Effect of environmental pH on the fermentation balance of Lactobacillus reuteri

The influence of environmental pH on the regulation of glucose catabolism by Lactobacillus reuteri was examined in anaerobic batch cultures. Under acidic conditions both glucose consumption and end-products formation were low. Maximum biomass was reached at pH 5·0, with a specific growth rate of µ= 0·78 h^-1. The shift in pH values from 4.3 to 6.5 reflected an increase in glucose uptake as well as in the yield ( Y _p/x) of acetate, lactate and ethanol after 12 h of incubation. Ethanol was the major metabolite produced at all pH values assayed.     

Growth of thermophilic obligately autotrophic hydrogen-oxidizing bacteria on thiosulfate

Thermophilic obligately autotrophic H₂-oxidizing bacteria from Icelandic hot springs were tested for growth on thiosulfate. Ten strains were tested and all grew on thiosulfate but not on sulfite or sulfur. The product of thiosulfate oxidation was sulfate. The growth rate on thiosulfate was slower (μ=0.12 h^-1) than on H₂ (μ=0.34 h^-1). Washed cells which had been grown on thiosulfate could oxidize thiosulfate rapidly but H₂-grown cells oxidized thiosulfate much more slowly and with about a 3 h lag time. The bacteria would not grow on agar medium under H₂ but grew on agar medium containing thiosulfate.     

Inhibition of Salmonella typhimurium by the products of tartrate metabolism by a Veillonella species

The inhibition of the growth of Salmonella typhimurium by a Veillonella species grown on media supplemented with tartrate was examined. Growth of Salmonella typhimurium was not inhibited by the concentrations of products metabolized by Veillonella cultures on media supplemented with 0 or 50 mmol 1^-1 of tartrate, but was inhibited on media supplemented with 100 or 150 mmol 1^-1 of tartrate. Inhibition of Salm. typhimurium was correlated with the increased production of acetate and propionate from tartrate by the Veillonella species.     

An extracellular β-galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall-polysaccharide hydrolysis

β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)^-1, h^-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co²⁴, Cu²⁺, Hg^2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.     

Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach

The genus Pectinatus has been often reported in beer spoilage with off-flavours. The bacteria are strictly anaerobic, Gram-negative rods. Propionate and acetate are the main fermentation products from glucose in the two species belonging to the genus, P. cerevisiiphilus and P. frisingensis. Amino acids routinely present at a high level in beer were not growth substrates for both species, and a significant accumulation of succinate was observed with lactate as growth substrate. Both Pectinatus ssp. showed almost identical fermentation balances on glucose. Growth kinetics of both glucose-grown species were unchanged under a N₂, H₂ or 20% CO₂-containing atmosphere. Combinations of culture medium pH values from pH 3·9 to pH 7·2, of glucose levels between 5 and 55 mmol l^-1, and of lactate concentrations varied from 4 to 40 mmol l^-1 demonstrated that biomass and volatile fatty acids production were proportional to glucose concentration for both Pectinatus species. A significant increase of volatile fatty acid production was measured for both species at the lowest pH values with a lactate or a glucose concentration increase. The maximum biomass production was observed at pH 6·2 for P. cerevisiiphilus , and between pH 4·5 and pH 4·9 for P. frisingensis. Glucose and lactate or pH value were dependent with regard to propionate and acetate production in P. frisingensis. On the other hand, the variations of these three parameters were independent with regard to biomass production for both strains, and to volatile fatty acids production for P. cerevisiiphilus. Addition of ethanol to glucose-grown cultures completely inhibited growth at 1·3 mol l^-1 ethanol for P. cerevisiiphilus , and at 1·8 mol l^-1 for P. frisingensis.     

Nickel and rubidium uptake by whole oat plants in solution culture

Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni²⁺ and Rb⁺ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni²⁺ and Rb⁺ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni²⁺ uptake by 93%. Nickel and Rb⁺ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. K_m (0.012 m M ) and V_max [2.72 μmol (g dry weight)^-1 h^-1] values for Ni²⁺ were nearly 7 times lower than those for Rb⁺ [0.09 m M and 19.2 μmol (g dry weight)^-1 h^-1]. In all experiments, Ni²⁺ and Rb⁺ showed qualitatively similar uptake patterns, but Rb⁺ uptake was quantitatively more sensitive than Ni²⁺ to experimental manipulations.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance

