Geosmin is an odorous secondary metabolite produced by some cyanobacteria during growth and released from the cells. Little is known about the biosynthesis of geosmin and the gene(s) required for its production have not been characterized. During late August and early September geosmin episodes due to planktonic cyanobacteria frequently occur in the northwest basin of Lake Ontario waters resulting in taste and odour episodes in drinking water that serves more than 5 million people. At high concentrations geosmin evades traditional drinking water treatment and reaches the tap. These episodes often elicit consumer concern and are wrongly construed to reflect impaired drinking water safety. Water quality managers in the region have generally been unable to prevent or control taste and odour episodes via a proactive approach due to the lack of knowledge of cyanobacterial communities in offshore waters as well as the inability to predict when geosmin will reach intake pipes due to downwelling, the process by which the surface waters mix with the hypolimnion. This study evaluated denaturing gradient gel electrophoresis (DGGE) as a molecular tool for proactive monitoring of potential taste and odour-causing cyanobacteria in environmental samples. The 16S rRNA gene was assessed for its ability to distinguish among geosmin-producing and non-producing strains. This study also examined the evolutionary relationships among geosmin-producing cyanobacteria using the full-length 16S rRNA gene and compared phylogenies with current taxonomy. <br /><br /> A DGGE standard using the V3 hypervariable region of the 16S rRNA gene was developed using geosmin-producing and non-producing isolates of cyanobacteria. Included in the standard was the suspected primary contributor to Lake Ontario taste and odour, <em>Anabaena lemmermannii</em> Richter. This standard was then applied to various environmental collections from Lake Ontario (August 2005) to examine the cyanobacterial community composition. DGGE profiles were consistent with the presence of <em>An. lemmermannii</em> at locations with increased geosmin concentrations (determined using gas chromatography-mass spectrometry), supporting hypothesis that <em>An. lemmermannii</em> is the primary contributor to northwestern Lake Ontario taste and odour. In addition, the application of DGGE in the identification of potential geosmin-producing species of cyanobacteria was deemed to be a potentially useful approach to monitoring cyanobacterial communities in source waters. The 16S rRNA-V3 region alone did not distinguish among geosmin-producing and non-producing strains, however with additional data (actual geosmin concentration) it was showed relationships. <br /><br /> In the phylogenetic analyses, geosmin-producing cyanobacteria did not group monophyletically and it was not possible to state that a single evolutionary event has led to the acquisition of the geosmin-producing trait. Phylogenies also showed that the taxonomy of the Cyanobacteria is largely unresolved. All five Sections (bacteriological classification)/four orders (Komárek & Anagnostidis classification) were paraphyletic, however the heterocystous cyanobacteria (Sections IV and V/Nostocales and Stigonematales) grouped separately from the non-heterocystous cyanobacteria (Sections I, III/Chroococcales and Oscillatoriales). Although both systems of classification compared in this study were similar, nomenclature and groupings were occasionally different among the groups. This demonstrates the incongruity between bacteriologists and phycologists and emphasizes the need for a consensus system of classification for the Cyanobacteria.

The animal phylogeny and feeding habits would affect the composition of gastrointestinal tract¡]GI tract¡^microbiota. GI tract microbiota plays an important role in host health and nutrient provision. In this study, we used PCR-DGGE and bacterial 16S rDNA sequencing to analyze the GI tract bacterial diversity of four animals with different feeding habits in Shou-Shan zoo, including one carnivore, one omnivore and two herbivores, in which one ruminant and one non-ruminant. The results show a great difference between GI tract bacterial diversity of the four animals. The abundance of GI tract bacterial diversity increased from carnivore, omnivore to herbivore. Comparing the similarity of the GI tract bacterial community structures of these four animals, the carnivore possessed the most different composition, to other animals, the next was the omnivore, while the two herbivores show the highest similarity to each other. Our results also indicated that the GI tract microbiota of these four different animals were very stable during the investigating period. We also found that two individuals of the same species had a very similar bacterial compositions in their GI tracts at different time point. This finding indicated that the bacterial compositions of GI tract in the four animals were affected mostly by the host phylogeny and their feeding habits. Moreover, according to bacterial 16S rDNA sequencing and idencification, results show that the Firmicutes were the dominant bacterial phyum in all four animals GI tracts, the amount of Bacteroides was much less than Firmicutes. This result might caused by the highly starch content in their feed. Large amount of carbohydrate-degrading, protein-degrading, lipid-degrading bacteria were found in all of these different animals. Fiber-degrading bacteria Fibrobacteres were identified in the GI tracts of the herbivores and omnivore, but not the carnivore, showing that GI tract microbiota plays an important role to provide nutrient and assist energy to the host.

