Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four-electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H(+) ions produced by some accumulated acidic substances, and the impedance thus increased.     

The growth of Bacillus stearothermophilus on stainless steel

AIMS: To determine the potential for Bacillus stearothermophilus cells to form biofilms of significance in dairy manufacture. METHODS AND RESULTS: The ability of isolates of B. stearothermophilus from dairy manufacturing plants to attach to stainless steel surfaces was demonstrated by exposing stainless steel samples to suspensions of spores or vegetative cells and determining the numbers attaching using impedance microbiology. Spores attached more readily than vegetative cells. The attachment of cells to stainless steel was increased 10-100-fold by the presence of milk fouling the stainless steel. The growth of B. stearothermophilus as a biofilm on stainless steel surfaces was determined using a continuously flowing experimental reactor. Vegetative cells were released in greater numbers than spores from biofilms of most strains studied. Biofilms of one strain (B11) were studied in detail. Biofilms of > 106 cells cm-2 formed in the reactor and released approximately 106 cells ml-1 into milk passing over the biofilm. A doubling time of 25 min was calculated for this organism grown as a biofilm. CONCLUSION: The formation of biofilms of thermophilic Bacillus species within the plant appears to be a likely cause of contamination of manufactured dairy products. Methods to control the formation of biofilms in dairy manufacturing plants are required to reduce the contamination of dairy products with thermophilic bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilms of B. stearothermophilus growing in dairy manufacturing plants can explain the contamination of dairy products with these bacteria.     

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.     

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans, five were B. circulans and four were B. licheniformis. Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65 degrees C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70 degrees C for 30 min.     

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans , five were B. circulans and four were B. licheniformis . Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65°C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70°C for 30 min.     

An investigation of indirect conductimetry for detection of some food-borne bacteria

B olton , F.J. 1990. An investigation of indirect conductimetry for detection of some food-borne bacteria. Journal of Applied Bacteriology 69 , 655–661.
Indirect conductimetry using a rapid automated bacterial impedance technique was investigated. Strains of Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and Salmonella spp. grown in Whitley Impedance broth all elicited indirect conductimetric changes. These indirect conductance responses were improved by the addition of 2 g/1 glucose to the medium and resulted in maximum changes of 2340–4300 μS with associated maximum rates of change of 520–1210 μS/h. Furthermore, the indirect conductimetric assay detected growth of staphylococci, listeria and salmonella in media containing high concentrations of salts used as selective agents in culture media for the isolation of these organisms.     

Development of defined and minimal media for the growth of Bacillus stearothermophilus.

Defined media, both solid and liquid, that support good growth of Bacillus stearothermophilus 1503 have been developed. Data are presented which indicate that manganese is required at relatively high concentrations for growth in a defined liquid medium. Phosphate concentrations higher than 5 times 10(-3) M have been shown to inhibit colony formation on solid media. Maximum viable counts of approximately 10(9) colony-forming units per ml were obtained in both the defined and minimal liquid media. Glucose, fructose, sucrose, glycerol, and starch support the growth of this obligate thermophile in the defined media, whereas citrate, alpha-ketoglutarate, succinate, fumarate, malate, acetate, and lactate do not. The described media have been utilized to isolate several amino acid-requiring mutants of B. stearothermophilus.     

Enantiopure derivatives of 1,2-alkanediols: substrate requirements of lipase B from Candida antarctica

Efficient methods for kinetic resolution of 1-phenoxy-2-butanol, 1-phenylmethoxy-2-butanol, and 1-phenoxy-2-pentanol were developed using lipase B from Candida antarctica as catalyst. Resolutions were performed in order to investigate the substrate requirements needed to obtain a high E-value. The effect of the substrate structure on E is different for transesterifications in organic media as compared to hydrolysis. The influence of different acyl donors on the E-value was also investigated.     

High performance affinity chromatography of Bacillus neutral proteases.

