Dissertations / Theses on the topic 'Bacteria isolation'

A unique, previously undescribed Gram-negative bacterium was isolated from several soils in Texas and extensively characterized in this study. The cells measured 1-2 by 4-6 μm. The distinguishing characteristic of the bacterium is the extraordinary capsular material which surrounds the cells. The new isolates are aerobic, mesophilic, non motile and have the ability to utilize a variety of organic compounds as the sole source of carbon and energy. The organism grows optimally at 30° C and the optimal pH lies between 7.0-8.0. The isolates produce catalase but oxidase is not produced. They do not produce indole or hydrogen sulfide. The organism can hydrolyze gelatin and Tween 80 but not starch, esculin and casein. The major cellular fatty acid is anteiso 15:0. The guanine and cytosine content is 58-62 mole%. The organism's taxonomic position was further established by specific gene probes, 16S rRNA homology, DNA homology and "ribotyping." These data showed that it was most closely related to members of the genus Paenibacillus, although somewhat divergent from other species classified in this genus. After careful evaluation of the results obtained during this study, it is proposed that this unique bacterium be named Paenibacillus velasolus sp. nov.

Ten bacteria capable of growth on the metal-cyano complex, tetracyanonickelate (II) {K2 [Ni(CN)J } (TCN), supplied as the sole nitrogen source, were isolated. Seven isolates were identified as pseudomonads while the remaining three were classified as Klebsiella species. In addition to TCN, all isolates were able to utilize KCN although it was significantly more toxic. The degradation of TCN was most complete when supplied at growth-limiting concentrations, did not occur when ammonia was present, and resulted in the formation of nickel cyanide [Ni(CN)2] as a degradation product.

