Inhibition of yeast by lactic acid bacteria in continuous culture: nutrient depletion and/or acid toxicity?

Lactic acid was added to batch very high gravity (VHG) fermentations and to continuous VHG fermentations equilibrated to steady state with Saccharomyces cerevisiae. A 53% reduction in colony-forming units (CFU) ml^–1 of S. cerevisiae was observed in continuous fermentation at an undissociated lactic acid concentration of 3.44% w/v; and greater than 99.9% reduction was evident at 5.35% w/v lactic acid. The differences in yeast cell number in these fermentations were not due to pH, since batch fermentations over a pH range of 2.5–5.0 did not lead to changes in growth rate. Similar fermentations performed in batch showed that growth inhibition with added lactic acid was nearly identical. This indicates that the apparent high resistance of S. cerevisiae to lactic acid in continuous VHG fermentations is not a function of culture mode. Although the total amount of ethanol decreased from 48.7 g l^–1 to 14.5 g l^–1 when 4.74% w/v undissociated lactic acid was added, the specific ethanol productivity increased ca. 3.2-fold (from 7.42×10^–7 g to 24.0×10^–7 g ethanol CFU^–1 h^–1), which indicated that lactic acid stress improved the ethanol production of each surviving cell. In multistage continuous fermentations, lactic acid was not responsible for the 83% (CFU ml^–1) reduction in viable S. cerevisiae yeasts when Lactobacillus paracasei was introduced to the system at a controlled pH of 6.0. The competition for trace nutrients in those fermentations and not lactic acid produced by L. paracasei likely caused the yeast inhibition.     

Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash

Two separate 4 (bacterial concentrations)×6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1×10⁶, 1×10⁷ and 1×10⁸ cells/ml mash. Yeast concentrations were 0, 1×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, and 4×10⁷ cells/ml mash. An increased yeast inoculation rate of 3×10⁷ cells/ml resulted in a greater than 80% decrease (P<0.001) and a greater than 55% decrease (P<0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1×10⁸ lactobacilli/ml. No differences (P>0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1×10⁷ or 1×10⁸ lactobacilli/ml, a reduction of 0.7–0.9% v/v (P<0.005) and a reduction of 0.4–0.6% v/v (P<0.005) in the final ethanol produced was observed in mashes inoculated with 1×10⁶ and 1×10⁷ yeast cells/ml, respectively. At higher yeast inoculation rates of 3×10⁷ or 4×10⁷ cells/ml, no differences (P>0.35) were observed in the final ethanol produced even when the mash was infected with 1×10⁸ lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3×10⁷ yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast.     

Urea Hydrogen Peroxide Reduces the Numbers of Lactobacilli, Nourishes Yeast, and Leaves No Residues in the Ethanol Fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from ~10⁷ to ~10² CFU/ml in a 2-h preincubation at 30°C of normal-gravity wheat mash at ~21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at ~10⁷ CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at ~10⁷ CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30°C whether the bactericidal agent was added as H₂O₂ or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H₂O₂) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H₂O₂. H₂O₂-resistant mutants were not expected or found when lethal levels of H₂O₂ or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.     

Fermentation of a milk-soymilk and Lycium chinense Miller mixture using a new isolate of Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum

A milk–soymilk mixture was fermented using Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum BCRC11847 at different inoculum ratios (1:1, 1:2, 1:5, 2:1, and 5:1). When the inoculum ratio was 1:2, the cell numbers of both strains were balanced after 12 h of cultivation. The pH and titratable acidity were very similar at the various inoculum ratios of cultivation. The milk–soymilk mixture was supplemented with 5, 10, 15, and 20% Lycium chinense Miller juice and fermented with Lactobacillus paracasei subsp. paracasei NTU101 and B. longum BCRC11847. Sensory evaluation results showed that supplementation with 5% Lycium chinense Miller juice improved the acceptability of the fermented milk–soymilk. The fermented beverage was stored at 4°C for 14 days; variations in pH and titratable acidity were slight. The cell numbers of L. paracasei subsp. paracasei NTU101 and B. longum BCRC11847 in the fermented beverage were maintained at 1.2×10⁹ CFU/ml and 6.3×10⁸ CFU/ml, respectively, after 14 days of storage.     