Abstract Lactic acid bacteria maintain a cytoplasm that is more alkaline than the medium, but whose pH decreases as the medium is acidified during growth and fermentation. Streptococci generally acidify the cytoplasm from approximately pH 7.6 to 5.7 (external pH 4.5) before growth and then fermentation cease. The internal enzyme machinery of these anaerobic fermenters thus tolerates a fairly wide range in internal proton concentration. Lactobacilli tolerate a significantly more acidic cytoplasmic pH of 4.4 (external pH 3.5). However, when the cytoplasmic pH decreases below a threshold pH, which depends on the organism cellular functions are inhibited. Fermentation end-products, such as organic acids or alcohols, exert their deleterious effects by bringing about acidification of the cytoplasm below the permissible pH. Organic acids, which act as protonophores, or solvents, which perturb membrane phospholipids, at high concentrations increase the inward leak of H⁺ so that H⁺ efflux is not rapid enough to alkalinize the cytoplasm. The membrane pH gradient is thus dissipated.
A specific strain of Lactobacillus acidophilus has been found to be unusually osmotolerant. The osmoresistance is due to the cells' capacity to accumulate glycine betaine by a transport carrier that is activated, but not induced, by high medium osmotic pressure.     

ETHYL ALCOHOL REDUCTION OF SALMONELLA TYPHIMURIUM IN POULTRY FEED

This study was designed to evaluate the effect of ethyl alcohol as a potential treatment for reduction of Salmonella populations in poultry feed. Growth rate of S. typhimurium in tryptic soy broth was significantly reduced by addition of greater than 0.3% volume/volume of ethyl alcohol and growth was completely inhibited by addition of 5% ethyl alcohol. Ethyl alcohol concentrations of 20% volume/weight and greater significantly reduced initial S.typhimurium populations in poultry feed (for 20% treated, 2.31 ± 0.31 vs 3.39 ± 0.29 for untreated; P < 0.05). When feed treatment was administered either before or after inoculation of S. typhimurium with 60% ethyl alcohol or 0.04% buffered propionic acid, populations in feeds treated after inoculation were decreased to a nondetection level (< 1.0 log₁₀ CFU/g) by ethyl alcohol treatment but not by other treatments. Ethyl alcohol treatment may have the potential for reducing Salmonella spp. contamination in poultry feed.     

Effects of glyphosate on nitrogen fixation of free-living heterotrophic bacteria

The effect of the herbicide glyphosate ( N -(phosphonomethyl)glycine) on the growth, respiration and nitrogen fixation of Azotobacter chroococcum and A. vinelandii was studied. Azotobacter vinelandii was more sensitive to glyphosate toxicity than A. chroococcum. Recommended dosages of glyphosate did not affect growth rates. More than 4 kg ha^-1 is needed to find some inhibitory effect. Specific respiration rates were 19.17 mmol O₂ h^-1 g^-1 dry weight for A. chroococcum and 12.09 mmol h^-1 g^-1 for A. vinelandii. When 20 kg ha^-1 was used with A. vinelandii , respiration rates were inhibited 60%, the similar percentage inhibition A. chroococcum showed at 28 kg ha^-1. Nitrogen fixation dropped drastically 80% with 20 kg ha^-1 in A. vinelandii and 98% with 28 kg ha^-1 in A. chroococcum. Cell size as determined by electron microscopy decreased in the presence of glyphosate, probably because glyphosate induces amino acid depletion and reduces or stops protein synthesis.     

Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Isolation and characterization of refuse methanogens

Four mesophilic, irregular, rod-shaped methanogenic bacteria were isolated from decomposing refuse recovered from laboratory-scale reactors and a municipal solid waste landfill. H₂/CO₂ was the only substrate on which the isolates could grow in a complex medium. Isolates grew between either 25° or 30° and 45°C and between pH 6 and 8. One isolate exhibited growth at pH 5. Growth of each isolate was enhanced by yeast extract and inhibited by anaerobic sewage sludge supernatant fluid. No isolate showed greater than 25% lysis on exposure to 1% sodium dodecyl sulphate (SDS) for 24 h, as is typical of methanogens with a proteinaceous cell wall. The physiological traits of the methanogens isolated here are similar to many previously characterized isolates.     

