Biodeterioration of Mayan archaeological sites in the Yucatan Peninsula, Mexico

Many of the monuments of the Mayan civilization are suffering deterioration caused by environmental factors (high temperatures and relative humidities), increasing contamination from natural and anthropogenic sources, and by the action of micro- and macro-biological communities. Archaeological sites and historical monuments in the Mayan area were constructed with different limestones which offer different resistances to degradation by the various types of contamination. Two different sampling sites were chosen at the archaeological site of Uxmal in the Yucatan Peninsula, Mexico. Heterotrophic bacteria, cyanobacteria and different fungi were isolated and classified taxonomically. The other archaeological site chosen for this study was the fortress of Tulum, located at the side of the Caribbean Sea and exposed to chloride of marine spray and sand erosion. In this case, heterotrophic aerobic and anaerobic bacteria, cyanobacteria and fungi were isolated from the four sampling areas selected. In both archaeological sites crust deposits were observed by using light microscopy, SEM and ESEM. Surface analyses were made by means of EDAX and electron microprobe. Possible mechanisms of stone decay, based on the type of microorganisms isolated, the physico-chemical characteristics of the constructional materials and environmental factors are discussed.     

Diagnosis of acute puerperal metritis by electronic nose device analysis of vaginal discharge in dairy cows

The objective of this study was to estimate the diagnostic accuracy of an electronic nose device using vaginal discharge samples to diagnose acute puerperal metritis (APM) in dairy cows. Uterine fluid was sampled manually with a gloved hand and under sterile conditions for electronic nose device analysis (day in milk (DIM) 2, 5, and 10) and bacteriologic examination (DIM 5), respectively, and on additional days, if APM was diagnosed during the daily clinical examinations. A dataset containing samples from 70 cows was used to create a model and to validate the APM status predicted by this model, respectively. Half of the dataset (n = 35; 14 healthy and 21 metritic cows) was provided with information regarding the APM diagnosis and contained all three measurements (DIM 2, 5, and 10) for each cow and was used as a training set whereas the second half was blinded (n = 35; 14 healthy and 21 metritic cows) and contained only the samples collected on DIM 5 of each cow and was used to validate the created prediction model. A receiver operating characteristic curve was calculated using the prediction results of the validation test. The best observed sensitivity was 100% with specificity of 91.6% when using a threshold value of 0.3. The calculated P-value for the receiver operating characteristic curve was less than 0.01. Overall, Escherichia coli was isolated in eight of 28 (28.6%) and 22 of 42 (52.4%) samples collected from healthy and metritic cows, respectively. Trueperella pyogenes and Fusobacterium necrophorum were isolated in 14 and six of 28 (50.0% and 21.4%) and 17 and 16 of 42 (40.5% and 38.1%) samples collected from healthy and metritic cows, respectively. The prevalence of Escherichia coli and Trueperella pyogenes was similar in the samples obtained from metritic cows used for the training set and the validation test. The results are promising especially because of the objective nature of the measurements obtained by the electronic nose device.     

Sensor systems,electronic tongues and electronic noses,for the monitoring of biotechnological processes

Production of biofuel is based on the conversion by microorganisms of complex organic substrates into the methane or ethanol, which are consequently used as energy sources. Real time monitoring of the fermented media composition is of paramount for the effectiveness of the whole process. However, despite the fact that products worth billions of dollars are produced through fermentation processes annually, analytical instruments used for these processes’ monitoring remain relatively primitive. Established laboratory techniques produce exhaustive information about media composition but analysis is often quite time-consuming, expensive, requires skilled personnel and hardly can be automated. Lack of on-line sensors for the fermentation monitoring is commonly stressed in the literature. One of the techniques particularly suitable for this purpose is chemical sensors. Such features as low prices, relatively simple instrumentation, minimal sample preparation and easy automation of measurements make chemical sensors an attractive tool for industrial process control. However, practical use of chemical sensors in complex media is often hindered by their insufficient selectivity. For example, only pH and oxygen probes are routinely used in bio-reactors. One of the emerging approaches permitting to overcome the selectivity problems is the use of systems instead of discrete sensors. Such systems for liquid and gas analysis were named electronic tongues and electronic noses correspondingly. They are capable to perform both quantitative analysis (components’ concentrations) and classification or recognition of multicomponent media. This review presents recent achievements in the R&D and applications of electronic tongues and noses to the monitoring of biotechnological processes. JIMB-2008: BioEnergy—Special issue.     

Microbial and enzymatic control of pitch in the pulp and paper industry

Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching.     