Cultivation ofLactobacillus crispatus KLB46 isolated from human vagina

Bacterial vaginosis can be treated by restoring the normal vaginal flora using lactobacilli.Lactobacillus crispatus KLB46 that was isolated from the human vagina has a strong antimicrobial activity and was grown in a batch and in a continuous fermentor. During batch cultivation, the maximum specific growth rate ofL. crispatus KLB 46 was 0.63 h⁻¹ and the highest viable cell count (1.9×10⁹ CFU/mL) was obtained at pH 5.5.L. crispatus KLB 46 did not grow well at either pH 3.5 or 7.5. During continuous cultivation, the highest viable cell count (1.53×10⁹ CFU/mL) was obtained at a dilution rate of 0.32 h⁻¹. However, the maximum productivity of viable cells was obtained at a dilution rate of 0.52 h⁻¹, and was 7.33×10¹¹ CFU L⁻¹ h⁻¹, that is approximately 5 times higher than that obtained from batch culture.     

A Novel GFP-Fused Eukaryotic Membrane Protein Expression System in Lactococcus lactis and Its Application to Overexpression of an Elongase

Ultraviolet (UV) irradiation has high potential to inactivate a wide range of biologic agents and is one of several nonadditive technologies being studied. The photoinactivation property of pulsed UV laser radiation (at wavelengths of 355 and 266 nm), used as an effective physical means to inactivate two typical microorganisms, prokaryotic (Escherichia coli K12) and eukaryotic (Saccharomyces cerevisiae), with respect to dose and exposure times, was examined. An E. coli population of 1.6 × 10⁴ colony-forming units (CFU)/ml was inactivated with a dose of 16.7 J/cm² energy at 355-nm wavelength. However, E. coli cells at higher concentrations were inactivated by only 98% using the same dose. Interestingly, an E. coli population of 2 × 10⁷ CFU/ml was completely inactivated using only 0.42 J/cm² at 266-nm wavelength (P ≤ 0.05). With respect to S. cerevisiae, the results were similar to those of E. coli irradiation considering that S. cerevisiae is 100 times larger than E. coli. A dose of 16.7 J/cm² completely inactivated an S. cerevisiae population of 6 × 10³ CFU/ml at 355-nm wavelength. Exposure to 266-nm wavelength, with energy doses of 1.67, 0.835, and 0.167 J/cm², successfully inactivated S. cerevisiae populations of 3 × 10⁶, 1.4 × 10⁵, and 1.5 × 10⁴ CFU/ml, respectively (P ≤ 0.05). In conclusion, compared with 355-nm wavelength, a pulsed UV laser at 266-nm wavelength inactivated a high titer of bacterial and yeast indicator standards suspended in phosphate-buffered saline-A.     

Seasonal abundance and distribution of <Emphasis Type="Italic">Vibrio</Emphasis> species in the treated effluent of wastewater treatment facilities in suburban and urban communities of Eastern Cape Province,South Africa

We assessed the seasonal abundance and distribution of Vibrio species as well as some selected environmental parameters in the treated effluents of two wastewater treatment plants (WWTP), one each located in a suburban and urban community of Eastern Cape Province, South Africa. Vibrio population density ranged from 2.1×10⁵ to 4.36×10⁴ CFU/ml in the suburban community and from 2.80×10⁵ to 1.80×10⁵ CFU/ml in the urban community. Vibrio species associated with 180 μ, 60 μ, and 20 μ plankton sizes were observed at densities of 0–136×10³ CFU/ml, 0–8.40×10² CFU/ml, and 0–6.80×10² CFU/ml, respectively at the suburban community’s WWTP. In the urban community, observed densities of culturable Vibrio were 0–2.80×10² CFU/ml (180 μ), 0–6.60×10² CFU/ml (60 μm), and 0–1.80× 10³ CFU/ml (20 μm). The abundance of free-living Vibrio species ranged from 0 to 1.0×10² and 1.0×10³ CFU/ml in the suburban and urban communities’ WWTPs, respectively. Molecular confirmation of the presumptive Vibrio isolates revealed the presence of V. fluvialis (41.38%), V. vulnificus (34.48%), and V. parahaemolyticus (24.14%) in the suburban community effluents. In the urban community molecular confirmation revealed that the same species were present at slightly different percentages, V. fluvialis (40%), V. vulnificus (36%), and V. parahaemolyticus (24%). There was no significant correlation between Vibrio abundance and season, either as free-living or plankton-associated entities, but Vibrio species abundance was positively correlated with temperature (r=0.565; p<0.01), salinity, and dissolved oxygen (p<0.05). Turbidity and pH showed significant seasonal variation (p<0.05) across the seasons in both locations. This study underscores the potential of WWTPs to be sources of Vibrio pathogens in the watershed of suburban and urban communities in South Africa.     

