Simultaneous enumeration of yeast and bacterial cells in the context of industrial bioprocesses.

In scenarios where yeast and bacterial cells coexist, it is of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared, mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries, and analyzed using bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter Counter (at 1:1 and 100:1 ratios). We observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter Counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (p > .05). In contrast, the Coulter Counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (p < .05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn't possible due to an overlap between yeast cell debris and bacterial cells. We conclude that each method has limitations, advantages, and disadvantages. ONE-SENTENCE SUMMARY: This study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision.

Removal and desorption of chromium in synthetic effluent by a mixed culture in a bioreactor with a magnetic field.

Two chromium removal experiments were performed in bioreactors with and without a magnetic field under the same conditions. The release of the chromium present in the biomass was tested in two experiments one with the initial pH of the medium and one with pH 4.0. The objective was to remove Cr(VI) and total Cr from the effluent, this was carried out by placing biological treatments of synthetic effluent contaminated with 100 mg/L of Cr(VI) in a bioreactor with neodymium magnets that applied a magnetic field (intensity 85.4 mT) to the mixed culture. The removal of Cr(VI) was approximately 100.0% for the bioreactor with a magnetic field and 93.3% for the bioreactor without a magnetic field for 9 hr of recirculation of the synthetic effluent by the bioreactor. The removal of total Cr was 61.6% and 48.4%, with and without a magnetic field, respectively, for 24 hr. The desorption of Cr(VI) in the synthetic effluent was 0.05 mg/L, which is below the limit established by Brazilian legislation (0.1 mg/L) for the discharge of effluent containing Cr(VI) into bodies of water. The results obtained for the removal of chromium in synthetic effluent suggested that there was no significant influence on the viable cell count of the mixed culture. The desorption of Cr(VI) in synthetic effluent after bioadsorption of chromium by the mixed culture in the process of removal of chromium in bioreactors with and without a magnetic field was not significant in either of the experiments with different initial pHs.

Obtaining, characterization and evaluation of two candidate preparations for probiotics developed with agroindustrial waste / Obtención, caracterización y evaluación de dos preparados candidatos a probióticos desarrollados con residuos agroindustriales

ABSTRACT Objective. Obtain, characterize and evaluate two bio-prepares developed from the sugar cane molasses - orange vinasse fermented with yeast and/or lactic acid bacteria. Materials and methods. A completely randomized design was used, with five repeats per treatment. The evaluated treatments were: T1, Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus y T2, the previous bacteria plus Saccharomyces cerevisiae and Kluyveromyces fragilis (L-4 UCLV). The previous mentioned microorganisms were inoculated in a substratum compounded by molasses - vinasse and these were incubated at 37°C for 24 hours. To the bioprepares, physiochemical, microbiological and in vitro tests was made to evaluate the probiotic capacity. Results. Both bioprepares presented a dark brown color, sweet and a pH lesser than 4. The bromatological and microbiologic development were higher (p>0.05) in T2. Both bioprepares the viability was higher than 92%. in vitro tests two bioprepares were resistant to an acid pH, bile salts, broad spectrum of microbial activity and inhibitory effect to E. coli, Salmonella spp. and S. aureus. Conclusions. The bioprepares obtained from sugar cane molasses - orange vinasse fermented with yeast and lactic acid bacteria manifested physiochemical and microbiologic properties appropriated to probiotic products. In in vitro tests, their potential was demonstrated as a probiotic.