Bacillus neutral proteases were purified using bacitracin-silica as an affinity medium. Several chromatographic procedures were investigated, including high speed runs on columns with 40- to 60-microns silica particles. The high speed procedure enabled the purification of 4.9 mg of B. subtilis neutral protease directly from 165-ml culture supernatant within 1.5 h. The neutral proteases of B. polymyxa and B. stearothermophilus were also purified. The latter enzyme was further concentrated by a second affinity chromatography step, using Sepharose with glycyl-D-phenylalanine as a ligand. During the purification procedures isopropanol was used to prevent autodigestion of the enzymes.     

Defined minimal media for the growth of prototrophic and auxotrophic strains of Bacillus stearothermophilus

Minimal chemically defined media for Bacillus stearothermophilus were developed at 60°C and quantitative requirements for each nutrient were determined. A prototrophic strain of B. stearothermophilus was grown in medium containing only glucose and mineral salts whereas auxotrophic strains in addition required biotin, thiamine, nicotinic acid and DL-methionine. Metabolic interaction between L-valine and L-leucine was observed with auxotrophic organisms. The presence of L-leucine in minimal medium necessitated the addition of L-valine. Growth took place in the absence of both amino acids.     

Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis.

A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance [Tcr]), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C. The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline. Bacillus subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.     

Sporulation of Clostridium perfringens (welchii) in Four Laboratory Media

S ummary . Sporulation of 7 strains of Clostridium perfringens ( welchii ) was investigated in 4 laboratory media. A method to induce rapid and simultaneous sporulation was attempted which involved obtaining a purely vegetative culture to inoculate the test media. Heat resistance of spores produced in the individual media by each of 4 selected strains was investigated. The clean spores for the heating tests were obtained by a special procedure which included chilling to 6° for a minimum of 1 week immediately following the usual incubation period, then centrifuging, resuspending to volume in 0.85% NaCl solution and pasteurizing at 75° for 20 min before subjecting to the heating tests. Morphology of each strain was studied using stained microscopic preparations from the 24 h sporulating cultures.
In the Ellner medium spore counts approaching 10⁷/ml were recorded and this medium appeared to be the most efficient when judged in terms of numbers of spores produced. In other media the counts were in the range 10⁴-10⁵ spores/ml. Cooked meat medium yielded slightly higher spore counts than did either SEC broth or modified Wagenaar & Dack medium, the latter contained in a dialysis sac apparatus. A period of chilling to 6° for a minimum of 1 week following incubation enhanced maturation in all cultures except those grown in SEC broth for 24 h or 15 days and those grown 15 days in the modified Wagenaar & Dack medium.
Considerable heat resistance, expressed as percentage spore survival, was recorded for spores of 4 strains when heated at 80°, and heat resistance generally increased with lengthening of incubation time for the culture. Survival of spores heated at 100° for 10 min was usually less than 0.01% but spores in SEC broth after 15 days showed a somewhat greater heat resistance than the others. In no instance did total destruction of spores occur at 100°.     

Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae

The dynamics of cyclic peptide destruxins (dtxs) produced by Metarhizium anisopliae strains V245 and V275 were monitored both on solid and in liquid media. The results showed that both strains did not produce dtxs in large-scale fermenter cultures or solid Czapek Dox (CD) agar. Production of the major dtxs A and B could be determined in both strains when grown on rice for up to 10-30 days. The main dtxs A, B, E, and E diol were detected in CD liquid culture filtrate from both strains after three days post-inoculation on. Parallel decrease of dtx E and increase of E diol in the culture medium were found, indicating that the latter is the hydrolytic product from the former. Production of dtxs A and B was significantly positively correlated. A negative correlation was observed between the production of the metabolites and pH value of the medium. The influence of different nutrient sources on dtx production was evaluated by using media with different carbon and nitrogen ratios as well as with different insect homogenates. The findings showed that the amount of dtxs A, B, and E increased with the increasing content of peptone in the medium. When insect homogenate was used as single nutrient source or added to CD medium, no toxins were detected in the culture filtrate. The potential risk posed by the toxic metabolites during mass production is discussed.     

