Effect of culture medium pH on bacterial gellan production.

The effect of the initial pH of the culture medium used in the production of the exopolysaccharide gellan by the bacterium Pseudomonas species ATCC 31461, when glucose or corn syrup served as the carbon source, was investigated. With glucose as the carbon source, exopolysaccharide formation was highest after 72 h of growth when the initial pH of the culture medium was 6.8 to 7.4. Polysaccharide production by the bacterial cells grown on corn syrup for 72 h was maximal when the initial pH of the medium was 7.0 or 7.2. Cell weights of the strain after 72 h tended to be higher for the glucose-grown cells than for the corn syrup-grown cells.     

Peptidomic analysis of blastocyst culture medium and the effect of peptide derived from blastocyst culture medium on blastocyst formation and viability

High‐quality in vitro human embryo culture medium can improve the blastocyst formation rate and blastocyst quality and be beneficial for the clinical application of single blastocyst transfer. Mammalian embryos can secrete protein products into the surrounding medium. As a group of bioactive molecules and degraded proteins, peptides have been shown to participate in various biological processes. Using liquid chromatography‐tandem mass spectrometry, we performed comparative peptidomic analysis of human culture medium in blastocyst formation and nonblastocyst‐formation groups. A total of 201 differentially expressed peptides originating from 157 precursor proteins were identified. Among these, a peptide derived from HERC2 (peptide derived from blastocyst culture medium [PDBCM]) passed through the zona pellucida, was distributed on the perivitelline space, was absent in arrest embryos and highly expressed in high‐quality blastocysts compared with low‐quality blastocysts, and significantly promoted blastocyst formation in a concentration‐dependent manner. These results indicate that PDBCM may be a novel biomarker for predicting blastocyst formation and viability. The mechanism remains unclear and needs to be explored in the future.     

Flow cytometric analysis of bacterial respiratory and enzymatic activity in the natural aquatic environment

The respiratory and enzymatic activity of bacteria in polluted and unpolluted river water was investigated by flow cytometry. Double staining with 6CFDA (6-carboxy fluorescein diacetate) and PI (propidium iodide) was used to examine bacterial esterase activity. CTC (5-cyano-2,3-ditolyl tetrazolium chloride) was employed as the indicator of bacterial respiration. The ratios of colony-forming units to total bacterial number were less than 2%, except highly polluted sites. The number of respiring bacteria determined by flow cytometry amounted to 10–15% of the total bacterial number at both unpolluted and polluted sampling stations, while it was 30% at the highly polluted station. Almost 50% of total bacteria demonstrated esterase activity in the unpolluted areas, whereas 70–90% of total bacteria retained this enzymatic activity in the polluted areas. Thus, many of the non-culturable bacteria in the natural aquatic environment have enzymatic activity regardless of the pollution level, and 6CFDA was more superior in the sensitivity for the evaluation of physiological activity of bacteria in the natural environment. The ratio of bacteria with esterase activity increased in direct proportion to the pollution level of river water and suggested that this ratio would be a useful indicator in evaluating the pollution levels in an aquatic environment.     

Introduction of air in the anaerobic culture of Streptococcus pneumoniae serotype 23F induces the release of capsular polysaccharide from bacterial surface into the cultivation medium

AIM: An approach to increase Streptococcus pneumoniae capsular polysaccharide (CPS) in the culture medium during fed-batch cultivation in bioreactor. METHODS AND RESULTS: Streptococcus pneumoniae serotype 23F was cultivated in a 5-l bioreactor with nitrogen-sparging and followed by addition of air in the stationary phase. The amount of CPS released in the supernatant progressively increased under air sparging. The profile of cellular viability and optical density was similar in both cultures. Immunoelectron microscopy showed that the amount of tightly cell-bound CPS was higher in bacteria cultivated under nitrogen than under air. CONCLUSIONS: The stress caused by the addition of air at the stationary phase promoted a large increase of free CPS into the medium, as a consequence of the morphologic change in the capsule. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of air in the stationary phase of the culture would greatly simplify the subsequent downstream process, allowing CPS purification from the supernatant. The direct consequence of this process improvement is the reduction of vaccine production costs.     

The effect of culture medium and aeration on growth of Listeria monocytogenes at pH 4.5

Listeria monocytogenes multiplied at 20°C in medium adjusted to pH 4.5 with HCl, and the lag before growth was eliminated when the inoculum was grown to log phase in the same medium. In a tryptone soya medium with yeast extract and added glucose, growth at pH 4.5 was more rapid than in a tryptose phosphate medium, and this difference was greater in air than under nitrogen. The results show that the bacterium was capable of more rapid growth in air than under nitrogen at this pH and suggest that the tryptose phosphate medium was nutritionally limiting for growth.     