Application of image analysis in the fungal fermentation of Trichoderma reesei RUT-C30

Trichoderma reesei was grown in a stirred-tank bioreactor (STB) and a reciprocating plate bioreactor (RPB) at four different agitation speeds. A semiautomatic image analysis protocol that was developed to characterize the mycelium morphology during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms) was applied to study the effect of agitation on the morphology of T. reesei. It was shown via statistical validation that broth samples used for image analysis represented the whole population of the fungi in the bioreactor. High shear was found to be damaging to T. reesei grown in the STB. The gentler shear produced in the RPB was not detrimental to the microorganism even at higher agitation speed. Better productivity was obtained for T. reesei grown in the STB and the highest productivity, 0.121 IU/mL h, was obtained at 400 rpm. The morphological parameter, the hyphal growth unit, was found to be correlated to the productivity. Understanding the effect of agitation on the morphology and productivity of T. reesei could lead to the design of better bioreactors and the selection of operating conditions of bioreactors to optimize the production of cellulase.     

Application of species richness estimators for the assessment of fungal diversity

Species richness and distribution patterns of wood-inhabiting fungi and mycetozoans (slime moulds) were investigated in the canopy of a Central European temperate mixed deciduous forest. Species richness was described with diversity indices and species-accumulation curves. Nonmetrical multidimensional scaling was used to assess fungal species composition on different tree species. Different species richness estimators were used to extrapolate species richness beyond our own data. The reliability of the abundance-based coverage estimator, Chao, Jackknife and other estimators of species richness was evaluated for mycological surveys. While the species-accumulation curve of mycetozoans came close to saturation, that of wood-inhabiting fungi was continuously rising. The Chao 2 richness estimator was considered most appropriate to predict the number of species at the investigation site if sampling were continued. Gray's predictor of species richness should be used if statements of the number of species in larger areas are required. Multivariate analysis revealed the importance of different tree species for the conservation and maintenance of fungal diversity within forests, because each tree species possessed a characteristic fungal community. The described mathematical approaches of estimating species richness possess great potential to address fungal diversity on a regional, national, and global scale.     

Application of beneficial rhizospheric microbes for the mitigation of seed-borne mycotoxigenic fungal infection and mycotoxins in maize

In the present investigation, seven rhizobacteria and nine Trichoderma spp. were evaluated to suppress seed-borne mycotoxigenic fungi (Aspergillus flavus and Fusarium verticillioides) and mycotoxin (aflatoxin and fumonisin) and to improve planting value of maize. Under in vitro conditions, these beneficial microorganisms suppressed the growth of A. flavus and F. verticillioides to various extents. Bacillus sp. (Bsp 3/aM), Pseudomonas putida (Has 1/c), Trichoderma asperellum (M5) and T. asperellum (T2) exhibited the greatest antagonistic effect on seed-borne mycotoxigenic fungi, and subsequently reduced mycotoxin concentrations in seeds. Under greenhouse conditions, these four biocontrol strains were also found to increase root length, shoot length, % germination, vigour index, fresh weight and dry weight of seedlings. Considering their overall performances, strains Bsp 3/aM, Has 1/c, M5 and T2 were selected for field studies as microbial talcum formulations. Among the tested microbial formulations, strain Bsp 3/aM significantly increased yield by 9.4% and 6.2% over the control in two maize cultivars Hema and Pearl, respectively. Increased plant growth and yield was also correlated with nutrient uptake in both the tested cultivars. All microbial formulation recorded significantly (p ≤ 0.05) reduced A. flavus infection and aflatoxin contamination in harvested seeds. But, none of the microbial formulations were found significant (p ≤ 0.05) in reducing F. verticilliodes incidence and fumonisin contamination. Our findings indicate that these microbial antagonists indirectly improve host health by suppressing seed-borne incidence of mycotoxigenic fungi and directly by facilitating nutrient uptake, thereby revealing their potential as both biofertilisers and biopesticides for maize production.     

Application of PCR for detection and identification of mycoplasma contamination in virus stocks

Summary A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.     

Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome

Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.     

The fluorescence lidar technique for the remote sensing of photoautotrophic biodeteriogens in the outdoor cultural heritage: A decade of in situ experiments

Fluorescence lidar is a non-invasive, remote sensing technique that makes it possible to extend the application of the laser-induced fluorescence technique to the outdoor environment, where uncontrolled external conditions must be met. Although initially developed for the investigation of marine environment and vegetation, in the past decade this technique has been successfully applied to the field of the cultural heritage. Among other applications, the detection and characterisation of photoautotrophic biodeteriogens has become very promising: the method is based on the detection of chlorophyll a fluorescence, while fluorescent accessory pigments can be exploited for a rough classification of the biodeteriogens present on the surface. Early experiments on monuments date back to the mid 1990s, when fluorescence lidar point measurements were conducted on the Cathedral and Baptistery of Parma, Italy. Subsequently, the technique has taken further advantage of the introduction of imaging capabilities in lidar instrumentation and this has led to the acquisition of hyperspectral fluorescence maps over extended areas of several monuments from distances as great as 80 m. Here, we present the main achievements obtained in the outdoor cultural heritage so far and the latest developments in the technique.     

