The fish disease epidemiology is urgent to be investigated for the surveillance and prevention. The diseased fish showed splenomegaly with diffusion of white nodules and microscopical granulomatous formation. It is important to develop a method of pathogens isolated from clinical samples with serial dilution method and disc diffusion method. Representative colonies were selected from diseased cobia on BHIA plate and were inoculated onto MacConkey agar, TCBS agar, and blood agar. The cage-culture of the different bacterial groups detected in the survey of bacteria isolated from THOD, HDSB, EMD with serial dilution method. 119 from 128 isolated strains were Gram¡¦s negative (93%), including pathogenic Vibrio spp. 57% (73/128) in THOD. 54 from 90 isolated strains were Gram¡¦s negative (60%), including pathogenic Vibrio spp. 12.2% (11/90) in HDSB. 61 from 104 isolated strains were Gram¡¦s negative (59%), including pathogenic Vibrio spp. 70.2% (77/90) in EMD. In different times diseased grouper, 104 from 139 isolated strains were Gram¡¦s negative (75%), including pathogenic Vibrio spp. 88% (123/139), in 2003. While 24 from 44 isolated strains were Gram¡¦s negative (55%), including pathogenic Vibrio spp. 73% (32/44), in 2004. 66 from 75 isolated strains were Gram¡¦s negative (88%), including pathogenic Vibrio spp. 97% (73/75) by disc dilution method in EMD. 9 from 31 isolated strains were Gram¡¦s negative (30%), including pathogenic Vibrio spp. 26% (8/31), by disc dilution method of grouper in PCG. A DGGE (denaturing gradient gel electrophoresis) technique can identify six groups of bacteria from cobia, and J6, R13, T29 have similarity 100%. Quantity One Version 4.5 (Bio-Rad) can identify six groups of bacteria from diffusion methods that F group diluted the bacterial strain from serial dilution method. B group and E group diluted the bacterial strain from disc diffusion method. Higher resistance rates of the different bacterial strains isolated from cobia and were £]-lactam and susceptible were observed in quinolones.

This study investigated the microbiota in ten different drinking water treatment pools, particles in the Biological Activated Carbon Filtration (BACF) bed, and two mimic columns in the Cheng-Ching Lake Water Treatment Plant. Assimilable organic carbon (AOC) is one of the main nutrition sources for microbes to survive in tap water. Over growing microbes not only decrease the water quality, but also contaminate the water treatment system and distribution system. In this study, we used two molecular biology techniques, the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE), to analyze the dynamic microbial communities and biodiversities in the drinking water cleaning system and the micorbiota that exist in the BAC and anthracite filtration pellets. The bacterial 16S rDNA sequences resulted from PCR-DGGE were compared with the data in the Ribosomal Database Project Bank to construct a phylogenetic tree which allowed us to understand the microbial communities and biodiversities in the drinking water treatment pools and the filtration pellets. The total bacterial count and PCR-DGGE profiles showed that the drinking water quality had been improved during the treating processes and most of the microbes in raw water were removed. The scanning electron microscopy clearly indicated the biofilms were developed on the pellet surface. From the mimic column studies, the PCR-DGGE profiles suggested that various microbial communities were present on different depth of the columns samples. In comparing the 16S rDNA sequences with Gene Bank, many are new category bacteria were found and most of them are unculturable. Most of these microbes belong to the beta-proteobacterium. Although many bacteria were located on the surface of the filtration pellet, the BAC and anthracite could still absorb AOC efficiently to enhance the bacteria growth. The over growing bacteria might release out and contaminate the drinking water. Therefore, we suggest that it is important to backwash the filter bed frequently in order to diminish microbes of the filtration pellet and avoid re-contaminate the drinking water.

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.

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.

<p>Allergic diseases, for example asthma and eczema, are nowadays considered belonging to the most common chronic diseases amongst children in the West, but the cause for this increase in allergy prevalence is unknown. Since studies have indicated a connection between children's exposure of microorganisms during infancy and risk of developing allergic disease, it is suggested that this exposure is a crucial factor in question of allergy development or not. Other studies have established differences in microflora composition between healthy children and children with allergic disease, and several studies have shown that probiotic therapy can give positive results in both prevention and treatment of allergic diseases.</p><p>The aim of this master's thesis was to develop a method, using PCR and denaturing gradient gel electrophoresis, to study the diversity of <em>Lactobacillus spp</em>. in fecal samples retrieved from a study of the probiotic strain<em> L. reuteri</em> ATCC 55730. The developed method was successful in detecting lactobacilli in fecal samples, but three other bacterial genera commonly found in humans were also amplified. Comparison of average numbers of detected bacterial strains and lactobacilli strains between samples belonging to the probiotics and placebo groups, respectively, showed higher numbers for the probiotics group. Also, the only fecal samples that contained <em>L. reuteri</em> belonged to the probiotics group. Although the results are far from statistically significant, they support the theories that probiotics may influence the intestinal microbiota.</p>

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.

