Mixed Pure Cultures Research Articles

<span><strong>Enhanced thiamethoxam degradation in water  and soil by </strong><em>Phanerochaete </em><strong>sp. Th1 and </strong><em>Ensifer </em><strong>sp. Th2</strong></span>

Thiamethoxam is widely applied to control pest insects, but it causes serious environmental contamination. In this study, the degradation processes of the compound in soil and water collected from a rice field by native microorganisms, by mixed culture of Phanerochaete sp. Th1 and Ensifer sp. Th2, and by both the mixed pure culture and native microorganisms were conducted. The result showed that the mixed culture inoculation increased thiamethoxam in all media. In liquid media, the degradation percentage of the substrate in collected water was 21.8±4.4%, collected water + inoculation was 44.2±5.0%, and mineral medium + inoculation was 98.0±0.4%. Degradation determination in media with 70% collected water and 30% dry soil showed that the main degradation occurred in the liquid phase. The degradation rates in the media with 50% collected water and 50% dry soil were higher in the surface layer, and lower in the bottom. Indigenous microorganisms also played an important role in the degradation process. This study provided valuable information on the thiamethoxam degradation in simulated media of a rice field and the role of Phanerochaete sp. Th1 and Ensifer sp. Th2 to enhance the degradation.

Degradation of pretilachlor and fenclorim and effects of these compounds on bacterial communities under anaerobic condition.

Pretilachlor and safener fenclorim are the main components of herbicides widely applied to control weeds. Although some pure cultures of bacteria and fungi which degraded these compounds under aerobic conditions were isolated, no isolated pretilachlor- and fenclorim-degrading bacterial strains under anaerobic condition had been available. In this study, the degradation of these compounds and the effects of them on bacterial community structures were investigated under anaerobic conditions. The dissipation rates of pretilachlor and fenclorim in slurries were in the order: soil from paddy field ≈ sediment from river > sediment from mangrove. Moreover, three pretilachlor-degrading bacterial strains (Pseudomonas sp. Pr1, Proteiniclasticum sp. Pr2 and Paracoccus denitrificans Pr3) and two fenclorim-degrading strains (Dechloromonas sp. Fe1 and Ralstonia pickettii Fe2) isolated from a slurry of paddy soil utilized the substrates as sole carbon and energy sources under anaerobic conditions. The degradation of pure pretilachlor and fenclorim at various concentrations by corresponding mixed pure cultures followed the Michaelis-Menten model, with the maximum degradation was 3.10 ± 0.31µM/day for pretilachlor, and 2.08 ± 0.18µM/day for fenclorim. During the degradation, 2-chloro-N-(2,6-diethylphenyl) acetamide and 2,6-dimethylaniline were produced in pretilachlor degradation, and benzene was a product of fenclorim degradation. The synergistic degradation of both substrates by all isolated bacteria reduced the metabolites concentrations accumulated in media. This study provides valuable information on effects of pretilachlor and fenclorim on bacterial communities in soil and sediments, and degradation of these substrates by isolated bacteria under anaerobic condition.

A Multicolor Fluorescence in situ Hybridization Approach Using an Extended Set of Fluorophores to Visualize Microorganisms.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a key method for the detection of (uncultured) microorganisms in environmental and medical samples. A major limitation of standard FISH protocols, however, is the small number of phylogenetically distinct target organisms that can be detected simultaneously. In this study, we introduce a multicolor FISH approach that uses eight fluorophores with distinct spectral properties, which can unambiguously be distinguished by confocal laser scanning microscopy combined with white light laser technology. Hybridization of rRNA-targeted DNA oligonucleotide probes, which were mono-labeled with these fluorophores, to Escherichia coli cultures confirmed that the fluorophores did not affect probe melting behavior. Application of the new multicolor FISH method enabled the differentiation of seven (potentially up to eight) phylogenetically distinct microbial populations in an artificial community of mixed pure cultures (five bacteria, one archaeon, and one yeast strain) and in activated sludge from a full-scale wastewater treatment plant. In contrast to previously published multicolor FISH approaches, this method does not rely on combinatorial labeling of the same microorganisms with different fluorophores, which is prone to biases. Furthermore, images acquired by this method do not require elaborate post-processing prior to analysis. We also demonstrate that the newly developed multicolor FISH method is compatible with an improved cell fixation protocol for FISH targeting Gram-negative bacterial populations. This fixation approach uses agarose embedding during formaldehyde fixation to better preserve the three-dimensional structure of spatially complex samples such as biofilms and activated sludge flocs. The new multicolor FISH approach should be highly suitable for studying structural and functional aspects of microbial communities in virtually all types of samples that can be analyzed by conventional FISH methods.

