A gene, umcel5N, was isolated from a metagenomic library constructed from the contents of buffalo rumen. Its putative product belongs to the glycosyl hydrolase family 5 and is most closely related to an endoglucanase (ABN54006.1) from Clostridium thermocellum with 44% identity and 60% similarity. Gene umcel5N was heterologously expressed in Escherichia coli. The purified recombinant Umcel5N hydrolyzed carboxymethyl cellulose with a rapid decrease in the viscosity of the solution but with little release of reducing sugars, suggesting an endo mode of action. The enzyme exhibited optimal activity toward p-nitrophenyl β-d-cellobioside at pH 5.5 and 55°C, and had a Km of 1.56 mM and a Vmax of 285.6 U/mg. Two glutamic acids (E144 and E285) of the wild-type Umcel5N were predicted as a proton donor and a nucleophile, respectively. Site-directed mutagenesis confirmed that they were required for the enzyme's activity.

A rapid and cost-effective technique for identification and classification of microorganisms was explored using fluorescence microscopy and image analysis. After staining the microorganisms with fluorescent dyes (diamidino-2-phenyl-indole [DAPI] and acridine orange [AO], images of the microorganisms were captured using a charge-coupled device camera attached to a light microscope. Textural features were extracted from the images. Fluorescence emission from Bacillus thuringiensis is the highest compared with other microbes, and the emission from Lactobacillus brevis is the lowest. Various microorganisms can be differentiated using various textural features from images using AO or DAPI dye. Many textural features of the images obtained from the two dyes were different. Conventional microbial detection methods take considerable time and are laborious. Rapid methods are required so that pathogens and spoilage microorganisms in foods and water can be identified and counted in a much shorter time. This work investigates image processing techniques particularly based on textural properties of the images of microorganisms. Images of microorganisms in samples can be captured using light microscopes after concentrating using centrifuge or membrane separation devices. This work will assist in developing a commercial method for rapid detection of microbes in food samples.

In vitro experiments were conducted to examine the characteristics and mode of action of a protease that increased the ruminal fiber digestibility of alfalfa hay. A commercial source of protease (Protex 6L, Genencor Int., Rochester, NY), already characterized for its main activities, was further analyzed to determine protease activity in response to pH, molecular size by SDS-PAGE, specificity to degrade model or feed substrates, response to autoclaving, and action of specific protease inhibitors in the absence or presence of ruminal fluid. In addition, batch culture in vitro incubations in buffered ruminal fluid were conducted to compare the enzyme product with purified protease sources, and dose-response studies (0 to 10 μL/g of forage DM) were carried out using alfalfa hay as a substrate. The enzyme product was shown to be an alkaline protease (optimum pH >8.5) of approximately 30 kDa. Specificity in the absence of ruminal fluid showed that the enzyme was active against gelatin and casein to the same extent, whereas it had limited (21% of the total) activity on BSA. In the presence of ruminal fluid and with the use of feed substrates, the protease increased (P < 0.05) 22-h IVDMD (%) of alfalfa hay, fresh corn silage, dry-rolled corn, and a total mixed ration composed of the 3 ingredients (39.5 vs. 44.7; 50.3 vs. 54.5; 63.8 vs. 68.4; and 55.4 vs. 56.4 for control vs. protease for each feed, respectively). Inhibitor studies in the absence of ruminal fluid indicated that the enzyme was inhibited most by a serine protease inhibitor but not by cysteine- or metalloprotease inhibitors (10 vs. 1.9 and 0.1%, respectively). In the presence of ruminal fluid, the serine protease inhibitor reversed (P < 0.05) the increase in alfalfa IVDMD achieved by the enzyme product, such that IVDMD was similar to that of the control treatment. Comparisons among different proteases revealed that only pure subtilisin achieved increases in IVDMD that were similar to those with protease, suggesting the serine protease was subtilisin-like (EC 3.4.1.62). Dose-response studies using alfalfa hay as substrate showed quadratic responses in IVDMD, NDF digestion, and hemicellulose and protein disappearance. It is postulated that this enzyme acts by removing structural proteins in the cell wall, allowing ruminal microbes to gain faster access to digestible substrates.

It has been conducted monitoring for the natural self-recovery of oil polluted loamy grounds by soil microorganisms as a basic factor and observing for recovering process in the soils recultivated by Carex hirta. Quantity of microorganisms most of the investigated physiological groups was reduced in the first months after oil pollution, the intensific development of hydrocarbon-utilizing microorganisms in ground was observed at the same time. The positive effect of C. hirta on the processes of microorganisms growth most of physiological groups and microbial destruction of hydrocarbon was revealed in the first months after oil pollution. Quantity of microorganisms of all physiological groups practically completely recovering and hydrocarbon contents in soil was remained in 18 months after oil pollution. An oil-resistant and long-rooted plant C. hirta favours the intensific development of oil-destructive microorganisms іn polluted soil and accordingly positively influences on the level of microbial destruction of oil hydrocarbons. Using of C. hirta and microorganisms in complex of bioremediation measures is a perspective method for soil purification from oil contamination.