Improvement of the multiple-stress tolerance of an ethanologenic <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain by freeze-thaw treatment

An effective, simple, and convenient method to improve yeast’s multiple-stress tolerance, and ethanol production was developed. After an ethanologenic Saccharomyces cerevisiae strain SC521 was treated by nine cycles of freeze-thaw, a mutant FT9-11 strain with higher multiple-stress tolerance was isolated, whose viabilities under acetic acid, ethanol, freeze-thaw, H₂O₂, and heat-shock stresses were, respectively, 23-, 26-, 10- and 7-fold more than the parent strain at an initial value 2 × 10⁷ c.f.u. per ml. Ethanol production of FT9-11 was similar (91.5 g ethanol l⁻¹) to SC521 at 30°C with 200 g glucose l⁻¹, and was better than the parent strain at 37°C (72.5 g ethanol l⁻¹), with 300 (111 g ethanol l⁻¹) or with 400 (85 g ethanol l⁻¹) g glucose l⁻¹.     

Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology

A fermentation system to test the merging of very-high-gravity (VHG) and multistage continuous culture fermentation (MCCF) technologies was constructed and evaluated for fuel ethanol production. Simulated mashes ranging from 15% to 32% w/v glucose were fermented by Saccharomyces cerevisiae and the dilution rates were adjusted for each glucose concentration to provide an effluent containing less than 0.3% w/v glucose (greater than 99% consumption of glucose). The MCCF can be operated with glucose concentrations up to 32% w/v, which indicates that the system can successfully operate under VHG conditions. With 32% w/v glucose in the medium reservoir, a maximum of 16.73% v/v ethanol was produced in the MCCF. The introduction of VHG fermentation into continuous culture technology allows an improvement in ethanol productivity while producing ethanol continuously. In comparing the viability of yeast by methylene blue and plate count procedures, the results in this work indicate that the methylene blue procedure may overestimate the proportion of dead cells in the population. Ethanol productivity (Yps) increased from the first to the last fermentor in the sequence at all glucose concentrations used. This indicated that ethanol is more effectively produced in later fermentors in the MCCF, and that the notion of a constant Yps is not a valid assumption for use in mathematical modeling of MCCFs. Journal of Industrial Microbiology & Biotechnology (2001) 27, 87–93. Received 20 January 2001/ Accepted in revised form 28 April 2001     

<Emphasis Type="Italic">In vitro</Emphasis> assessment of immunomodulating activity of the two <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from traditional fermented milk

The objective of this study was to evaluate the in vitro immunomodulating capacity of Lactobacillus coryniformis subsp torquens T3L (L. coryniformis T3L) isolated from traditional fermented yak’s milk in Tibet, China, and Lactobacillus paracasei supsp. paracasei M5L (L. paracasei M5L)isolated from kumiss in Sinkiang, China was used as control. The effects of live bacteria, cell wall and genomic DNA of the two Lactobacillus strains on human peripheral blood mononuclear cells (PBMCs) proliferation, production of interleukin-12 (IL-12 p70), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and natural killer (NK) cell activity were assessed. The live bacteria, cell wall and genomic DNA of the two lactobacilli exerted proliferative effects on PBMCs. Live bacteria at 1 × 10⁶ c.f.u. ml⁻¹, cell wall at 20 μg protein ml⁻¹ and DNA at 50 μg DNA ml⁻¹ of the strainS induced the secretion of IL-12 (p70), IFN-γ and TNF-α by PBMCs. NK cell activities increased after cultivation of PBMCs with live bacteria, cell wall and DNA of the strains. Overall, these results demonstrate that the live bacteria, cell wall and genomic DNA of the two Lactobacillus strains exhibit immunomodulating activity.     

Spore-forming bacteria in soil cultivated with GM white poplars: Isolation and characterization

The impact of transgenic white poplars (Populus alba L. cv. ‘Villafranca’) was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 10⁶−2.5 × 10⁸ CFU/g dry soil and of 10⁴−5 × 10⁸ CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 10³−5 × 10⁴ CFU/g dry soil and 10²−2 × 10⁵ CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide-resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil level.     