Dose responses of tsetse flies (Glossina) to carbon dioxide, acetone and octenol in the field

ABSTRACT Studies were conducted in Zimbabwe of the catch of Glossina pallidipes Austen from an electric net plus target baited with mixtures of acetone plus carbon dioxide or 1-octen-3-ol (octenol) plus carbon dioxide. For acetone dispensed alone at 5–50, 000 mg h^-1, ten-fold increments in the dose increased the catch 1.7 times. For carbon dioxide dispensed alone, dose increments from 12 to 1201 h^-1 doubled the catch, but the catch was not further increased by dispensing carbon dioxide at 600–1200 1 h^-1. For mixtures of these two odours, ten-fold increments in the dose of carbon dioxide between 12 and 12, 0001 h^-1 increased the catch c . 2.5 times if acetone was also dispensed at >50 mg h^-1; changes in the dose of acetone between 50 and 50 000 mg h^-1 did not affect the catch. The addition of octenol (0.05 mg h^-1) to carbon dioxide (12–12001 h^-1) doubled the catch. Ten-fold increments in the dose of octenol between 0.05 and 5 mg h^-1 did not increase the catch significantly and the catch was independent of changes in the dose of carbon dioxide between 120 and 12001 h^-1. The behavioural basis of the dose-response curves was investigated using an incomplete ring of electric nets to assess the flight orientation of tsetse in different odours. Upwind flight was not elicited by acetone or octenol alone, or by carbon dioxide unless it was at very high doses, however, mixtures of carbon dioxide with acetone or octenol elicited upwind flight. It is suggested that the attractiveness of mixtures of acetone and carbon dioxide is a function of the region of overlap of these two odours at above threshold concentration. Acetone and octenol on their own appear to increase the responsiveness of flies to visual cues.     

COMBINATION OF DITHIOTHREITOL AND SULFIDE AS REDUCTANTS IN NITROGEN-FREE MINIMAL MEDIA FOR GROWTH OF ANAEROBIC RUMINAL BACTERIA AND PREVOTELLA RUMINICOLA STRAIN B14 ON AMMONIA

Cysteine is commonly employed as the medium reductant for ruminal bacteria, but many ruminal bacteria can use cysteine as a source of nitrogen as well as sulfur. The objective of the present study was to test a combination of dithiothreitol and sulfide as possible reductant substitutes for cysteine in anaerobic media containing ammonia as the nitrogen source. The type of reductant (cysteine versus dithiothreitol-sulfide) and ammonia concentration did not alter growth rates of Prevotella ruminicola strain B,4 (P>0.15). However, growth rates in dithiothreitol-sulfide reduced media varied tremendously between individual organisms ranging from 0.10 h⁻¹ for Ruminococcus flavefaciens to 1.6 h⁻¹ for Streptococcus bovis grown in 1 mM NH₃-N. At both 1 and 11 mM NH₄Cl, Str. bovis strain JB1 exhibited the greatest growth rate followed by Str. bovis strain C277. Megasphaera elsdenii strain T81 and Ruminococcus flavefaciens strain FD1 had the lowest growth rates at both NH₄Cl concentrations. Increasing NH₄Cl concentration from 1 to 11 mM resulted in increased growth rates for Ruminobacter amylophilus strains H18 and 70 and Str. bovis strain C277 (P<0.05), and decreased growth rates for S. ruminantium subsp. lactilytica strain HD₄ and Str. bovis strain JB1 (P<0.01). These results indicate that dithiothreitol and sulfide can be combined as reductants in nitrogen-free basal media for most ruminal bacterial species.     

Osmoadaptation of Thiocapsa roseopersicina OP-1 in batch and continuous culture: Accumulation of K+ and sucrose in response to osmotic stress

Abstract Increasing growth medium NaCl concentration inhibited the growth of Thiocapsa roseopersicina OP-1 due to both an increase in the lag phase of the growth cycle and a reduction in specific growth rate. Addition of 0.05% w/v acetate to the growth medium stimulated growth at all NaCl concentrations, but this stimulation was greatest at supra-optimal NaCl concentrations. Optimal growth under all conditions tested in both batch and continuous culture was recorded at a salt concentration of 0.3 M NaCl. The intracellular concentrations of both K⁺ and sucrose increased linearly with increasing growth medium NaCl concentration indicating as osmoregulatory role for these solutes. Time courses of osmoadaptation in batch culture demonstrated a biphasic response to osmotic stress. The initial phase consisted of a rapid accumulation (within 30 min) of K⁺ from the growth medium. This was followed by a slower synthesis of sucrose which partially replaced intracellular K⁺ during the second phase of osmoadaptation.     