Restoring the selectivity of modified charcoal cefoperazone deoxycholate agar for the isolation of Campylobacter species using tazobactam, a β-lactamase inhibitor

Extended spectrum β-lactamase (ESBL) producing Escherichia coli have emerged as a contaminant on modified charcoal cefoperazone deoxycholate agar (mCCDA) when attempting to selectively isolate Campylobacter spp. from poultry. E. coli are particularly problematic given their ability to grow under microaerophilic conditions and have been shown to outcompete Campylobacter species making Campylobacter detection or enumeration difficult. This paper recommends a novel method for restoring the selectivity of mCCDA using tazobactam, a β-lactamase inhibitor. The method significantly inhibited ESBL E. coli growth in spiked or naturally contaminated broiler caecal samples (p≤0.01) when compared to conventional mCCDA. This effect was seen at concentrations as low as 1mg/L tazobactam. TmCCDA11TmCCDA (tazobactam modified cefoperazone deoxycholate agar). was found to inhibit up to 8log10CFU/mL of ESBL E. coli in mixed pure cultures and 7.5log10CFU/mL in caecal samples. Furthermore TmCCDA concentrations up to 10mg/L had no statistically significant inhibitory effect (p≥0.05) on the recovery of a panel of 27 Campylobacter jejuni and 5 Campylobacter coli isolates when compared to conventional mCCDA. From this study it is suggested that tazobactam, which is more chemically stable than clavulanic acid or sulbactam, is more suitable for restoring the selectivity of mCCDA for the detection or isolation of campylobacters.

Methanogenic archaea and sulfate reducing bacteria co-cultured on acetate: teamwork or coexistence?

Acetate is a major product of fermentation processes and an important substrate for sulfate reducing bacteria and methanogenic archaea. Most studies on acetate catabolism by sulfate reducers and methanogens have used pure cultures. Less is known about acetate conversion by mixed pure cultures and the interactions between both groups. We tested interspecies hydrogen transfer and coexistence between marine methanogens and sulfate reducers using mixed pure cultures of two types of microorganisms. First, Desulfovibrio vulgaris subsp. vulgaris (DSM 1744), a hydrogenotrophic sulfate reducer, was cocultured together with the obligate aceticlastic methanogen Methanosaeta concilii using acetate as carbon and energy source. Next, Methanococcus maripaludis S2, an obligate H2- and formate-utilizing methanogen, was used as a partner organism to M. concilii in the presence of acetate. Finally, we performed a coexistence experiment between M. concilii and an acetotrophic sulfate reducer Desulfobacter latus AcSR2. Our results showed that D. vulgaris was able to reduce sulfate and grow from hydrogen leaked by M. concilii. In the other coculture, M. maripaludis was sustained by hydrogen leaked by M. concilii as revealed by qPCR. The growth of the two aceticlastic microbes indicated co-existence rather than competition. Altogether, our results indicate that H2 leaking from M. concilii could be used by efficient H2-scavengers. This metabolic trait, revealed from coculture studies, brings new insight to the metabolic flexibility of methanogens and sulfate reducers residing in marine environments in response to changing environmental conditions and community compositions. Using dedicated physiological studies we were able to unravel the occurrence of less obvious interactions between marine methanogens and sulfate-reducing bacteria.

Screening and monitoring microbial xenobiotics’ biodegradation by rapid, inexpensive and easy to perform microplate UV-absorbance measurements