Development of emulsion from rhizobial fermented starch industry wastewater for application as Medicago sativa seed coat

Starch industry wastewater was efficiently employed for the production of Sinorhizobium meliloti and the concentrated culture was used for the development of a biofertilizer formulation. Tween‐80 (0.02 g/L) acted as the best emulsifier for a Sinorhizobium–canola oil emulsion. The stability of the emulsion and survival of the organism was enhanced by supplementation of xanthan gum at pH 8. The refrigerated condition was most favorable for stability and survival of the microorganism. The survival of microorganism at 4±1°C was 2.78×10¹⁰ and 2.01×10¹⁰ CFU (colony forming unit)/mL on storage for 1 and 2 months, respectively. The values were higher than the prescribed cell count (×10³ CFU/mL) for field application. At 40°C, the survival of bacteria reduced from 3×10¹⁰ CFU/mL to 8.1×10⁹ and 8.8×10⁶ CFU/mL in 1 and 2 months, respectively. Emulsion‐coated seed was incubated at different temperatures and a cell count of 10⁵ CFU/seed was observed after 2 months of storage at 4°C, which was equal to the highest level of the described requirement (10³–10⁵ CFU/seed). Emulsion supplemented with xanthan gum improved the shelf‐life under optimized conditions (Sinorhizobium concentrate – canola oil (1:1) emulsion with 0.02 g/L Tween‐80; storage at pH 8 and temperature 4±1°C) and this emulsion with the required cell count and prolonged viability was used for the pre‐inoculation of seed or for in situ soil application.     

Ethanol production in a hollow fiber bioreactor using Saccharomyces cerevisiae

Summary A new approach for continuous production of ethanol was developed using a Hollow fiber fermentor (HFF). Saccharomyces cerevisiae cells were packed into the shell-side of a hollow fiber module. Using 100 g/l glucose in the feed gave an optimum ethanol productivity, based on total HFF volume, of 40 g ethanol/l/h at a dilution rate of 3.0 h^-1. Under these conditions, glucose utilization was 30%. However, at 85% glucose utilization the productivity was 10 g ethanol/l/h. This compares to batch fermentor productivity of 2.1 g ethanol/l/h at 100% glucose utilization.     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

Experimental Pathogenicity of a Clinical Isolate of <Emphasis Type="Italic">Trichosporon dermatis</Emphasis> in a Murine Model

The pathogenicity of Trichosporon dermatis isolated from skin lesions of a patient has been examined in mice. Balb/c mice were treated with two intraperitoneal injections of 100 mg/kg cyclophosphamide on days 4 and 1 and one subcutaneous injection of 10 mg/kg dexamethasone on day 1 pre-inoculation, and then challenged with 0.2 ml T. dermatis inoculum (1 × 10⁸ CFU/ml) by topical application on an abrasive wound in the dermabrasive group and by hypodermic injection in the subcutaneous group. In the intravenous group, 0.2 ml of high (1 × 10⁸ CFU/ml) or low (1 × 10⁷ CFU/ml) inoculum was injected into the tail vein. Histopathology and inverse fungal culture were performed on the skin lesion and viscera, and renal fungal burden was also determined. Inoculated sites developed localized infections after dermabrasive and subcutaneous challenge in all mice, but the maximum area of skin lesions, and number of positive cultures from the lesions, were higher for immunocompromised mice. In the intravenous group, all immunocompetent animals survived during the four-week period, whereas 100 and 70% of immunocompromised animals died by 3 and 5 days in the high and low-inoculum groups, respectively. The incidence of disseminated infection and the renal fungal burden of immunocompromised mice were higher than those of immunocompetent mice. Our results demonstrate that subcutaneous and intravenous injection of T. dermatis can successfully establish cutaneous and systemic infection models in immunocompromised mice, with the kidney and lung being most susceptible.     

Identification of a replicon from pCC3, a cryptic plasmid from <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> C4 derived from kimchi,and development of a new host–vector system

Analysis of the structural properties of pCC3, a cryptic plasmid from Leuconostoc citreum C4 isolated from kimchi, determined its length as 3,338 bp and revealed three open reading frames (ORFs): ORF1–ORF3. ORF3 showed high homology with a replication initiation protein of the theta-type plasmid pTXL1. The fragment encompassing ORF3 and its upstream sequences (nt 1,299–1,634) was found to contain a functional plasmid replicon. A new shuttle vector, pUCC3E1, was constructed based on pCC3. Using Southern hybridization analysis, no single-stranded DNA intermediate was detected from Leu. citreum harboring pUCC3E1, which indicates that pCC3 replicated via the theta mechanism. The pUCC3E1 could be replicated in E. coli TG1 (5.8 × 10⁴ CFU/μg DNA) and the developed cloning hosts, Leu. citreum C16 (2.1 × 10² CFU/μg DNA) and Leu. citreum GJ7 (8.0 × 10¹ CFU/μg DNA). pUCC3E1 was stably maintained in Leu. citreum C16 (for 100 generations, ca. 94.2%) in the absence of erythromycin (5 μg/ml).     