Leaking of petroleum products from storage tanks is a commonly found cause of soil contamination. Among those petroleum products, diesel-oil contaminated soils are more difficult to treat compared to gasoline (a more volatile petroleum product). With the growing interest in environmental remediation, various approaches have been proposed for treating petroleum-hydrocarbon (PH) contaminated sites. Given that it is often not possible to remove the released oil or remediate the site completely within a short period of time, using the in situ remedial technology, soil excavation followed by more cost-effective technology should be applied to accelerate the efficiency of site cleanup. In the first-part of this study, laboratory degradation experiments were conducted to determine the optimal operational conditions to effectively and economically bioremediate diesel-fuel contaminated soils. In the second part of this study, a combined full-scale landfarming and biopile system was operated to cleanup diesel fuel-contaminated soils. In the laboratory study, except of frequent soil tilling for air replacement, different additives were added in the laboratory bioreactors to enhance the total petroleum hydrocarbon (TPH) removal efficiency. The additives included nutrients, TPH-degrading bacteria, activated sludge, fern chips, and kitchen waste composts. PH-degrading bacteria were isolated from PH-contaminated soils and activated sludge was collected from a wastewater treatment plant containing PH in the influent. PH-degrading bacteria and sludge were added to increase the microbial population and diversity. Fern chips and kitchen waste composts were added to increase the soil permeability. Results indicate that the bioreactor with kitchen waste compost addition had the highest TPH removal rate. The observed TPH-removal ratios for the compost, activated sludge, PH-degrading bacteria, fern chips, nutrients, TPH-degrading bacteria addition, and control (with HgCl2 addition) groups were 80.5%, 78.6%, 77.4%, 75.1%, 73.3%, 66.1%, and 1.6% respectively. In the field study, activated sludge was selected as the additive from the engineering point of view. With the addition of activated sludge, an increase of 20% was observed for TPH removal ratio. Results from the denaturing gradient gel electrophoresis (DGGE) tests show that the detected PH-degrading bacteria in the activated sludge included the following: Pseudomonas sp., Pseudoxanthomonas sp., Rhodocyclaceae bacterium, Variovorax sp., Acidovorax sp., Leptothrix sp., Alcaligenaceae bacterium, and Burkholderia sp. Some of these bacteria became dominant species in the field after a long-term operation, which was beneficial to the soil bioremediation. Results indicate that the in situ bioremediation has the potential to be developed into an environmentally and economically acceptable remediation technology.

Abstract Based on maintaining healthy soil for sustainable agriculture and enhancing banana disease resistance, Taiwan Banana Research Institute began to conduct organic cultivation on a trial basis in 1998. It had been proved that the morbidity of banana Fusarial wilt disease at organic cultivation plots was significantly lower than that of conventional farming. In order to study the differences of soil microbiota between the organic cultivation plots and the conventional farming areas, physical and chemical properties of the rhizosphere and non- rhizosphere soil samples were assayed during the period of Aug. 2010 to May 2011. The bacterial diversity was analyzed by molecular biology methods, including PCR-DGGE to separate the 16S rDNA V6 ~ V8 region of various bacteria and the recombinant DNA technology by using pGEM-T Easy Vector System to separate and sequence the DNA fragments. The results showed that organic plots was loam soil, but the conventional farming soil was sandy loam with higher sand content. The soil pH in 13 years organic area was mildly alkaline, but in conventional farming area was mildly acidic to slightly acidic. The content of various nutrients in organic 13-year area soil was not necessarily higher than the conventional farming area soil. The available nutrient contents in organic areas trend to be more stable than that in the conventional areas. Fertilization may affect the content of available nutrients in the soil. No bacterial DNA could be extracted from the organic fertilizer. The bacterial microbiota in soil was very stable, and was not related to the sampling seasons. The Banana strains had little effect on soil bacterial microbiota. There was no difference on the bacterial microbiota between the rhizosphere and non-rhizosphere soil samples. It is not sure whether there were any differences on the bacterial microbiota between the nearby soil of banana Fusarial wilt plants and the nearby soil of the healthy plants. By analyzing the DNA fragment clone library, 43 strains correspond to known category, of which 28 belonged to the Proteobacteria, and 34 were uncultured strains. The role of these microbial strains might involve in various element cycles, such as N cycles, C cycles, and S cycles (including some photosynthetic bacteria). The systematic cladogram showed that organic 13-year areas, organic 3-year areas and conventional farming areas represented three major categaries. The organic 13-year area and conventional area possessed the highest difference on the microbiota composition.