Some bacteria have a higher tendency to produce biofilm than others. Especially, Pseudomonas and Aeromonas strains are acknowledged to be pioneer colonizers and are predominant in biofilm formation. We examined biofilm formation and first attachment maintance of biofilms of Pseudomonas spp., Pseudomonas aeruginosa, Aeromonas spp, sulphate reducing bacteria and filamentous fungi. A simulated recirculating cooling-water system was used. Heterotrophic bacteria counts on galvanized steel and glass surfaces rose during the tidy period of 720 hours. In addition, we determined that although Pseudomonas spp., Pseudomonas aeruginosa and Aeromonas spp. were the pioneer colonizers, they surprisingly could not be determined in the biofilms on both types of surface after 456 hours. Sulphate reducing bacteria were observed in biofilms on both surfaces from the outset of the experiments. Filamentous fungi were seen on the galvanized steel and glass surfaces after 0.5 h. (© 2009 WILEY-VCH Verlag GmbH '' Co. KGaA, Weinheim)

1. In natural grassland ecosystems, root-feeding nematodes and insects are the dominant below-ground herbivores. In coastal foredunes, the ectoparasitic nematode Tylenchorhynchus ventralis would be a major root herbivore if not strongly controlled by soil microorganisms. Here, we examined if the suppressive effects of the microbial enemies of T. ventralis act by local interactions such as predation, parasitism or antagonism, or local induction of plant defence, or by non-local interactions, such as systemic effects when microorganisms in one section of the plant roots can affect nematode control in another section of the root system. We show that abundance of T. ventralis in the root zone of the grass Ammophila arenaria is suppressed by local interactions. 2. We compared local vs. non-local control of nematodes by a natural community of soil microorganisms in a split-root experiment, where nematodes and microbes were inoculated to the same, or to opposite root compartments. 3. The split-root experiment revealed that microorganisms affected T. ventralis numbers only when present in the same root compartment. Therefore, the effects of microorganisms on T. ventralis are due to local interactions and not due to induction of a systemic defence mechanism in the plant host. 4. When inoculated together with microorganisms, the nematodes were heavily infected with unknown bacteria and with fungi that resembled the genus Catenaria, suggesting that microorganisms control nematodes through parasitism. However, local defence induction cannot be completely excluded. 5. Besides microbial enemies of nematodes, the root zone of A. arenaria also contains plant pathogens. Root biomass was reduced by nematode infection, but also by the combination of nematodes and microorganisms, most likely because the soil pathogens overwhelmed the effects of nematode control on plant production. 6. We conclude that there may be a trade-off between beneficial effects of soil microorganisms on the plant host due to nematode control vs. pathogenic effects of soil microorganisms on the plant host. We propose that such trade-offs require more attention when studying below-ground multitrophic interactions

Androstenedione (AD) is a steroid intermediate used in the pharmaceutical industry for the production of several important anabolic drugs. An important route for producing AD is by the microbial transformation of sterols. Microbes capable of efficiently transforming sterols to AD are few and newer strains need to be isolated. Conventional procedures for screening and isolation are time consuming. A new procedure was used for screening and isolation of fungal microorganisms capable of biotransformation of sitosterol to androstenedione. In this procedure, Basic Alignment Search Tool (BLAST) and Position-Specific Iterative BLAST were employed to obtain a parent set of candidate microorganisms. The parent set was reduced using heuristics and constraints to obtain a manageable number of microorganisms that may be tested experimentally. For this work, screening of the entire NCBI database yielded a parent set containing 64 microorganisms. Among these, only two microorganisms, Aspergillus oryzae and Aspergillus nidulans FGSC A4, qualified to the experimental stage. Sitosterol biotransformation experiments were carried out using A. oryzae and the production of AD in culture medium was confirmed.

Nutrient management plans (NMPs) for application of wastewater from concentrated animal feeding operations are designed to meet crop water and nutrient requirements, but implicitly assume that pathogenic microorganisms in the wastewater will be retained and die-off in the root zone. A NMP was implemented on a field plot to test this assumption by monitoring the fate of several fecal indicator microorganisms (Enterococcus, fecal coliforms, somatic coliphage, and total Escherichia coli). When well-water and wastewater were applied to meet measured evapotranspiration (ET), little advective transport of the indicator microorganisms occurred below the root zone and the remaining microorganisms rapidly died-off (within 1 mo). Additional experiments were conducted in the laboratory to better quantify microorganism transport and survival in the field soil. Batch survival experiments revealed much more rapid die-off rates for the bacterial indicator microorganisms in native than in sterilized soil, suggesting that biotic factors controlled survival. Saturated column experiments with packed field soil, demonstrated much greater transport potential for somatic coliphage than bacterial indicators (Enterococcus and total E. coli) and that the retention rates for the indicator microorganisms were not log-linear with depth. A worst case transport scenario of ponded infiltration on a large undistributed soil column from the field was also initiated and indicator microorganisms were not detected in the column outflow or in the soil at a depth of 65 cm. All of these observations support the hypothesis that a NMP at this site will protect groundwater supplies from microorganism contamination, especially when applied water and wastewater meet ET.

The effect of some pulsed electric field (PEF) processing parameters (electric field strength, pulse frequency and treatment time), on a mixture of microorganisms (Kloeckera apiculata, Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus hilgardii and Gluconobacter oxydans) typically present in grape juice and wine were evaluated. An experimental design based on response surface methodology (RSM) was used and results were also compared with those of a factorially designed experiment. The relationship between the levels of inactivation of microorganisms and the energy applied to the grape juice was analysed. Yeast and bacteria were inactivated by the PEF treatments, with reductions that ranged from 2.24 to 3.94 log units. All PEF parameters affected microbial inactivation. Optimal inactivation of the mixture of spoilage microorganisms was predicted by the RSM models at 35.0 kV cm⁻¹ with 303 Hz pulse width for 1 ms. Inactivation was greater for yeasts than for bacteria, as was predicted by the RSM. The maximum efficacy of the PEF treatment for inactivation of microorganisms in grape juice was observed around 1500 MJ L⁻¹ for all the microorganisms investigated. The RSM could be used in the fruit juice industry to optimise the inactivation of spoilage microorganisms by PEF.