Application of oscillation for efficiency improvement of continuous ethanol fermentation with Saccharomyces cerevisiae under very-high-gravity conditions

Compared with steady state, oscillation in continuous very-high-gravity ethanol fermentation with Saccharomyces cerevisiae improved process productivity, which was thus introduced for the fermentation system composed of a tank fermentor followed by four-stage packed tubular bioreactors. When the very-high-gravity medium containing 280 g l⁻¹ glucose was fed at the dilution rate of 0.04 h⁻¹, the average ethanol of 15.8% (v/v) and residual glucose of 1.5 g l⁻¹ were achieved under the oscillatory state, with an average ethanol productivity of 2.14 g h⁻¹ l⁻¹. By contrast, only 14.8% (v/v) ethanol was achieved under the steady state at the same dilution rate, and the residual glucose was as high as 17.1 g l⁻¹, with an ethanol productivity of 2.00 g h⁻¹ l⁻¹, indicating a 7% improvement under the oscillatory state. When the fermentation system was operated under the steady state at the dilution rate of 0.027 h⁻¹ to extend the average fermentation time to 88 h from 59 h, the ethanol concentration increased slightly to 15.4% (v/v) and residual glucose decreased to 7.3 g l⁻¹, correspondingly, but the ethanol productivity was decreased drastically to 1.43 g h⁻¹ l⁻¹, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h⁻¹.     

Study of Adhesion and Survival of Lactobacilli and Bifidobacteria on Table Olives with the Aim of Formulating a New Probiotic Food

With the aim of developing new functional foods, a traditional product, the table olive, was used as a vehicle for incorporating probiotic bacterial species. Survival on table olives of Lactobacillus rhamnosus (three strains), Lactobacillus paracasei (two strains), Bifidobacterium bifidum (one strain), and Bifidobacterium longum (one strain) at room temperature was investigated. The results obtained using a selected olive sample demonstrated that bifidobacteria and one strain of L. rhamnosus (Lactobacillus GG) showed a good survival rate, with a recovery of about 10⁶ CFU g⁻¹ after 30 days. The Lactobacillus GG population remained unvaried until the end of the experiment, while a slight decline (to about 10⁵ CFU g⁻¹) was observed for bifidobacteria. High viability, with more than 10⁷ CFU g⁻¹, was observed throughout the 3-month experiment for L. paracasei IMPC2.1. This strain, selected for its potential probiotic characteristics and for its lengthy survival on olives, was used to validate table olives as a carrier for transporting bacterial cells into the human gastrointestinal tract. L. paracasei IMPC2.1 was recovered from fecal samples in four out of five volunteers fed 10 to 15 olives per day carrying about 10⁹ to 10¹⁰ viable cells for 10 days.     

Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

Modeling bacterial contamination of fuel ethanol fermentation

The emergence of antibiotic‐resistant bacteria may limit the effectiveness of antibiotics to treat bacterial contamination in fuel ethanol plants, and therefore, new antibacterial intervention methods and tools to test their application are needed. Using shake‐flask cultures of Saccharomyces cerevisiae grown on saccharified corn mash and strains of lactic acid bacteria isolated from a dry‐grind ethanol facility, a simple model to simulate bacterial contamination and infection was developed. Challenging the model with 10⁸ CFU/mL Lactobacillus fermentum decreased ethanol yield by 27% and increased residual glucose from 6.2 to 45.5 g/L. The magnitude of the effect was proportional to the initial bacterial load, with 10⁵ CFU/mL L. fermentum still producing an 8% decrease in ethanol and a 3.2‐fold increase in residual glucose. Infection was also dependent on the bacterial species used to challenge the fermentation, as neither L. delbrueckii ATCC 4797 nor L. amylovorus 0315‐7B produced a significant decrease in ethanol when inoculated at a density of 10⁸ CFU/mL. In the shake‐flask model, treatment with 2 µg/mL virginiamycin mitigated the infection when challenged with a susceptible strain of L. fermentum (MIC for virginiamycin ≤2 ppm), but treatment was ineffective at treating infection by a resistant strain of L. fermentum (MIC = 16 ppm). The model may find application in developing new antibacterial agents and management practices for use in controlling contamination in the fuel ethanol industry. Biotechnol. Bioeng. 2009;103: 117–122. Published 2008 Wiley Periodicals, Inc.