Growth and Grazing Rates of the Marine Planktonic Ciliate Strombidinopsis sp. on Red-Tide and Toxic Dinoflagellates

ABSTRACT We investigated growth and grazing rates of Strombidinopsis sp. when feeding on several species of red-tide and/or toxic dinoflagellates. Strombidinopsis sp. one of the largest aloricate choreotrichs so far reported, grew well on Lingulodinium polyedrum, Gymnodinium sanguineum, Scrippsiella trochoidea, Cochlodinium polykrikoides , and Prorocentrum minimum , but failed to grow on Amphidinium carterae. Specific growth rates of Strombidinopsis sp. increased rapidly with increasing prey density up to ca. 100 ng C ml^-1, but were saturated or increased slightly at higher concentrations. Maximum specific growth rates of Strombidinopsis sp. on various prey species were 1.38 day^-1 for C. polykrikoides , 1.27 for G. sanguineum , 1.06 for P. minimum , 0.83 for L. polyedrum , and 0.67 for S. trochoidea. Threshold prey concentrations (where net growth = 0) were 12–38 ng C ml^-1. Maximum ingestion and clearance rates of Strombidinopsis sp. were 353 ng C grazer^-1 day^-1 and 110 μ, l grazer^-1 h^-1, respectively. Strombidinopsis sp. exhibited higher maximum growth, ingestion, and clearance rates than the mixotrophic dinoflagellate Fragilidium cf. mexicanum or the heterotrophic dinoflagellates Protoperidinium cf. divergens and P. crassipes , when grown on the same prey species. In addition, the sequence of prey species arranged according to growth response of Strombidinopsis sp. differed considerably from those of Fragilidium cf. mexicanum, Protoperidinium cf. divergens , and P. crassipes.     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

Coupling of fermentation and microfiltration for α-amylase production from Bacillus amyloliquefaciens

Abstract: A strain of Bacillus amyloliquefaciens secreting α-amylase was cultivated continuously in a fermentor coupled to a filtration unit. Under the tested operating conditions, the maximum flux was 91 m^-2 h^-1 during the first day and 61 m^-2 h^-1 during the 2nd day. The α-amylase retention was around 30%. Compared to a batch process, continuous cultivation with cell recycling led to lower α-amylase concentrations but to a doubling of volumetric productivities.     

Effects of Extracellular Potassium on Acid Release and Motility Initiation in Toxoplasma gondii

The internal pH (pH_i) of Toxoplasma gondii was estimated by measuring the accumulation of the weak base 9-aminoacridine in buffers with various ionic compositions. The pH_i of the metabolizing parasite increased when the extracellular K⁺ was elevated in alkaline medium or when the external pH (pH_c) was substantially increased in medium employing high external K⁺ (90 mM). The parasite in mouse peritoneal fluid, or in potassium sulfate buffer (pH 8.2), where the pH_i was demonstrated to be increased to 7.9, became motile when acidic buffer was substituted for the original suspension medium. This acid-induced independent movement subsided within 5 min but was repeatedly induced if the pH_c was serially lowered to 6.0. Basic buffers, on the other hand, abolished motility when applied to the moving parasites. Nigericin, which is known to collapse pH gradients across the membrane, also abolished motility.     

Survival of a Salmonella typhimurium poultry isolate in the presence of propionic acid under aerobic and anaerobic conditions

Propionic acid is commonly found as a fermentation product in the gastrointestinal tracts of food animals and has also been used to limit the microbial contaminants in animal feeds. Because propionic acid is known to have antibacterial activity, the propionic acid encountered by foodborne pathogens during their life cycles may play an important role in inhibiting the survival of the pathogens. The survival patterns of Salmonella typhimurium poultry isolate were determined both in aerobic and anaerobic tryptic soy broth (TSB; pH 5.0 or 7.0) containing various concentrations of propionic acid (0-200 mM). The levels of recovered cells were consistently greater at pH 7.0 compared to those at pH 5.0. For the first 4 days, the levels were significantly decreased by incubation under anaerobic conditions as compared to aerobic condition at pH 7.0 (P<0.05). However, there were fluctuations of cell populations with different patterns depending on both concentrations and growth conditions. To characterize the nature of the capability which allowed the cell multiplication following decreases in cell population during incubation at pH 7.0, the cells isolated from the outgrowth cultures were tested for survival in aerobic or anaerobic TSB (pH 5.0 or pH 7.0) containing propionic acid (50 mM). The outgrowth isolates did not show significant differences in the level of recovered cells in the presence of propionic acid when compared to the wild type strain (P>0.05), suggesting that the cells in the outgrowth cultures did not harbour mutation(s) conferring increased resistance to propionic acid. In addition, the level of recovered cells of isogenic rpoS mutant strain of S. typhimurium was not significantly different from that of the wild type strain in the same assay conditions (P<0.05). The results of this study show that the bactericidal activity of propionic acid on S. typhimurium can be affected by environmental conditions such as acidic pH levels and anaerobiosis in food materials and gastrointestinal tracts. However, S. typhimurium is also able to multiply in the presence of sublethal concentrations of propionic acid at neutral pH during prolonged incubation under both aerobic and anaerobic conditions.