RESUMEN Objetivo. Obtener, caracterizar y evaluar dos biopreparados desarrollados a partir de melaza de caña de azúcar - vinaza de naranja fermentados con levaduras y/o bacterias ácido lácticas. Materiales y métodos. Se utilizó un diseño completamente aleatorizado con cinco repeticiones por tratamiento. Los tratamientos evaluados fueron: T1, Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus y T2, las bacterias anteriores más Saccharomyces cerevisiae y Kluyveromyces fragilis (L-4 UCLV). En un sustrato compuesto por melaza- vinaza se inocularon los microorganismos anteriormente mencionados y estos fueron incubados a 37ºC por 24 h. Se les determinaron a los biopreparados los parámetros fisicoquímicos, microbiológico y se realizaron las pruebas in vitro para evaluar la capacidad probiótica. Resultados. Ambos biopreparados presentaron un color marrón oscuro, dulzón y con pH inferior a 4. El comportamiento bromatológicos y microbiológicos fueron mayores (p>0.05) en el T2. En ambos biopreparados la viabilidad fue superior a 92%. En pruebas in vitro, ambos biopreparados fueron resistentes a pH ácido, sales biliares, amplio espectro de actividad antimicrobiana y efecto inhibitorio a la E. coli, Salmonella spp. y S. aureus. Conclusiones. Los biopreparados obtenidos a partir de melaza de caña de azúcar-vinaza de naranja fermentados con levaduras y/o bacterias ácido lácticas demostraron propiedades físicoquímicas, microbiológicas apropiadas para productos probióticos. En las pruebas in vitro, se demostró su efecto potencial como probiótico.

Chemotaxis detection towards chlorophenols using video processing analysis.

To our knowledge, this communication is the first report of chemotaxis towards chlorophenols by any bacteria. We used a recently published method based on the agarose in-plug assay combined with video processing analysis and we also present a new index of bacterial mean speed for these assays.

Formulación mixta de bacterias lácticas para el control de Listeria monocytogenes / Mixted formulation of lactic bacterium for the control of Listeria monocytogenes

Resumen La combinación de la actividad metabólica de cepas bacterianas potencializa la actividad antimicrobiana contra microorganismos patógenos, en comparación con la actividad que pueden presentar las cepas microbianas en forma individual. La formulación mixta de bacterias acido lácticas ha sido estudiada para la producción de preparados probióticos con actividad antimicrobiana contra patógenos. Listeria monocytogenes es considerado un microorganismo patógeno para el hombre y animales, causando principalmente, la enfermedad conocida como listeriosis. Se evaluó la actividad antimicrobiana de una formulación mixta de Lactobacillus brevis y Weisella cibaria frente a Listeria monocytogenes. L. brevis y W. cibaria se reprodujeron por fermentaciones en discontinuo durante 48 horas. Se midió la cinética de la actividad antimicrobiana contra L. monocytogenes en los siguientes tiempos de fermentación, 0, 1, 2, 6, 12, 24 y 48 horas. En cada tiempo, la actividad antimicrobiana de la mezcla de cepas se comparó con la actividad antimicrobiana de las cepas en forma individual. La actividad antimicrobiana se midió mediante el diámetro de Feret, utilizando un software de evaluación de imágenes. Se encontró que la actividad antimicrobiana de la mezcla de cepas contra L. monocytogenes fue estable desde la segunda hora de fermentación hasta las 48 horas. A partir de 18 horas de fermentación la mezcla de cepas presentó actividad antimicrobiana superior, comparada con las cepas individuales. Los resultados indican que la formulación mixta de L. brevis y W. cibaria podría ser una opción biotecnológica para el desarrollo de antimicrobianos naturales para el control y prevención de L. monocytogenes.

Abstract The combination of the metabolic activity of bacterial strains potentiates the antimicrobial activity against pathogenic microorganisms, in comparison with the activity that the microbial strains can present individually. The mixed formulation of lactic acid bacteria has been studied to the production of probiotic preparations with antimicrobial activity against pathogens. Listeria monocytogenes is considered a pathogenic microorganism for man and animals, causing the disease known as listeriosis. The antimicrobial activity of a mixed formulation of Lactobacillus brevis and Weisella cibaria was evaluated against Listeria monocytogenes. L. brevis and W. cibaria were reproduced by discontinuous fermentations for 48 hours. The kinetics of antimicrobial activity against L. monocytogenes were measured at the next fermentation times, 0, 1, 2, 6, 12, 24 and 48 hours. At each time, the antimicrobial activity of the mixed formulation was compared with the antimicrobial activity of the strains individually. The antimicrobial activity was measured by Feret's diameter, using image evaluation software. It was found that the antimicrobial activity of the mixed formulation against L. monocytogenes was stable from the second hour of fermentation to 48 hours of fermentation. After 18 hours of fermentation the mixed formulation presented superior antimicrobial activity, compared to the individual strains. The results indicate that the mixed formulation of L. brevis and W. cibaria could be a biotechnological option for the development of natural antimicrobials for the control and prevention of L. monocytogenes.