BackgroundEvaluation of xenobiotics biodegradation potential, shown here for benzotriazoles (corrosion inhibitors) and sulfamethoxazole (sulfonamide antibiotic) by microbial communities and/or pure cultures normally requires time intensive and money consuming LC/GC methods that are, in case of laboratory setups, not always needed.ResultsThe usage of high concentrations to apply a high selective pressure on the microbial communities/pure cultures in laboratory setups, a simple UV-absorbance measurement (UV-AM) was developed and validated for screening a large number of setups, requiring almost no preparation and significantly less time and money compared to LC/GC methods. This rapid and easy to use method was evaluated by comparing its measured values to LC-UV and GC-MS/MS results. Furthermore, its application for monitoring and screening unknown activated sludge communities (ASC) and mixed pure cultures has been tested and approved to detect biodegradation of benzotriazole (BTri), 4- and 5-tolyltriazole (4-TTri, 5-TTri) as well as SMX.ConclusionsIn laboratory setups, xenobiotics concentrations above 1.0 mg L-1 without any enrichment or preparation could be detected after optimization of the method. As UV-AM does not require much preparatory work and can be conducted in 96 or even 384 well plate formats, the number of possible parallel setups and screening efficiency was significantly increased while analytic and laboratory costs were reduced to a minimum.

Reverse Transcription of 16S rRNA To Monitor Ribosome-Synthesizing Bacterial Populations in the Environment

Identification and quantification of phylogenetically defined bacterial populations in the environment are often performed using molecular tools targeting 16S rRNA. Fluorescence in situ hybridization has been used to monitor the expression and processing of rRNA by targeting the 3' tail of precursor 16S rRNA. To expand this approach, we employed reverse transcription of total RNA using primer S-D-Bact-0338-a-A-18. Length heterogeneity detected by slab gel analysis, denaturing high-performance liquid chromatography (DHPLC) was used to differentiate the 5' tail of the precursor from mature 16S rRNA, and the relative abundance of the precursor compared to the abundance of mature 16S rRNA was shown to be a sensitive indicator of the physiologic state of Acinetobacter calcoaceticus ATCC 23055(T). Our results demonstrate that this is a sensitive and reliable method with a detection limit of 10 ng of single-stranded DNA. The assay was also used to differentiate among precursor 16S rRNA levels with mixed pure cultures, as well as to examine the response of a mixed activated sludge culture exposed to fresh growth medium and the antibiotic chloramphenicol. The results of this study demonstrate that this assay is a novel reverse transcription assay that simultaneously measures the mature and precursor 16S rRNA pools for mixed bacterial populations in an engineered environment. Furthermore, collection of the reverse transcription products derived from activated sludge samples by the DHPLC approach enabled identification of the active bacterial genera. Comparison of 16S and precursor 16S rRNA clone library results indicated that the precursor 16S rRNA library is a more sensitive indicator for active bacteria in engineered environmental samples.

Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 +/- 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 +/- 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 +/- 1.5 to 14 +/- 2 and from 36 +/- 6 to 54 +/- 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).

Microbiological safety of kinema, a fermented soya bean food

Kinema is a fermented soya bean food of Nepal and the hilly regions of North-eastern States of India. Generally, the fermentation is dominated by Bacillus spp. that often cause alkalinity and desirable stickiness in the product. The present study was undertaken in a limited number of commercial (market) kinema samples to test for the presence of foodborne pathogens and their properties. Bacillus cereus was present in numbers exceeding 10 4 cfu/g product in five of the tested 15 market samples. Enterobacteriaceae and coliform bacteria exceeded 10 5 cfu/g in 10 of the 15 samples. Escherichia coli exceeding 10 5 cfu/g was found in two samples. Staphylococcus aureus was not detected in any of the tested samples. Of 31 isolated typical and atypical strains of B. cereus, 18 representative strains were tested qualitatively for the ability to produce diarrhoeal type enterotoxin (BCET) using an Oxoid BCET-RPLA test kit. Overall, BCET was formed by 12 strains in BHIG (brain heart infusion broth +1% glucose), by seven strains on sterilized cooked rice, and by five strains on sterilized cooked soya beans. Semi-quantitative tests on BCET revealed that levels exceeding 256 ng/g soya beans, produced by single pure culture inoculation with the isolated B. cereus strains, were reduced to ≤ 8 ng/g by frying kinema in oil, a common procedure when making kinema curry. It was also shown in a mixed pure culture experiment that a kinema strain B. Subtilis DK-W1, is able to suppress growth and BCET formation by a selected toxin producing strain (BC7-5) of B. cereus. It is concluded that the traditional way of making kinema and its culinary use in curries is safe. However, for novel applications of kinema, safety precautions are advisable.