Changes in bacterial communities from swine feces during continuous culture with starch

Bacteria from swine feces were grown in continuous culture with starch as the sole carbohydrate in order to monitor changes during fermentation and to determine how similar fermenter communities were to each other. DNA extracted from fermenter samples was analyzed by denaturing gradient gel electrophoresis (DGGE). A significant decrease in diversity was observed, the Shannon–Weaver index dropped from 1.92 to 1.13 after 14 days of fermentation. Likewise, similarity of fermenter communities to those in the fecal inoculum also decreased over time. Both diversity and similarity to the inoculum decreased most rapidly in the first few days of fermentation, reflecting a period of adaptation. Sequencing of DGGE bands indicated that the same species were present in replicate fermenters. Most of these bacteria were placed in the Clostridium coccoides/Eubacterium rectale group (likely saccharolytic butyrate producers), a dominant bacterial group in the intestinal tract of pigs. DGGE proved useful to monitor swine fecal communities in vitro and indicated the selection and maintenance of native swine intestinal bacteria during continuous culture.     

Effect of culture medium on the in vitro secretion activity of prothoracic glands from Pseudaletia separata

The prothoracic glands (Pgs) taken from the last instar of the common armyworm, Pseudaletia separata, were cultured in various media for the purpose of finding a suitable medium for relatively long-term culture of Pgs. Among the tested culture media, MGM-450 medium without serum was the best to maintain PG cells viable for relatively long periods, and to continue to secrete ecdysteroids. Secretion of ecdysteroid by the PG in vitro became marked when the PG was taken from last instar larvae older than 2 days after the last molt. PGs cultured in any of the media secreted ecdysteroid only within the first 2 h after placing them in culture, however, in the MGM-450 medium, the PGs secreted ecdysteroid even after 5 days of culture. Arch. Insect Biochem. Physiol. 38:147–154, 1998. © 1998 Wiley-Liss, Inc.     

Time-periodic oscillations in a model for the respiratory process of a bacterial culture

Summary Deterministic (analytical), stochastic and computer-aided methods have been used to construct the time-periodic (limit cycle) solution of a model suggested by H. Degn to account for the qualitative features of the respiratory process in a Klebsiella Aerogenes bacterial culture.     

Changes in the amount of fatty acids produced by Melosira dastei culture on synthetic medium

Some of fatty acids contents of the marine Diatom, Melosira dastei, were characterized. The major constituents of them are palmitic, palmitoleic and eicosapentaenoic acids. Linolenic acid type appeared to be a minor constituent. When that Diatom is cultured on an artificial medium, it might be considered as a potential source of valuable eicosapentaenoic acid. Even when the cells concentration is low, in batch cultures, eicosapentaenoic acid constitutes up to 5% of the dry weight and reaches about 20% of the triglycerids content. This level might be improved.     

Identification of aggrecanase activity in medium of cartilage culture.

Erosion of cartilage is a major feature of joint diseases, i.e., osteoarthritis and rheumatoid arthritis, which leads with time to a loss of joint function. Proteolytic cleavage of the aggrecan core protein is a key event in the progress of these joint diseases. Aggrecan degradation has been believed to be mediated by a putative proteinase, aggrecanase. We identified aggrecanase activity in conditioned medium from explant culture of bovine nasal cartilage stimulated by retinoic acid. The activity was partially purified more than 10,000-fold. The enzyme cleaves at the aggrecanase site (Glu(373)-Ala(374)) but not at the MMP site (Asn(341)-Phe(342)) in the interglobular domain of the aggrecan. It also cleaves at Glu(1971)-Leu(1972), which is located in the gap region in the chondroitin sulfate attachment region prior to the aggrecanase site. The enzyme is a typical Ca(2+)-dependent metalloproteinase with a unique salt-dependency and is inhibited by several hydroxamate-based inhibitors for matrix metalloproteinases. Heparin and chondroitin sulfate inhibited the enzyme in a dose-dependent manner, suggesting that the large carbohydorate in aggrecan is important for substrate recognition by aggrecanase.     

Changes in the properties of Bacillus thuringiensis after prolonged culture in a rich medium

Aim:  To investigate the effect of repeated culture in a rich medium on certain genetic, metabolic, pathogenic and structural characteristics of fresh isolates of Bacillus thuringiensis.
Methods and Results:  Four strains of B. thuringiensis , which had been isolated in vegetative form from leaf surfaces, were grown for 500 generations in batch culture in a rich medium. One of the strains, S4g, differed from the parent in the following respects: greater cell width; changed plasmid profile; complete loss of ability to produce δ-endotoxins; loss of ability to produce β-exotoxin and disruption of vip 3 gene; radically different fatty acid composition; and altered metabolic activity. Two of the other evolved strains (S1g and S6g) showed differences in fatty acid profiles compared with the parents. Genetic finger-printing showed that there were also mutations in the cry genes of two of the evolved strains (S1g and S2g). The δ -endotoxins of strain S6g were significantly less toxic to the larvae of Pieris brassica compared with those of the parent and it also differed in the plasmid content.
Conclusion:  Radical and unpredictable changes can occur in fresh isolates of B. thuringiensis when subjected to growth in the laboratory.
Significance and Impact of the Study:  This is the first analysis of a Gram positive and biotechnologically significant bacterium after repeated laboratory culture. It is of great relevance to the biotechnological exploitation of B. thuringiensis that prolonged growth of environmental isolates on laboratory culture media can have profound effects on their structure, genome and virulence determinants.