Mestrado em Biotecnologia
Bioremediation has proven to be an effective strategy in the recuperation of oil contaminated ecosystems. However most bacteria used in this processes, while being able to degrade a wide range of the oil hydrocarbons, have limited action due to the low water solubility of these compounds. Hence, a possible solution for this problem would be the use of biosurfactant-producing bacteria, since the presence of surfactants help improve the hydrocarbons dispersal, solubilization and bioavailability. The objective of this work was to assess the biotechnological potential of Ria de Aveiro estuarine system regarding the presence of hydrocarbonoclastic biosurfactant-producing bacteria and to evaluate different combinations of environmental inocula and carbon sources for the isolation of biosurfactants producing bacteria. Selective cultures (diesel, crude and paraffin) were prepared using inocula from different environmental matrixes: samples from the surface microlayer (SML), bulk estuarine sediments and sediments of the rhizosphere of Halimione portulacoides, a characteristic halophyte from the salt marshes of Ria de Aveiro. During the incubation period, the development of the selective cultures was assessed by quantification of colony forming units (CFU). The highest value of CFU was obtained in the crude-sediment culture, while the lowest value was found with the diesel-rhizosphere combination. The DGGE profiles of the 16s rRNA gene fragments of the total community DNA extracted at the end of the incubation of the selective cultures, show that communities were different in terms of structural diversity. The values of the Shannon-Weaver index of diversity indicate that the higher diversity was achieved in the selective cultures with paraffin as carbon source (2.5231), followed by the crude oil (2.2509), and diesel (1.6726) selective cultures. From the selective cultures, 111 presumably hydrocarbonoclastic isolates were obtained after isolation and purification. Of these, 66 were tested for biosurfactant production by the atomized oil assay, with positive results for 17 isolates (25.8%). The environmental matrix with best results was the SML water and diesel was the most effective carbon source. Having in consideration the high number of isolates obtained from the selective cultures and the percentage of biosurfactant producers, the estuarine system of Ria the Aveiro, and in particular the SML, can be regarded as an interesting seedbank for the prospection of hydrocarbonoclastic and biosurfactants producing bacteria. The SML microhabitat shows particularly high biotechnological potential for the isolation of bacterial strains with interesting properties for application in bioremediation strategies in coastal and estuarine areas.
A biorremediação é tida como uma possível estratégia na recuperação de ecossistemas contaminados com hidrocarbonetos. A aplicação eficaz desta tecnologia é, no entanto, muitas vezes limitada pela natureza hidrofóbica dos contaminantes. O recurso a estirpes bacterianas simultaneamente degradadoras de hidrocarbonetos e produtoras de biossurfactantes apresenta um enorme potencial na reciclagem de compostos hidrofóbicos. Assim, o objectivo deste trabalho consistiu em avaliar o potencial biotecnológico do sistema estuarino da Ria de Aveiro quanto à presença de bactérias hidrocarbonoclásticas produtoras de biossurfactantes e a avaliação de várias combinações de inóculos ambientais e fontes de carbono para a obtenção de isolados bacterianos de interesse. Para tal foram realizadas experiências em meios selectivos (diesel, crude e parafina) a partir de inóculos de diferentes matrizes ambientais: amostras da microcamada superficial (SML), sedimentos estuarinos e rizosfera de bancos de Halimione portulacoides, uma planta halófita dos sapais da Ria de Aveiro. O desenvolvimento da cultura ao longo do período de incubação foi avaliado pela contagem de unidades formadoras de colónias (CFUs). A cultura selectiva com maior teor de bactérias cultiváveis foi a de crude-sedimento e aquela em que a abundância bacteriana foi mais baixa foi a de diesel-rizosfera. A partir da análise dos perfis de DGGE dos fragmentos do gene 16s rRNA do DNA total extraído das culturas selectivas verificou-se que no fim do período de incubação, o grau de semelhança entre as comunidades bacterianas das culturas selectivas é relativamente baixo. Pelo índice de diversidade de Shannon-Weaver a maior diversidade estrutural das comunidades bacterianas encontra-se nas culturas selectivas de parafina (2,5231), seguidas das de crude (2.2509) e das de diesel (1.6727). Das culturas selectivas, foi obtido um conjunto de isolados que foi testado quanto à capacidade de produção de biossurfactantes pelo método atomized oil. De 66 isolados testados, 17 produziram resultado positivo (25,8%), sendo a água da SML a matriz ambiental com melhores resultados e o diesel a melhor fonte de carbono para o isolamento de bactérias produtoras de biossurfactantes. Tendo em conta o elevado número de isolados obtidos e a percentagem de produtores de biossurfactantes, pode concluir-se que na Ria de Aveiro, particularmente na SML, existem comunidades bacterianas adaptadas à utilização se substratos hidrofóbicos, com uma boa representação de produtores de biossurfactantes. Os resultados confirmam a perspectiva de que a SML da Ria de Aveiro é um microhabitat com elevado potencial biotecnológico para isolamento de estirpes de bactérias hidrocarbonoclásticas produtoras de biossurfactantes com promissoras aplicações em processos de biorremediação de regiões estuarinas e costeiras após contaminação acidental com hidrocarbonetos de petróleo.

A seliberia-like bacterium (SLO), isolated from reverse osmosis membranes was characterized by morphological, physiological and DNA studies. The helically twisted cells of this organism were often observed in star-shaped clusters. Depending on nutritional conditions, cells ranged from 0.5 to 21 um in length and possessed prosthecae. Small motile cells were produced by asymmetric fission or by a budding process. Ovoid "generative" cells were observed in mixed culture conditions or when the pure culture isolate was grown in the presence of humic acid. The SLO oxidatively utilized glucose, maltose, xylose, cellobiose, and several amino acids as sole carbon and energy sources. The organism is a strict aerobe and does not anaerobically respire. The moles percent guanine plus cytosine (mol% G + C) of the SLO DNA was 38% as compared with 63-67% for Seliberia stellata. Although the cellular morphology and physiology of the SLO closely resembles that of S. stellata, the SLO is considered to be a new species of Seliberia based on the presence of prosthecae and the mol% G + C.