There is currently growing concern about the presence of heterotrophic plate count (HPC) bacteria in drinking water. These HPC may have potential pathogenic features, enabling them to cause disease. It is especially alarming amongst individuals with a weakened immune system. South Africa, the country with the highest incidents of HIV positive individuals in the world, mainly uses these counts to assess the quality of drinking water in terms of the number of micro-organisms present in the water. These micro-organisms may be present in the bulk water or as biofilms adhered to the surfaces of a drinking water distribution system. The current study investigated the pathogenic potential of HPC bacteria occurring as biofilms within a drinking water distribution system and determined the possible presence of these micro-organims within the bulk water. Biofilm samples were taken from five sites within a drinking water distribution system. Fifty six bacterial colonies were selected based on morphotypes and isolated for the screening of potential pathogenic features. Haemolysin production was tested for using sheep-blood agar plates. Of the 56, 31 isolates were ?-haemolytic. Among the 31 ?-haemolytic positive isolates 87.1% were positive for lecithinase, 41.9% for proteinase, 19.4% for chondroitinase, 9.7% for DNase and 6.5% for hyaluronidase. All of the ?-haemolytic isolates were resistant to oxytetracycline 30 ?g, trimethoprim 2.5 ?g and penicillin G10 units, 96.8% were resistant to vancomycin 30 ?g and ampicillin 10 ?g, 93.5% to kanamycin 30 ?g, 74.2% to chloramphenicol 30 ?g, 54.8% to ciprofloxacin 5 ?g, 22.6% to streptomycin 300 ?g and 16.1% to erythromycin 15 ?g. Nineteen isolates producing two or more enzymes were subjected to Gram staining. The nineteen isolates were all Gram-positive. These isolates were then identified using the BD BBL CRYSTALTM Gram-positive (GP) identification (ID) system. Isolates were identified as Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus and Kocuria rosea. 16S rRNA gene sequencing was performed to confirm these results and to obtain identifications for the bacteria not identified with the BD BBL CRYSTALTM GP ID system. Additionally identified bacteria included Bacillus thuringiensis, Arthrobacter oxydans and Exiguobacterium acetylicum. Morphological properties of the different species were studied with transmission electron microscopy (TEM) to confirm sequencing results. All the isolates displayed rod shaped cells with the exception of Arthrobacter oxydans being spherical in the stationary phase of their life cycle. Bulk water samples were taken at two sites in close proximity with the biofilm sampling sites. The DNA was extracted directly from the water samples and the 16S rRNA gene region was amplified. Denaturing gradient gel electrophoresis (DGGE) was performed to confirm the presence of the isolates from the biofilm samples in the bulk water samples. The presence of Bacillus pumilus and Arthrobacter oxydans could be confirmed with DGGE. This study demonstrated the presence of potentially pathogenic HPC bacteria within biofilms in a drinking water distribution system. It also confirmed the probable presence of two of these biofilm based bacteria in the bulk water. / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2010.

