scholarly journals Diversity of methanogens in ruminants in Queensland

2008 ◽  
Vol 48 (7) ◽  
pp. 722 ◽  
Author(s):  
D. Ouwerkerk ◽  
A. F. Turner ◽  
A. V. Klieve
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Rumen Fluid ◽  
Bos Indicus ◽  
Methane Emissions ◽  
Molecular Techniques ◽  
Feed Conversion ◽  
Methanogen Community ◽  
Gradient Gel Electrophoresis ◽  
Reduce Emissions ◽  
Cattle And Sheep

Methane emissions from ruminant livestock represent a loss of carbon during feed conversion, which has implications for both animal productivity and the environment because this gas is considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in different breeds of cattle and sheep, as well as in response to different diets, is required. A study was undertaken using the molecular techniques denaturing gradient gel electrophoresis, DNA cloning and DNA sequence analysis to define the extent of diversity among methanogens in ruminants, particularly Bos indicus cross cattle, on differing forages in Queensland. It was found that the diversity of methanogens in forage-fed cattle in Queensland was greater than in grain-fed cattle but there was little variability in methanogen community composition between cattle fed different forages. The species that dominate the rumen microbial communities of B. indicus cross cattle are from the genus Methanobrevibacter, although rumen-fluid inoculated digestors fed Leucaena leucocephala leaf were populated with Methanosphaera-like strains, with the Methanobrevibacter-like strains displaced. If ruminant methane emissions are to be reduced, then antimethanogen bioactives that target both broad groups of ruminant methanogens are most likely to be needed, and as a part of an integrated suite of approaches that redirect rumen fermentation towards other more useful end products.

scholarly journals Rumen protozoa and methanogenesis: not a simple cause–effect relationship

2011 ◽  
Vol 107 (3) ◽  
pp. 388-397 ◽  
Author(s):  
Diego P. Morgavi ◽  
Cécile Martin ◽  
Jean-Pierre Jouany ◽  
Maria José Ranilla
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Methane Emissions ◽  
Rumen Protozoa ◽  
Methanogen Community ◽  
Gradient Gel Electrophoresis ◽  
Fermentation Parameters ◽  
Methyl Coenzyme M Reductase ◽  
Butyrate Production

Understanding the interactions between hydrogen producers and consumers in the rumen ecosystem is important for ruminant production and methane mitigation. The present study explored the relationships between rumen protozoa, methanogens and fermentation characteristics. A total of six donor sheep harbouring (F, faunated) or not (D, defaunated) protozoa in their rumens (D animals were kept without protozoa for a period of a few months (D − ) or for more than 2 years (D+)) were used inin vitroandin vivoexperiments.In vitrothe absence of protozoa decreased NH3and butyrate production and had no effect on methane. In contrast, the liquid-associated bacterial and methanogens fraction of D+ inocula produced more methane than D −  and F inoculum (P < 0·05).In vivofermentation parameters of donor animals showed the same trend on NH3and butyrate and showed that D+ animals were high methane emitters, while D −  were the lowest ( − 35 %). The concentration of dissolved dihydrogen measured after feeding followed the opposite trend. Methane emissions did not correlate with the relative abundance of methanogens in the rumen measured by quantitative PCR, but there was a trend for higher methanogens concentration in the solid-associated population of D+ animals compared with D −  animals. In contrast, PCR-denaturing gradient gel electrophoresis profiles of methanogens' methyl coenzyme-M reductase A gene showed a clear clustering in liquid-associated fractions for all three groups of donors but fewer differences in solid-associated fractions. These results show that the absence of protozoa may affect differently the methanogen community and methane emissions in wethers.

A bromochloromethane formulation reduces enteric methanogenesis in cattle fed grain-based diets

2009 ◽  
Vol 49 (12) ◽  
pp. 1053 ◽  
Author(s):  
N. W. Tomkins ◽  
S. M. Colegate ◽  
R. A. Hunter
Keyword(s):  
Methane Production ◽  
Bos Indicus ◽  
Methane Emissions ◽  
Control Group ◽  
Maximum Residue Limit ◽  
Australian Government ◽  
Feed Conversion ◽  
Enteric Fermentation ◽  
Edible Tissue ◽  
Australian Greenhouse