Constructed wetlands had been used for water treatment worldwide. The efficiency of wastewater treatment in a constructed wetland depends on its design, types of aquatic plants and microbial community present in this wetland. The goal of this study is to analyze the microbial populations in KaoPing River Rail Bridge constructed wetland which was designed to remove the polluted material from municipal sewage and industrial wastewater. Sediment and water samples were collected every 3 months from April, 2007 to April, 2008. The bacterial community diversities were analyzed by PCR-DGGE of the bacterial 16S rRNA gene. Results show approximately 60% BOD, 41% COD, 46% nitrate, 22% total nitrogen, and 97% coliforms were removed by this wetland system. DGGE profiles revealed the bacterial community diversities shifted progressively from the entry to the exit of both A and B systems in this wetland. The microbial populations in water, sediment, biofilms on plants, and soil were quite different from each others. The fecal indicator Escherichia coli was used as a marker to monitor the fecal contamination in all samples. From PCR-DGGE profiles, E. coli could be successfully removed by this wetland system. In conclusion, this constructed wetland is a very successful system for wastewater treatment and is able to remove most of the pollutants before they are discharged into KaoPing River. The results of this study provided useful suggestions for the government to assess the bacterial diversities and the efficiency of this wetland system, to protect people from hazardous risks, and to manage a constructed wetland in the future.

Degradation of the several million tons of solid waste produced in the U.S. annually is microbially mediated, yet little is known about the structure of prokaryotic communities actively involved in the waste degradation process. In the first study, leachates generated during degradation of municipal solid waste (MSW) in the presence (co-disposal) or absence of biosolids were analyzed using laboratory-scale bioreactors over an eight-month period. Archaeal and bacterial community structures were investigated by denaturing gradient gel electrophoresis (DGGE) targeting 16S rRNA genes. Regardless of waste composition, microbial communities in bioreactor leachates exhibited high diversity and temporal trends. Methanogen sequences from a co-disposal bioreactor were predominantly affiliated with the orders Methanosarcinales and Methanomicrobiales. Effect of moisture content on indicator organism (IO) survival during waste degradation was studied using culture-based methods. Fecal coliform and Enterococcus concentrations in leachate decreased below detection limits within fifty days of bioreactor operation during the hydrated phase. IOs could be recovered from the bioreactor leachate even after a prolonged dry period. This study advances the basic understanding of changes in the microbial community during solid waste decomposition. The purpose of the second study was to compare the ability of BOX-PCR to determine genetic relatedness with that of the "gold standard" method, 16S rRNA gene sequencing. BOX-PCR typing could clearly differentiate the strains within different Enterococcus species but closely related genera were not as distinguishable. In contrast, 16S rRNA gene sequencing clearly differentiates between closely related genera but cannot distinguish between different strains of Enterococcus species. This study adds to our knowledge of genetic relationships of enterococci portrayed by two separate molecular methods. The incidence of vancomycin resistant enterococci (VRE) in environmental matrices, residential and hospital wastewater was also investigated. Low-level VRE ( vanC genotype) were isolated from environmental matrices and residential wastewater. VRE isolates from hospital wastewater were identified as E. faecium and demonstrated resistance to ampicillin, ciprofloxacin and vancomycin (vanA genotype), but sensitivity to chloramphenicol and rifampin. Although no high-level VRE were isolated from surface waters, the high proportion of low-level VRE in environmental matrices is a cause for concern from the public health perspective.