Lactic acid production from submerged fermentation of broken rice using undefined mixed culture.

The present work aimed to characterize and optimize the submerged fermentation of broken rice for lactic acid (LA) production using undefined mixed culture from dewatered activated sludge. A microorganism with amylolytic activity, which also produces LA, Lactobacillus amylovorus, was used as a control to assess the extent of mixed culture on LA yield. Three level full factorial designs were performed to optimize and define the influence of fermentation temperature (20-50 °C), gelatinization time (30-60 min) and broken rice concentration in culture medium (40-80 g L-1) on LA production in pure and undefined mixed culture. LA production in mixed culture (9.76 g L-1) increased in sixfold respect to pure culture in optimal assessed experimental conditions. The optimal conditions for maximizing LA yield in mixed culture bioprocess were 31 °C temperature, 45 min gelatinization time and 79 g L-1 broken rice concentration in culture medium. This study demonstrated the positive effect of undefined mixed culture from dewatered activated sludge to produce LA from culture medium formulated with broken rice. In addition, this work establishes the basis for an efficient and low-cost bioprocess to manufacture LA from this booming agro-industrial by-product.

Characterization of Ferroplasma acidiphilum growing in pure and mixed culture with Leptospirillum ferriphilum.

Biomining is defined as biotechnology for metal recovery from minerals, and is promoted by the concerted effort of a consortium of acidophile prokaryotes, comprised of members of the Bacteria and Archaea domains. Ferroplasma acidiphilum and Leptospirillum ferriphilum are the dominant species in extremely acid environments and have great use in bioleaching applications; however, the role of each species in this consortia is still a subject of research. The hypothesis of this work is that F. acidiphilum uses the organic matter secreted by L. ferriphilum for growth, maintaining low levels of organic compounds in the culture medium, preventing their toxic effects on L. ferriphilum. To test this hypothesis, a characterization of Ferroplasma acidiphilum strain BRL-115 was made with the objective of determining its optimal growth conditions. Subsequently, under the optimal conditions, L. ferriphilum and F. acidiphilum were tested growing in each other's supernatant, in order to define if there was exchange of metabolites between the species. With these results, a mixed culture in batch cyclic operation was performed to obtain main specific growth rates, which were used to evaluate a mixed metabolic model previously developed by our group. It was observed that F. acidiphilum, strain BRL-115 is a chemomixotrophic organism, and its growth is maximized with yeast extract at a concentration of 0.04% wt/vol. From the experiments of L. ferriphilum growing on F. acidiphilum supernatant and vice versa, it was observed that in both cases cell growth is favorably affected by the presence of the filtered medium of the other microorganism, proving a synergistic interaction between these species. Specific growth rates were obtained in cyclic batch operation of the mixed culture and were used as input data for a Flux Balance Analysis of the mixed metabolic model, obtaining a reasonable behavior of the metabolic fluxes and the system as a whole, therefore consolidating the model previously developed. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1390-1396, 2016.

Complete genome sequence of Burkholderia caribensis Bcrs1W (NBRC110739), a strain co-residing with phenanthrene degrader Mycobacterium sp. EPa45.

Complete genome sequence of Burkholderia caribensis Bcrs1W, isolated from a phenanthrene-degrading mixed culture, was determined. The genomic information of Bcrs1W will be beneficial to elucidating the mechanisms of its positive effects on phenanthrene degradation by co-residing Mycobacterium sp. Epa45, and to exploiting their degradation potentials.