Enteric fermentation has been estimated to be responsible for 64.2 Mt CO2-e, or ~16% of Australia’s greenhouse gas emissions (Australian Greenhouse Office 2007). A bromochloromethane (BCM) formulation, previously shown to inhibit methanogenesis, was included in the diet of Brahman (Bos indicus) cross steers, twice daily in three separate experiments, to determine the effect on methane production, daily feed intake, liveweight (LW) gain and accumulation of residues of BCM in edible tissue. In the first experiment, the BCM formulation was fed at rates of 0, 0.15, 0.30, or 0.60 g/100 kg LW, twice daily, for 28 days. Methane production (mean ± s.e.), measured over 11 h after feed was first consumed on day 28, was 0.3 ± 0.13 and 0.1 ± 0.03 L/h for animals treated at a rate of 0.30 and 0.60 g/100 kg LW, respectively. This was significantly less (P < 0.05) than for control animals (4.6 ± 0.46 L/h) and animals treated at a rate of 0.15 g/100 kg LW (2.1 ± 0.28 L/h). The dose rate of 0.30 g/100 kg LW was associated with a decrease in methanogenesis by ~93% compared with the control group and was used in subsequent experiments. The second experiment evaluated the efficacy of the BCM formulation fed at rates of 0 or 0.30 g/100 kg LW, twice daily, for 90 days. Methane production was measured over 24-h periods, on days 30, 60 and 90. For days 30 and 90, methane production was reduced by 60% (P < 0.05) to 4.2 ± 1.82 L/h and by 50% (P < 0.05) to 6.1 ± 0.63 L/h, respectively, for treated animals compared with the control group. The final experiment determined the effect on LW gain and detectable residues in edible tissue, with animals given the BCM formulation at rates of 0 or 0.30 g/100 kg LW, twice daily, for 85 days. Liver, kidney, depot fat and muscle samples collected 1 and 10 days after the last day of treatment had concentrations of BCM that did not exceed 0.015 mg/kg and were less than the temporary maximum residue limit (0.02 mg/kg BCM), which applies to bovine meat, fat and edible offal. There were no significant differences in LW gain (1.4 ± 0.10 v. 1.5 ± 0.07 kg/day), feed conversion ratio (5.7 ± 0.32 v. 5.4 ± 0.09), hot carcass weight (235 ± 5.0 v. 250 ± 6.5 kg) or P8 fat depth (6.4 ± 0.89 v. 8.1 ± 1.15 mm) between control and treated animals. The experiments reported here were completed in 2004 before the Australian Government prohibited the manufacture and use of BCM. It is unlikely that the BCM formulation will be available for commercial use to mitigate livestock methane emissions in Australia. Nevertheless, the study has demonstrated that methane emissions were substantially reduced over a 90-day feedlot finishing period. This indicates that alternative antimethanogens with a similar mechanism of action may have practical commercial relevance.

Mitochondrial Mutations as Molecular Markers for Minimal Reidual Disease (MRD) in Acute Myeloid Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2996-2996
Author(s):  
Alexander A. Morley ◽  
Scott A. Grist ◽  
Xiao J. Lu ◽  
Katrina Patsouris
Keyword(s):  
Acute Myeloid Leukemia ◽  
Molecular Markers ◽  
Myeloid Leukemia ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Residual Disease ◽  
Molecular Techniques ◽  
Leukemic Cells ◽  
Mitochondrial Mutations ◽  
Gradient Gel Electrophoresis ◽  
Acute Myeloid

Abstract Measurement of minimal residual disease (MRD) is being increasingly used in haematological malignancies in order to assess prognosis and decide on treatment. However for some patients, including many patients with acute myeloid leukemia (AML), molecular techniques for MRD measurement cannot be performed owing to lack of a molecular marker. We have detected mitochondrial mutations (MM) in the D loop of the mitochondrial genome of the leukemic cells in approximately 40% of patients with AML and have investigated 2 techniques - denaturing gradient gel electrophoresis (DGGE) and single nucleotide primer extension (SNUPE) - to detect and quantify them. Mixing experiments showed that DGGE had a sensitivity of approximately 0.5% for detection of MM, and it was able to detect leukemia in remission marrow in 5 of 6 patients with AML. When present in a patient, MM are usually multiple. We therefore tested a 2-step strategy, as shown in the figure, which involved initial enrichment by PCR using primers directed at 1 or 2 flanking mutations followed by DGGE to detect an inner mutation. In 2 mixing experiments this 2-step strategy increased sensitivity of detection down to 0.0001% (this level of detection was evident in the original gel photo). Figure Figure SNUPE was more sensitive than DGGE and in 3 mixing experiments it showed a sensitivity of 0.02–0.05%. Four patients with AML have now been studied by SNUPE and the levels of MRD in the marrow documenting morphological remission at the end of induction therapy were 2.8%,16.1%,40.8% and 15.7%. The relatively high levels of MRD as measured by PCR in 3 patients suggest that some of the leukaemic cells at the end of induction may be more mature than blasts and thus not identifiable by morphology. While questions remain to be answered, mitochondrial mutations are promising molecular markers for quantifying MRD in AML and possibly other haematological disorders

scholarly journals Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques

2001 ◽  
Vol 67 (7) ◽  
pp. 2942-2951 ◽  
Author(s):  
Beatriz Dı́ez ◽  
Carlos Pedr�s-Ali� ◽  
Terence L. Marsh ◽  
Ramon Massana
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Phylogenetic Groups ◽  
Gradient Gel Electrophoresis ◽  
Phylogenetic Identification ◽  
Phylogenetic Composition ◽  
Primer Sets