Novel fermentation media for production of Bacillus thuringiensis subsp. israelensis

The production of Bacillus thuringiensis subsp. israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology. In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti. Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani). Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium. These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs.     

Study of stability of recombinant plasmids during the continuous culture of Bacillus stearothermophilus NUB3621 in nonselective medium

The optimal culture conditions for Bacillus stearothermophilus NUB3621 (BGSC 9A5) in chemostat were studied. The results obtained showed that the optimal culture conditions in terms of biomass concentration and maximum growth rate were 65 degrees C, pH 6.8 to 7.2. Dissolved oxygen became growth limiting at pO(2) levels below 10%. Furthermore, this strain was transformed with three new hybrid vectors (pPAM2, pPCH2, or pPLY2) constructed by cloning in pRP9, a plasmid based on the thermophilic replicon, pBC1, and three heterologous genes: the alpha-amylase gene from Bacillus licheniformis, the cholesterol oxidase gene from Streptomyces sp., and the lipase gene from Pseudomonas fluorescens. The influence of several fermentative conditions on segregational and structural stability of the recombinant B. stearothermophilus NUB3621 transformants was studied.The parameters of plasmid loss, that is, rate of plasmid loss (R) and specific growth rate difference (deltamu), were calculated. B. stearothermophilus NUB3621 carrying pRP9 showed great segregational stability in all the assayed conditions, exceeding more than 300 generations without significant plasmid loss, whereas NUB3621 carrying pPAM2, pPCH2, or pPLY2 exhibited relatively low plasmid stability. The segregational instability of the recombinant constructs increased by increasing the fermentation temperature, decreased by increasing the dilution rate, and was not affected by the level of dissolved oxygen. On the other hand, plasmid maintenance decreased in minimal medium if compared with the results obtained in complex medium. Restriction analyses carried out on cultures of NUB3621 carrying pRP9, pPAM2, pPCH2, or pPLY2, grown for 200 generations on nonselective media, revealed that all the clones tested contained the parental plasmids. These results indicate that the heterologous inserts did not affect the structural stability of the recombinant plasmids. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 507-514, 1997.     

Micropropagation protocol for Antigonon leptopus an important ornamental and medicinal plant

The effect of some factors on in vitro consecutive micropropagation behavior of Antigonon leptopus was examined including those of culture establishment, shootlets multiplication, rooting and acclimatization stages. The highest percent of aseptic cultures and survival of explants (100%) were obtained as a result of using Clorox 10% for 3?min followed by MC 0.1% for 2?min while, using each of them individually (Clorox 20% or MC 0.1%) for 5?min caused the highest percent of shoot formation. During the multiplication stage, the highest percent of shoot formation was reached to 100% with repeating culture of explants (two times) on MS medium supplemented with 2ip at 1.0 and IBA at 0.2?mg/l. The highest numbers of shootlets/explant were obtained when 2.0?mg/l of BAP or 0.5?mg/l BA?+?0.2?mg/l of IBA were added to MS culture medium. Culturing the explants on MS medium supplemented with 2ip at 0.5 or 1.0?mg/l each combined with 0.2?mg/l of IBA showed the longest shootlets. Reducing the strength of culture media to ½ or ¾ had promotion effect on rooting formation of shootlets. The best results of plant acclimatization (survival percent, plant height and root length) were obtained by using sand or peat moss soil. The amplified DNA fragments using B7, B9 and C19 primers for mother and micropropagated plants showed that the produced pattern by primer B7 had a maximum number of 10 bands of DNA fragments with molecular size ranging between 1025.57 and 176.36?bp, micropropagated plants showed 95.2% similarity in relation to mother plant.     