<p>The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity (&gt / 1%) was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study.</p>

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While forage plays an important role in equine nutrition, little research has been conducted evaluating fiber utilization by young horses. Therefore, studies were conducted to compare in vivo digestibility and digesta passage in weanlings and mature horses (Exp 1) and yearlings and mature horses (Exp 2). All horses were fed forage-based diets at the same rate (on a metabolic BW basis; Exp 1: 67% alfalfa cubes, 33% concentrate; Exp 2: 75% timothy cubes, 25% concentrate). Ytterbium labeled hay and cobalt-ethylenediaminetetraacetic acid were used to estimate digesta mean retention time (MRT), while in vivo digestibility (DM, OM, and NDF) was measured using a total fecal collection method. Feed and water intake was similar between young and mature horses in both experiments. In Exp 1, there were no differences in digestibility or MRT due to age. The results suggested that weanling horses are capable of digesting a relatively high quality diet as efficiently as mature horses and that most of the development of the gastrointestinal tract occurs before 6 mo of age. In Exp 2, digestibility estimates were greater (P < 0.0311) for the yearlings than for the geldings. The increased digestive capacity of the yearlings was likely due to the longer MRT observed for the particulate phase in the yearlings (P = 0.0190). A third study was conducted to compare the microbial profiles of the feces of mares and foals. Fecal samples were collected from mare-foal pairs as the foal matured. The profiles of each pair, obtained using polymerase chain reaction-denaturing gradient gel electrophoresis, were compared and used to describe bacterial colonization in the foal. Mean similarity between mares and their foals on the day of parturition was low, but rapidly increased. Within 2 wk of parturition, similarity among mares and their foals was higher than among mature mares, suggesting that by 2 wk of age the bacterial species found in the foals’ gut are similar to those found in the mature horse. Collectively, the results from this series of experiments describe the early development of the foal’s digestive capacity.

Toxic cyanobacterial blooms constitute a threat to the safety and ecological quality of aquatic environments worldwide. Cyclic hepatotoxin, especially microcystin, is the most widely occurring of the cyanotoxins. The aim of this study was to identify the cyanobacterial genotypes present including how many toxic genotypes were present in two North American lakes and one African Lake. All three lakes are prone to cyanobacterial blooms and were sampled in 2005 and 2006: Lake Ontario (Bay of Quinte, Canada), Lake Erie (Maumee Bay, Canada) and Lake Victoria (Nyanza Gulf, Kenya). The cyanobacterial genotypic community was assessed using DNA based analyses of the hypervariable V3 region of the 16S rRNA gene. In addition, the aminotransferase (AMT) domain in modules mcyE and ndaF of the microcystin and nodularin gene cluster respectively was used to detect the presence of hepatotoxic genotypes. Denaturing gradient gel electrophoresis (DGGE) results from this study suggested that hepatotoxin producers were present in all study sites sampled and were most likely members of the genus Microcystis. This study was the first to report the potential for microcystin production in the in-shore and off-shore open lake of Nyanza Gulf in Kenya. A seasonal study of the Bay of Quinte and Maumee Bay showed differences in the cyanobacterial genotypic community from early to late summer. In addition, the cyanobacterial genotypic community from the Bay of Quinte differed from 2005 to 2006 and quantification of the North American samples revealed an increase in cyanobacterial cells from early to late summer. The Bay of Quinte saw relatively no change in hepatotoxic cells from early to late summer but in Maumee Bay hepatotoxic cells increased from undetectable in early summer to dominating the cyanobacterial community by late summer. This study demonstrated the use of DGGE and qPCR of the 16S rRNA-V3 and AMT gene region in monitoring the cyanobacterial community of waterbodies susceptible to toxic cyanobacterial blooms.