Application of leftover sample material from waterborne protozoa monitoring for the molecular detection of Bacteroidales and fecal source tracking markers

In this study, we examined the potential for detecting fecal bacteria and microbial source tracking markers in samples discarded during the concentration of Cryptosporidium and Giardia using USEPA Method 1623. Recovery rates for different fecal bacteria were determined in sewage spiked samples and environmental waters using different group-specific and host-specific PCR assays. Bacteroidales DNA recovery ranged from 59 to 71% for aliquots of supernatant collected after the elution step. The recovery of human-specific Bacteroidales DNA from sewage spiked samples was 54% in the elution step. An additional 1-7% Bacteroidales DNA was recovered after the immunomagnetic separation step, while recovery from the pellet left after the immunomagnetic separation of protozoa parasites was substantially lower. Comparison of Bacteroidales 16S rRNA gene sequences from elution and immunomagnetic separation discarded samples indicated that the distribution of clones was not statistically different, suggesting that there were no recovery biases introduced by these steps. Human- and cow-specific Bacteroidales and fecal indicator bacteria (i.e., enterococci,) were also detected in the discarded fractions of environmental samples collected from different geographic locations. Overall, the results of this study demonstrated the potential application of leftover sample fractions that are currently discarded for the PCR detection of fecal bacterial indicators and molecular source tracking.     

Simple and easy method for the determination of fungal growth and decolourative capacity in solid media

A technique was developed for studying the biodegradative ability of white rot fungi in different solid media. This technique enables the gravimetric determination of fungal growth (increase of biomass) and the spectrometric measurement of fungal decolourization ability (both by the determination of the production of the extracellular enzyme manganese-dependent peroxidase (MnP) and by the rate of decolourization of dyes). Bjerkandera sp., strain BOS55, was grown in different solid media. Its growth rate, decolourization of solophenil blue 2BL (azoic dye), neutral red (eurhodin dye), methyl green and crystal violet (triphenylmethane dyes) and the production of MnP were determined. Application of this technique enabled a spectrometric quantification of enzymatic activity. Assays indicate that greater amounts of MnP were present in agar plate cultures of Bjerkandera sp. than in liquid cultures.     

A novel PCR-based approach for the detection and classification of potential cellulolytic fungal strains isolated from museum items and surrounding indoor environment

Aims: To develop a novel PCR‐based method able to detect potential cellulolytic filamentous fungi and to classify them exploiting the amplification of the cellobiohydrolase gene (cbh‐I) and its polymorphism. Methods and Results: A mixed approach including the combination of (i) fungal cultivation and isolation, (ii) classification of fungal isolates through the amplification of the cbh gene using a fluorescently labelled primer (f‐CBH‐PCR) and (iii) final fungal identification based on amplification and sequencing of the ITS1‐5.8S rDNA‐ITS2 region of the selected fungal strains was developed. By this approach, it was possible to screen 77 fungal strains belonging to 14 genera and 26 species. Conclusions: The f‐CBH‐PCR permitted the discrimination of fungal species, producing typical f‐CBH profiles. Significance and Impact of the Study: In this study, the cbh gene was used as a preliminary classification tool able to differentiate among themselves the fungal members isolated from indoor museum items and surrounding environment. Such mixed approach consented the fast identification of all isolated fungal strains. The f‐CBH‐PCR method demonstrated its discrimination power, and it can be considered as a new molecular system suitable for the classification of fungal strains isolated from different environments.     

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of na?ve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.     

Colorimetric paper bioassay by horseradish peroxidase for the detection of catechol and resorcinol in aqueous samples

Abstract

Phenolic compounds such as catechol and resorcinol are toxic and persistent pollutants in the aqueous environment. Detection procedures such as chromatographic and spectrophotometric methods are time-consuming and require sophisticated instruments with skilled manpower. Development of a simple, cost effective, portable and disposable paper based biosensor could be a better alternative to the conventional methods. The present study attempted to develop a paper based biosensor by immobilizing horseradish peroxidase enzyme to detect catechol and resorcinol in aqueous samples. Horseradish peroxidase catalyzes the oxidation of phenolic compounds to semiquinones, which on reaction with a chromogen, 3-methyl 2-benzothiazolinone hydrazine (MBTH) gives faint pink to red color depending on the compound and its concentration in the sample is the basis for biosensing application. Different methods of enzyme immobilization on filter paper like physical adsorption, covalent coupling, and polysaccharide entrapment were executed. The performance of the various enzyme immobilization methods was evaluated by analyzing the developed color intensity using ImageJ software. Entrapment technique is the most effective method of immobilizing enzyme on the filter paper that produces the highest color intensity with better stability. The visible limit of detection (LoD) was observed as 0.45?mM (50?mg/L) for catechol and 0.09?mM (10?mg/L) for resorcinol in aqueous samples.     

Application of dried whole blood collected on filter paper disks to ELISA for the detection of Sendai virus and mouse hepatitis virus antibodies in mice.

This study was undertaken to simplify the preparation procedures for test specimens by applying whole blood collected on filter paper disks. The results of ELISA obtained using specimens collected in this way for the detection of Sendai virus and mouse hepatitis virus antibodies in mice were comparable to those for ordinary ELISA using serum samples.