An investigation of indirect conductimetry for detection of some food-borne bacteria

Indirect conductimetry using a rapid automated bacterial impedance technique was investigated. Strains of Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and Salmonella spp. grown in Whitley Impedance broth all elicited indirect conductimetric changes. These indirect conductance responses were improved by the addition of 2 g/l glucose to the medium and resulted in maximum changes of 2340-4300 microS with associated maximum rates of change of 520-1210 microS/h. Furthermore, the indirect conductimetric assay detected growth of staphylococci, listeria and salmonella in media containing high concentrations of salts used as selective agents in culture media for the isolation of these organisms.     

Monitoring viral-induced cell death using electric cell-substrate impedance sensing

Using an electrical measurement known as electric cell-substrate impedance sensing (ECIS), we have recorded the dynamics of viral infections in cell culture. With this technique, cells are cultured on small gold electrodes where the measured impedance mirrors changes in attachment and morphology of cultured cells. As the cells attach and spread on the electrode, the measured impedance increases until the electrode is completely covered. Viral infection inducing cytopathic effect results in dramatic impedance changes, which are mainly due to cell death. In the current study, two different fish cell lines have been used: chinook salmonid embryonic (CHSE-214) cells infected with infectious pancreatic necrosis virus (IPNV) and epithelioma papulosum cyprini (EPC) carp cells infected with infectious hematopoeitic necrosis virus (IHNV). The impedance changes caused by cell response to virus are easily measured and converted to resistance and capacitance. An approximate linear correlation between log of viral titer and time of cell death was determined.     

Production of Bacillus thuringiensis Berliner var. kurstaki Grown in Alternative Media

Agro - industrial residues and by - products available in southeastern Brazil were used as ingredients for low - cost culture media for liquid fermentation of Bacillus thuringiensis var. kurstaki. Highest spore yield was obtained with a medium containing cheese whey , soya bean milk and molasses (WSM) . Crystals and spores were produced in all media and potency of the final product was highest for nutrient broth + yeast extract medium (NBY) . There was no correlation between the number of spores in the fermented media and the potency of the preparations . Considering all three factors , the potencies , costs and yields of the final products , lowest relative cost was obtained with BMM medium ( Bombyx mori pupae + molasses) . NBY and WSM had intermediate relative cost approximately nine times higher than BMM . The cost analysis suggests that BMM medium should be preferred for local production of B. thuringiensis var . kurstaki in comparison to other media tested . The results also demonstrate the importance of considering yields , cost and potency of the B. thuringiensis preparations in selecting the production medium .     

Development and viability of in vitro derived porcine blastocysts cultured in NCSU23 and G1.2/G2.2 sequential medium

Porcine embryo development in vitro is relatively inefficient compared to other domestic species. Currently, a single culture medium (NCSU23) is the standard for porcine in vitro systems. However, the G1.2/G2.2 sequential culture system has been beneficial for embryo development in other species. The objective of this study was to compare porcine preimplantation embryo development in vitro and subsequent blastocyst viability and metabolic activity using NCSU23 and G1.2/G2.2 culture media. Oocytes were matured in defined TCM199 base medium for 45 to 47 h and fertilized in mTBM for 4 h. Embryos were cultured in either NCSU23 for 146 h or G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 74 h. Blastocyst substrate use was measured using a modification of the hanging drop technique. Culture in NCSU23 resulted in a higher percentage (P < 0.05) of embryo cleavage (74.0%) and blastocyst development (14.6%) than culture in G1.2/G2.2 (67.8% and 7.8%, respectively). Both NCSU23 and G1.2/G2.2 produced blastocysts with similar mean cell numbers (51.5 +/- 4.3 and 47.1 +/- 4.3, respectively), similar glucose use (10.81 +/- 1.39 and 10.12 +/- 1.72 pmol/embryo/3 h, respectively) and pyruvate use (1.08 +/- 0.056 and 0.88 +/- 0.048 pmol/embryo/3 h, respectively). These data indicate that a sequential culture system can support porcine embryo development in vitro without compromising embryo viability. However, the G1.2/G2.2 system was not as effective as NCSU23 in supporting blastocyst development. Sequential media should be formulated specifically for porcine embryos to improve embryonic cleavage and blastocyst development.