     

Biodegradation of Metal-EDTA Complexes by an Enriched Microbial Population

A mixed culture utilizing EDTA as the sole carbon source was isolated from a mixed inoculum of water from the River Mersey (United Kingdom) and sludge from an industrial effluent treatment plant. Fourteen component organisms were isolated from the culture, including representatives of the genera Methylobacterium, Variovorax, Enterobacter, Aureobacterium, and Bacillus. The mixed culture biodegraded metal-EDTA complexes slowly; the biodegradability was in the order Fe>Cu>Co>Ni>Cd. By incorporation of inorganic phosphate into the medium as a precipitant ligand, heavy metals were removed in parallel to EDTA degradation. The mixed culture also utilized a number of possible EDTA degradation intermediates as carbon sources.     

Cultivation of syntrophic anaerobic bacteria in membrane-separated culture devices

A dialysis culture device was used for growth of syntrophic fatty acid-oxidizing and ethanol-oxidizing anaerobic bacteria. A pure culture of the fatty acid oxidizer Clostridium bryantii was grown inside dialysis tubing which was surrounded by a pure culture of Desulfovibrio vulgaris. The same apparatus was used for the syntrophic cultivation of Pelobacter acetylenicus and Acetobacterium woodii with ethanol as substrate. In both cases, substrate degradation and product formation were about half as fast as with the homogeneously mixed control cultures. In the compartment of the hydrogen producer, the concentration of free hydrogen during syntrophic ethanol degradation was about 10 times as high as in that of the hydrogen utilizer, whereas the homogeneously mixed culture exhibited an intermediate hydrogen partial pressure.     

In vitro culture of Plasmodium berghei using a new suspension system

Attempts were made to develop techniques for the continuous in vitro culture of Plasmodium berghei. The candle jar method (Trager &; Jensen, 1976) proved to be insufficient for the culture of this rodent malaria parasite. Parasitaemia decreased rapidly after the first schizogonic cycle in culture. A simple suspension technique was developed using a newly designed culture apparatus which can be placed in the laminar-flow. All manipulations necessary for the refreshment of medium and cells can be made with almost no disturbance of the culture conditions. With this system it was possible to culture P. berghei repeatedly for more than a week, completing at least four schizogonic cycles with considerable mcrozoite invasion and a 2–6-fold multiplication. Infection rates of up to 6.0% were achieved and cultures were maintained for 9 days. Several specific properties of P. berghei and the differences between the candle jar method and the new suspension method are discussed to explain the results obtained in both systems.     

Production of salmosin, a snake venom-derived disintegrin, in recombinant Pichia pastoris using high cell density fed-batch fermentation

Salmosin, a snake venom-derived disintegrin, was successfully expressed in the methylotrophic yeast Pichia pastoris and secreted into the culture supernatant, as a 6 kDa protein. High-cell density fermentation of recombinant P. pastoris was optimized for the mass production of salmosin. In a 5 L jar fermentor, recombinant P. pastoris was fermented in growth medium containing 5% (w/v) glycerol at the controlled pH of 5.0. After culturing for 21 h, glycerol feeding medium was fed at one time into the culture broth. After 7 h (a total of 28 h), induction medium that contained methanol was increasingly added until the culture time totaled 75 h. Finally, these optimized culture conditions produced a high cell density of recombinant P. pastoris (dry cell weight of 113.38 g/L) and led to the mass production of salmosin (a total protein concentration of 369.2 mg/L). The culture supernatant containing salmosin inhibited platelet aggregation, resulting in a platelet aggregation of 9% compared to that of 94% in the control experiment, without culture supernatant. These results demonstrate that recombinant salmosin in culture supernatant from high cell density fed-batch fermentation can serve as a platelet aggregation inhibitor.     

A Novel Acidimicrobium Species in Continuous Cultures of Moderately Thermophilic, Mineral-Sulfide-Oxidizing Acidophiles

A novel species of Acidimicrobium appeared to be the predominant ferrous iron oxidizer in a mixed culture that effected the continuous, efficient extraction of nickel from a mineral concentrate at 49°C, but it was not isolated in pure culture. It outcompeted Acidimicrobium ferrooxidans, which was expected to have a major role in iron oxidation in reactors gassed with air, and was outnumbered at 49°C only by the sulfur-oxidizing Acidithiobacillus caldus. Sulfobacillus species were expected to compete with Acidimicrobium species when culture aeration was enriched with carbon dioxide, but they were a minor component of the populations with and without this enrichment. Sulfobacillus thermosulfidooxidans replaced the Acidimicrobium species and Acidithiobacillus caldus when the temperature was increased to 55°C.     

Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae

The DNA (glaB) and a cDNA-encoding glucoamylase produced in solid-state culture of Aspergillus oryzae were cloned using oligodeoxyribonucleotide probes derived from internal amino acid sequences of the enzyme. Comparison of the nucleotide sequences of a genomic DNA fragment with its cDNA showed the glaB gene carried three exons interrupted by two introns and had an open reading frame encoding 493 aa residues. The 5′-flanking region had a TATA box at nt −87 from the start codon and two putative CAAT sequences at nt −276 and −288. The glaB gene shared 57% homology at the aa level with the glaA gene which was cloned previously from A. oryzae. Interestingly, the glucoamylase encoded by the glaB gene had no C-terminal domain such as that proposed to have starch binding activity in Aspergillus glucoamylases. Introduction of cDNA of the glaB gene to Saccharomyces cerevisiae caused the secretion of active glucoamylase to culture medium and introduction of the glaB gene to A. oryzae increased glucoamylase productivity in solid-state culture. Northern blot analysis showed the glaB gene was expressed in solid-state culture, but not in submerged culture.