Selection of an actinobacteria mixed culture for chlordane remediation. Pesticide effects on microbial morphology and bioemulsifier production.

Chlordane bioremediation using actinobacteria mixed culture is an attractive clean-up technique. Their ability to produce bioemulsifiers could increase the bioavailability of this pesticide. In order to select a defined actinobacteria mixed culture for chlordane remediation, compatibility assays were performed among six Streptomyces strains. The strains did not show growth inhibition, and they were assayed for chlordane removal, either as pure or as mixed cultures. In pure cultures, all of the strains showed specific dechlorination activity (1.42-24.20 EU mg(-1)) and chlordane removal abilities (91.3-95.5%). The specific dechlorination activity was mainly improved with cultures of three or four microorganisms. The mixed culture consisting of Streptomyces sp. A2-A5-A13 was selected. Their ability to produce bioemulsifiers in the presence of glucose or chlordane was tested, but no significant differences were observed (p > 0.05). However, the stability of the emulsions formed was linked to the carbon source used. Only in chlordane presence the emulsions retained 100% of their initial height. Finally, the selected consortium showed a high degree of sporulation in the pesticide presence. This is the first study on the effects that chlordane exerts on microbe morphology and emulsifier production for a defined mixed culture of Streptomyces with ability to remediate the pesticide.

Stoichiometric model and flux balance analysis for a mixed culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum.

The oxidation process of sulfide minerals in natural environments is achieved by microbial communities from the Archaea and Bacteria domains. A metabolic reconstruction of two dominant species, Leptospirillum ferriphilum and Ferroplasma acidiphilum, which are always found together as a mixed culture in this natural environments, was made. The metabolic model, composed of 152 internal reactions and 29 transport reactions, describes the main interactions between these species, assuming that both use ferrous iron as energy source, and F. acidiphilum takes advantage of the organic compounds secreted by L. ferriphilum for chemomixotrophic growth. A first metabolic model for a mixed culture used in bacterial leaching is proposed in this article, which pretends to represent the characteristics of the mixed culture in a simplified manner. It was evaluated with experimental data through flux balance analysis (FBA) using as objective function the maximization of biomass. The growth yields on ferrous iron obtained for each microorganism are consistent with experimental data, and the flux distribution obtained allows understanding of the metabolic capabilities of both microorganisms growing together in a bioleaching process. The model was used to simulate the growth of F. acidiphilum on different substrates, to determine in silico which compounds maximize cell growth, and which are essential. Knockout simulations were carried out for L. ferriphilum and F. acidiphilum metabolic models, predicting key enzymes of central metabolism. The results of this analysis are consistent with experimental data from literature, showing a robust behavior of the metabolic model.

Mixed Cultures Fermentation for the Production of Poly- ß-hydroxybutyrate

Poly-ß-hydroxybutyrate (PHB) is a biodegradable intracellular microbial product produced by many bacteria and it is comparable to some of the petrochemical derived thermoplastics such as polypropylene. One of the main barriers for the commercial exploitation is the high cost of the substrate for the production of biopolymer. The utilization of mixed microbial cultures facilitates the use of complex substrates thereby reducing the cost of PHB production. In the present study, mixed culture systems were evaluated for PHB production. Bacillus firmus NII 0830 was used for the production of PHB since it accumulates a large amount of PHB and a second organism Lactobacillus delbrueckii NII 0925 was used to provide lactic acid. FTIR and 1H NMR analyses revealed that the PHB extracted from pure culture and mixed culture showed exact match to that of standard PHB. Biodegradation studies of the PHB blends showed 87% degradation. It was also found that a consortium of organisms degraded the films faster than a single organism.