Despite emerging water shortages, most water is only used once, and often with low efficiency. However, with appropriate treatment, water can be re–used to reduce the demand on freshwater sources. The Department of Water Affairs, South Africa, promotes industries to reduce discharges into water resources in order to sustain an overall good water quality of all water systems. All of this ultimately leads to industries striving towards zero effluent discharge. Primary Column Bottoms (PCBs) is a wastewater stream derived from the Fischer–Tropsch Gas to Liquid process and consists mainly of organic acids, but no nitrogen or phosphorous, which by implication excludes possible biodegradation. In the operation of cooling towers in industrial processes, cooling water quality has a direct impact on the cooling performance of the system, where nutrient levels may affect fouling, scaling and corrosion observed in the cooling towers. Fouling, scaling and corrosion affect the operating efficiency of cooling water systems and may necessitate the addition of chemical agents to control these phenomena. This has a financial and labour time impact on the operation of these systems. In this study a mini cooling tower test rig was operated with a synthetic PCB effluent as cooling water and various cycles of concentration, pH and linear flow velocities (LFVs). A constant delta temperature of 10 °C was maintained. Cycles of concentration (COC) evaluated included 2, 4 and 6 cycles of concentration and linear flow velocities evaluated was 0.6 m/s, 0.9 m/s and 1.2 m/s. Fouling, scaling and corrosion rates were determined using corrosion coupons and heat exchanger tubes for mild steel and stainless steel. Besides the evaluation of the various operational parameters for fouling, scaling and corrosion, the possibility for chemical oxygen demand (COD) removal by operating the cooling tower as a bioreactor was also evaluated. To this end nutrient correction was applied to the reactor to allow for a CNP ratio of 100:10:1. With regard to fouling, scaling and corrosion, mild steel was more affected by fouling, scaling and corrosion compared to stainless steel where almost no fouling, scaling and corrosion was observed. Overall increased linear flow velocities resulted in higher fouling and scaling rates, whereas lower linear flow velocities resulted in decreased corrosion rates. In terms of cycles of concentration, increased COC resulted in higher fouling, scaling and corrosion rates. Despite the high nutrient removal levels, the accompanying fouling, scaling and corrosion was still below the particular industry’s guidelines. Besides physical–chemical evaluation of the towers under the various operational conditions, culture–dependent and culture–independent methods were also employed. Concerning culture–dependent approaches the study demonstrated that aerobic and anaerobic organisms are present in both the planktonic and sessile phase of the cooling tower reactors. Heterotrophic aerobes were found to be the most abundant under all the operating conditions. Sulphate reducing bacteria were more abundant in the sessile phase of the cooling towers, and the presence of high sulphate levels in the experiments could be indicative of the sulphate reducing bacteria actively participating in the microbial community. Lower than expected corrosion levels, however, suggest that a combination of the organisms in the biofilm rather than sulphate reducing bacteria alone, contributed to the corrosion rates observed. Culture–independent methods, specifically phospholipid fatty acid analysis supported the results from the culture–dependent methods. Furthermore results demonstrated that linear flow velocity had a greater effect on the community structure than cycles of concentration. Finally molecular methods, specifically denaturing gradient gel electrophoresis, found that increasing cycles of concentration resulted in increased microbial community diversity, while increasing linear flow velocity resulted in decreased microbial community diversity. Regarding COD removal, nutrient correction of the synthetic PCB effluent achieved 89.35 % COD removal at 2 COC and 1.2 m/s LFV, while 80.85 % COD removal was achieved at 4 COC at 1.2 m/s LFV. From these results it was recommended that the operation of the cooling tower should be at 4 COC and 1.2 m/s, which despite slightly lower % COD removal, were characterised by fouling, scaling and corrosion rates well within guidelines. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2012.

There is currently growing concern about the presence of heterotrophic plate count (HPC) bacteria in drinking water. These HPC may have potential pathogenic features, enabling them to cause disease. It is especially alarming amongst individuals with a weakened immune system. South Africa, the country with the highest incidents of HIV positive individuals in the world, mainly uses these counts to assess the quality of drinking water in terms of the number of micro-organisms present in the water. These micro-organisms may be present in the bulk water or as biofilms adhered to the surfaces of a drinking water distribution system. The current study investigated the pathogenic potential of HPC bacteria occurring as biofilms within a drinking water distribution system and determined the possible presence of these micro-organims within the bulk water. Biofilm samples were taken from five sites within a drinking water distribution system. Fifty six bacterial colonies were selected based on morphotypes and isolated for the screening of potential pathogenic features. Haemolysin production was tested for using sheep-blood agar plates. Of the 56, 31 isolates were ?-haemolytic. Among the 31 ?-haemolytic positive isolates 87.1% were positive for lecithinase, 41.9% for proteinase, 19.4% for chondroitinase, 9.7% for DNase and 6.5% for hyaluronidase. All of the ?-haemolytic isolates were resistant to oxytetracycline 30 ?g, trimethoprim 2.5 ?g and penicillin G10 units, 96.8% were resistant to vancomycin 30 ?g and ampicillin 10 ?g, 93.5% to kanamycin 30 ?g, 74.2% to chloramphenicol 30 ?g, 54.8% to ciprofloxacin 5 ?g, 22.6% to streptomycin 300 ?g and 16.1% to erythromycin 15 ?g. Nineteen isolates producing two or more enzymes were subjected to Gram staining. The nineteen isolates were all Gram-positive. These isolates were then identified using the BD BBL CRYSTALTM Gram-positive (GP) identification (ID) system. Isolates were identified as Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus and Kocuria rosea. 16S rRNA gene sequencing was performed to confirm these results and to obtain identifications for the bacteria not identified with the BD BBL CRYSTALTM GP ID system. Additionally identified bacteria included Bacillus thuringiensis, Arthrobacter oxydans and Exiguobacterium acetylicum. Morphological properties of the different species were studied with transmission electron microscopy (TEM) to confirm sequencing results. All the isolates displayed rod shaped cells with the exception of Arthrobacter oxydans being spherical in the stationary phase of their life cycle. Bulk water samples were taken at two sites in close proximity with the biofilm sampling sites. The DNA was extracted directly from the water samples and the 16S rRNA gene region was amplified. Denaturing gradient gel electrophoresis (DGGE) was performed to confirm the presence of the isolates from the biofilm samples in the bulk water samples. The presence of Bacillus pumilus and Arthrobacter oxydans could be confirmed with DGGE. This study demonstrated the presence of potentially pathogenic HPC bacteria within biofilms in a drinking water distribution system. It also confirmed the probable presence of two of these biofilm based bacteria in the bulk water. / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2010.