Microbial Systems for Enhancement of Oil Recovery Used in Romanian Oil Fields

Microbial Enhanced Oil Recovery (MEOR) as a new Enhanced Oil Recovery (EOR) technology became mainly after the petroleum crisis in 1973, an important goal of research and development. The right microbial systems involved by this technology play an important role for the applications success. In Romania an intensive R and D activity took place during two “generations” of MEOR field trials (1975-1982 and 1986-1992 respectively). The investigations were focussed on several types of bacterial inoculum, good production of acids, gases, solvents and biosurfactants on nutrient support based on molasses (2-4%). It was found that bacterial consortia represented by Adapted Mixed Enrichment Cultures (AMEC) are much more efficient than the pure or mixed pure cultures. Finally, it was established a simple methodology to obtain AMEC for any oil reservoir with temperature up to 55-60°C, salinity up to 70-100 g NaCl/1, porosity and permeability more than 20%, and 150-300 mD respectively. The AMEC inoculum is based mainly on sporogenic bacteria of Bacillus and Clostridium type and nonsporogenic bacteria of Gram-negative rods type belonging mainly to Pseudomonas and Enter obacteriaceae groups.

Screening and characterization of fungal cultures for a milk-clotting enzyme for use in an oriental style dairy product

SummaryThe aim of this work was to develop new oriental style dairy products coagulated with culture filtrates from lao-chao, a traditional fermented rice product. The fermenting fungal cultures screened wereRhizopus javanicus, Rhiz. oryzae, Rhiz. chinensis, Rhiz. oligosporus, Aspergillus oryzae, Mucor racemosusand chiu-yao, a commercial starter. All the inocula liquefied a steamed glutinous rice base and produced culture filtrates with both milk-clotting and proteolytic activities. Of the fungal products tested,Rhiz. javanicuswas the most successful, producing a good yield of culture filtrate with the desired milk-clotting activity, and the resultant yogurt-like product was more acceptable to consumers than any except that from chiu-yao. The firmness was less acceptable, but this could be improved by using mixed pure cultures ofRhiz. javanicusandM. racemosus.

Effects of Germination and Pure Culture Fermentation by Yeasts and Lactobacilli on Phytic Acid and Polyphenol Content of Pearl Millet

ABSTRACTGermination of pearl millet at 30°C for 24 hr reduced phytic acid significantly (P<0.05) which, on fermentation with mixed pure cultures of Saccharomyces diastaticus, Saccharomyces cerevisiae, Lactobacillus brevis, and Lactobacillus fermentum at 30°C for 72 hr, was eliminated or reduced to negligible levels in fermented sprouts. Concentration of polyphenols did not change on germination. The fermentation of sprouts by combinations of S. cerevisiae with L. brevis and S. cerevisiae and L. fermentum did not change polyphenol whereas S. diastaticus with L. brevis (SdLb) increased and S. diastaticus with L. fermentum combinations decreased the polyphenol in the sprouts.

Effect of Fermentation by Pure Cultures of Yeasts and Lactobacilli on Phytic Acid and Polyphenol Content of Pearl Millet

ABSTRACTSingle and mixed pure culture fermentations of pearl millet flour with yeasts and lactobacilli, namely, Saccharomyces diastaticus, Saccharomyces cerevisiae, Lactobacillus brevis and Lactobacillus fermentum, at 30oC for 72 hr brought about a significant reduction in phytic acid and polyphenols which were present in considerable amounts in pearl millet flour. Reduction in phytic acid was more pronounced than that in the polyphenols.

Proficiency test sample media for single and mixed pure cultures of water pollution indicator bacteria

Two transport media, NYSDH-1 and NYSDH-2, were developed for use in a split bacteriological water sample program. The media maintained 88% viability of inoculated organisms for at least 48 h, and the samples do not require special handling or reconstitution. Procedures for preparing and shipping the samples to participating laboratories were developed. A reference set of samples was analyzed in laboratories certified by either New York State or the Environmental Protection Agency. A statistical analysis was performed, and the results indicate that the media are suitable for integration into a laboratory quality control program.

An apparatus for studying syntrophic interactions between microorganisms

An apparatus has been designed to grow pure axenic cultures of different microorganisms in the same liquid phase; the organisms are separated by Millipore membranes which prevent cross contamination but permit cross feeding. It is possible with this mixed pure culture apparatus (MPCA) to duplicate the syntrophic effects observed in mixed cultures of algae and bacteria and at the same time to measure physiological changes in homogeneous axenic cultures.