Biodegradation of oil refinery residues using mixed-culture of microorganisms isolated from a landfarming

In this study, the potential for using an inoculum composed of a mixed-culture of bacteria and fungi, isolated from a landfarming at the Paulínia Oil Refinery, Brazil, to degrade oil residues generated in the process of petroleum refinement was investigated. The isolation of these microorganisms was carried out beforehand, assuming that they would be better adapted to petroleum hydrocarbons, as the landfarming consisted of an area impacted by the deposit of such compounds. The Bartha and Pramer respirometric test was used to measure the rate of biodegradation of the hydrocarbons by the mixed-culture of microorganisms via the evolution of CO2. The results obtained with respect to the efficiency of biodegradation showed no significant differences (P>0.05), indicating no increase in the biodegradation process using the inoculum. The addition of nutrients (N, P, K) also did not contribute to an increase in biodegradation of the oil residue studied.

Neste estudo foi investigado o potencial de um inóculo composto de cultura mista de bactérias e fungos, isolados do landfarming da Refinaria de Paulínia, Brasil, em degradar resíduos oleosos gerados no processo de refinamento de petróleo. O isolamento desses microrganismos foi realizado previamente, supondo-se que estejam melhor adaptados aos hidrocarbonetos de petróleo uma vez que o landfarming consiste em área impactada por deposição de tais compostos. Utilizou-se o teste respirométrico de Bartha e Pramer no intuito de verificar a taxa de biodegradação dos hidrocarbonetos pela cultura mista de microrganismos através da evolução de CO2. Os resultados obtidos para a eficiência da biodegradação não apresentaram diferença estatisticamente significativa (P>0.05) indicando que não houve aumento do processo de biodegradação com o uso do inóculo. A adição de nutrientes (N, P, K) tampouco contribuiu para aumentar a biodegradação do resíduo oleoso estudado.

Mixed mycobacterial cultures: is it important to separate and to identify multiple species? / Culturas mistas de micobactérias: é importante isolar e identificar?

Careful examination of occurrence of mixed mycobacterial cultures is important because most of them include at least one pathogenic agent. Among 8, 036 mycobacterial cultures samples received between 1999 and 2000 at the Mycobacteria Laboratory of Instituto Adolfo Lutz, 21 (0.26%) classified as mixed cultures were selected for being investigated. For this, the use of 7H11 plate was included for colonies isolation. The isolated colonies were then subcultured into Lowenstein-Jensen media and incubated at 37ºC. The isolates were identified by phenotypic tests and by PCR restriction enzyme analysis of hsp65 gene. The analysis of those 21 mixed cultures showed the occurrence of M. tuberculosis and non-tuberculous mycobacteria (NTM) in eight samples, three cultures with two species of NTM , and ten with only one specie of mycobacteria. The results of this study suggest that microbiology technicians should be aware of mixed cultures and apply every practical method available to separate and to identify the involved mycobacteria species.

A investigação de culturas mistas de micobactérias é importante pois geralmente estas incluem ao menos uma espécie patogênica ou potencialmente patogênica. Dentre 8.036 culturas recebidas entre 1999 e 2000, pelo Setor de Micobactérias do Instituto Adolfo Lutz, foram selecionadas 21 (0,26%) com resultados sugestivos de culturas mistas. Após o isolamento em meio 7H11 as colônias foram repicadas em Löwenstein Jensen e incubadas à 37º C. A identificação dos 32 subcultivos foi feita por métodos fenotípicos e pela analise do perfil de restrição do produto da amplificação de 440 pares de base do gene hsp65. Em oito subcultivos foi encontrada a espécie M. tuberculosis associada com MNT, em 3 subcultivos foram encontradas 2 espécies de MNT e nos demais foi identificado apenas um tipo de micobacteria. O tempo de crescimento lento das micobactérias inviabiliza o plaqueamento de todas as culturas pois este procedimento acarretaria demora na liberação do resultado final dos testes, além de representar gastos excessivos em áreas endêmicas, geralmente com escassos recursos econômicos. Embora as dificuldades mencionadas, os microbiologistas devem estar atentos quanto à presença de culturas mistas de micobactérias e usar todos os métodos disponíveis para separar e identificar as espécies.