ABSTRACT We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.

scholarly journals Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions

2000 ◽  
Vol 66 (10) ◽  
pp. 4372-4377 ◽  
Author(s):  
Bo Normander ◽  
Jim I. Prosser
Keyword(s):  
Community Composition ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Community Composition ◽  
Pcr Amplification ◽  
Molecular Techniques ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Populations ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis

ABSTRACT An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.

scholarly journals Elucidation of the microbial community structure within a laboratory scale activated sludge process using molecular techniques

2006 ◽  
Author(s):  
◽  
Pamela Padayachee
Keyword(s):  
Microbial Community ◽  
Activated Sludge ◽  
Denaturing Gradient Gel Electrophoresis ◽  
In Situ Hybridisation ◽  
Molecular Techniques ◽  
Laboratory Scale ◽  
Oligonucleotide Probes ◽  
Mass Balances ◽  
Nitrogen Levels ◽  
Gradient Gel Electrophoresis

The microbial community present in a laboratory-scale modified Ludzack-Ettinger activated sludge system was investigated using a combination of novel molecular techniques. The parent system was investigated for a duration of one year and samples were taken at regular intervals to determine the profile and structure of the microbial community present within the anoxic and aerobic zones of the MLE system. The combination of molecular techniques included fluorescent in situ hybridisation (FISH) and denaturing gradient gel electrophoresis (DGGE). FISH was performed using oligonucleotide probes, which were complementary to conserved regions of the rRNA for the alpha, beta and gamma subclasses of the gram negative family Proteobacteria as well as a group-specific HGC oligonucleotide probe as a representative of the gram positive actinomycetes branch. The total eubacteria present was determined using the EUB oligonucleotide probes, EUB388, EUB388-II and EUB388-III. The DGGE analysis of PCR-amplified 16S rDNA gene segments was used to examine the microbial community profile in the anoxic and aerobic zones. The profile for each of the zones revealed a number of consistent bands throughout the duration of the laboratory-scale process. However, the profiles obtained suggested that a diverse microbial community existed within the aerobic and anoxic zones. The bands also indicated the presence of dominant and less dominant species of bacteria. Hybridisations obtained from the FISH analyses indicated that the alpha and gamma subclasses were predominant within the anoxic zone and the aerobic zone showed a dominance of the beta subclass of Proteobacteria. The steady state behaviour of the MLE system was confirmed with the results obtained from COD, TKN, nitrates and OUR analytical tests. COD and nitrogen mass balances were conducted to confirm the acceptance of the results obtained for each batch as an indication of the system performance for the MLE model. Nitrogen mass balances indicated an upset in the nitrogen levels for batches two and seven.

scholarly journals A Brief History of Endophyte Detection Techniques in Grasses

2019 ◽  
Vol 8 (3) ◽  
pp. 66
Author(s):  
Rana Zaheer Ahmad ◽  
Fuad Ameen ◽  
Rida Khalid ◽  
Mousa A. Alghuthaymi ◽  
Reem Alsalmi ◽  
...  
Keyword(s):  
Molecular Phylogenetics ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Economic Value ◽  
Molecular Techniques ◽  
Morphological Identification ◽  
Detection Techniques ◽  
Molecular Approaches ◽  
Gradient Gel Electrophoresis ◽  
History Of ◽  
Traditional Approaches

Endophytes are the plant mutualists that live asymptomatically inside plant tissue and are found in nearly whole plant kingdom. Endophytic fungi receive shelter and nutrition from host plants and in return provide great advantages to the host. Grasses are a useful forage species and are of great agricultural and socio-economic value. The presence of endophytes in these grasses provide protection, persistence and improved yield against herbivores, insects, pathogens, drought and several other biotic and abiotic stresses. This review summarizes traditional and modern molecular techniques to identify endophytes from turf and forage grasses. Traditional approaches include direct observation, staining, laser micro dissection and pressure catapulting and cultivation-dependent methods that provide a morphological identification of endophytic mycobiota in grass tissues. Earlier studies on endophytes using these methods resulted in several technical implications which molecular approaches are able to solve now-a-days. Molecular approaches include DNA extraction, PCR based DNA Fingerprinting techniques, Denaturing Gradient Gel Electrophoresis, Sanger sequencing, Pyrosequencing, Immunoblot assay, Biosensors, DNA Barcoding and Molecular Phylogenetics etc. A comparison of these detection techniques will facilitate other researchers as well to develop new ways for the detection of endophytes that will contribute to the improvement of grassland in future.

Design of PCR Primers for the mcrA Genes Detection of Methanogen Communities

2011 ◽  
Vol 135-136 ◽  
pp. 408-413 ◽  
Author(s):  
Nguyen Ngoc Tuan ◽  
Shir Ly Huang
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Biogas Production ◽  
Pcr Primers ◽  
Molecular Techniques ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Mcra Gene ◽  
Catabolic Genes ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis

Methanogens play an important role to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent green house gases, during organic matter degradation in anaerobic environments. Therefore, it is necessary to better understand microorganisms that produce natural gas. Indeed, methanogens are difficult to perform through culture based methods. In addition, the culture independent methods using the 16S rRNA gene also revealed some disadvantages. For these reasons, the culture independent molecular techniques using the specific catabolic genes such as methyl coenzyme M reductase (MCR) were studied. In this study, a primer set which can amplify specific fragments from a wide variety of mcrA gene was designed based on the homologous regions of 100 mcrA genes listed in the GenBank. PCR with the mcrA primers amplified DNA fragments of the expected size from all the six samples which obtained from biogas production reactors. In addition, denaturing gradient gel electrophoresis PCR analysis using our designed primers also revealed the diversity of mcrA gene in each sample. These results revealed that our primers were successfully to detect the mcrA genes and it is also helpful to know the diversity of mcrA genes in methanogen communities.

Biofilm dynamics studied with microsensors and molecular techniques

1998 ◽  
Vol 37 (4-5) ◽  
pp. 125-129 ◽  
Author(s):  
Cecilia M. Santegoeds ◽  
Gerard Muyzer ◽  
Dirk de Beer
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Treatment Plant ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Biofilm Growth ◽  
Sulfide Production ◽  
Gradient Gel Electrophoresis ◽  
Anoxic Zones ◽  
Biofilm Community

Here we present preliminary data on the development of a biofilm from a wastewater treatment plant studied with microsensors and molecular techniques. The development during biofilm growth of oxygen, sulfide and pH profiles was measured with microsensors. Anoxic zones developed within one week and further increased during the following weeks. However, sulfide production was delayed and was first detected in a six-week-old biofilm. With denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments the sequence of the bacterial community was followed showing an increasing complexity of the biofilm community during development. In a mature biofilm the influence of nitrate on sulfide production was studied by measuring oxygen, sulfide, pH, nitrite and nitrate profiles with microsensors. Sulfide production was detected deeper in the biofilm and in lower concentrations, when nitrate was added to the medium. The DGGE pattern of the mature biofilm showed both differences and similarities with the DGGE pattern of the 12-week-old biofilm. In particular the RNA pattern changed when nitrate was added to the medium, indicating a change in activity of certain strains.

Impact of oxygen on the coexistence of nitrification, denitrification, and sulfate reduction in oxygen-based membrane aerated biofilm

2015 ◽  
Vol 61 (3) ◽  
pp. 237-242 ◽  
Author(s):  
Hong Liu ◽  
Shuying Tan ◽  
Zhiya Sheng ◽  
Tong Yu ◽  
Yang Liu
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Aerobic Oxidation ◽  
Bulk Water ◽  
Biofilm Reactor ◽  
Molecular Techniques ◽  
Bulk Liquid ◽  
Rrna Gene ◽  
Ammonia Oxidizing Bacteria ◽  
Microbial Composition ◽  
Gradient Gel Electrophoresis

Membrane aerated biofilms (MABs) are subject to “counter diffusion” of oxygen and substrates. In a membrane aerated biofilm reactor, gases (e.g., oxygen) diffuse through the membrane into the MAB, and liquid substrates pass from the bulk liquid into the MAB. This behavior can result in a unique biofilm structure in terms of microbial composition, distribution, and community activity in the MAB. Previous studies have shown simultaneous aerobic oxidation, nitrification, and denitrification within a single MAB. Using molecular techniques, we investigated the growth of sulfate-reducing bacteria (SRB) in the oxygen-based MAB attached to a flat sheet membrane. Denaturing gradient gel electrophoresis of the amplified 16S rRNA gene fragments and functional gene fragments specific for ammonia-oxidizing bacteria (amoA), denitrifying bacteria (nirK), and SRB (dsrB) demonstrated the coexistence of nitrifiers, denitrifiers, and SRB communities within a single MAB. The functional diversities of SRB and denitrifiers decreased with an increase in the oxygen concentration in the bulk water of the reactor.

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