Mestrado em Biotecnologia - Biotecnologia Industrial e Ambiental
Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible biopolymers. PHAs emerge as a possible solution as substitutes of petroleum based plastics, being produced under the Biorefinery concept, in which wastes and by-products of numerous industries may be used as carbon source. This project aimed the isolation and characterization of organisms able to store PHAs from Hardwood Sulphite Spent Liquor (HSSL), a by-product of the pulp and paper industry. Isolation was performed from a Mixed Microbial Culture (MMC) selected under feast and famine conditions, using some components present in HSSL as substrates, such as acetic acid and xylose. Five pure isolates able to produce PHAs resulted from the successive streaking in solid medium containing HSSL. The purity of the isolates was evaluated through Gram staining and FISH analysis and the PHAs accumulation by Nile Blue staining. Two strains were identified as Rhohococcus spp. and three as Pseudomonas spp.. One isolate of each genus was selected and further studied in terms of growth and PHAs accumulation capability from three distinct carbon sources (HSSL, acetic acid and xylose). Both isolates, Rhodococcus spp. and Pseudomonas spp., were able to grow and use the three carbon sources as well as to produce PHAs. However, both strains showed a higher maximum specific growth rate (μmax) when HSSL was used as carbon source, 0.212 ± 0.0219 h-1 and 0.251 ± 0.0526 h-1, respectively. A qualitative evaluation of the PHAs accumulation through Nile Blue staining exhibited a higher accumulation when acetic acid was used as sole carbon source. In an attempt to identify some of the species responsible for PHAs accumulation of the selected MMC, belonging to the dominant class, Alphaproteobacteria, a 16S rDNA clone library was constructed. It was possible to identity Novosphingobium spp., Sphingobium spp. and Pleomorphomonas spp.
Polihidroxialcanoatos (PHAs) são biopolímeros biodegradáveis e biocompatíveis. Os PHAs são considerados uma solução possível como substitutos dos plásticos derivados do petróleo, podendo ser produzidos no âmbito do conceito de Biorefinaria utilizando resíduos como fonte de carbono. Este trabalho teve como objectivo o isolamento e a caracterização de bactérias produtoras de PHAs a partir de licor de cozimento ao sulfito ácido (HSSL), um sub-produto da indústria papeleira. Os isolamentos foram realizados partindo de uma cultura mista seleccionada para a acumulação de PHAs por imposição de ciclos de fome e fartura, utilizando alguns dos componentes do HSSL como substrato, nomeadamente a xilose e o ácido acético. Após repicagens sucessivas em meio sólido contendo HSSL, foi possível obter cinco isolados puros capazes de acumular PHAs. A pureza dos isolados foi avaliada através de coloração de Gram e análise FISH e a capacidade de acumulação de PHAs por coloração de Azul do Nilo. Duas estirpes foram identificadas como Rhohococcus spp. e três como Pseudomonas spp.. Um isolado de cada género foi seleccionado e estudado em termos de crescimento e capacidade de acumulação de PHAs, a partir de três fontes de carbono distintas (HSSL, ácido acético e xilose). Verificou-se que ambos os isolados, Rhodococcus spp. e Pseudomonas spp., foram capzes de crescer nos três meios e produziram PHAs. Contudo, ambas as estirpe apresentaram uma taxa específica de crescimento (μmax) superior com HSSL como fonte de carbono, 0.212 ± 0.0219h-1 e 0.251 ± 0.0526h-1 respectivamente. Uma avaliação qualitativa da acumulação de PHAs utilizando coloração Azul do Nilo mostrou uma acumulação maior nos ensaios em que o ácido acético era a única fonte de carbono. Numa tentativa de identificar algumas das espécies responsáveis pela acumulação de PHAs da cultura mista seleccionada pertencentes à classe dominante, Alfaproteobactéria, recorreu-se à construção de uma biblioteca de clones 16S rDNA. Foram identificadas as espécies Novosphingobium spp., Sphingobium spp e Pleomorphomonas spp.

In  this  study,  tolerance  of  soil  bacteria  to  hexavalent  chromium  (Cr(VI))  was  investigated.  First,  influence  of  high chromium levels of anthropogenic and geogenic origin on the  soil cultivable  bacterial community was examined. Next, a number  of  bacterial  strains  with  high  Cr(VI)  tolerance  were  isolated  from  diverse   environmental  samples  such  as  soil, sediment, water and waste material.  Strains were  identified  and  tested for  the  level of  Cr(VI) tolerance  and  the  ability toreduce toxic Cr(VI) to more innocuous Cr(III). Selected  Bacillus cereus  group strains  were further characterized  -  their morphological  and  biochemical  characteristics,  16S  rRNA  and  pycA  gene  sequences,  biofilm  formation  potential  and resistance to other heavy metals were determined. Also, more detailed study of their tolerance level and  Cr(VI) reduction was  conducted.  Strain  with  the highest  resistance  together  with the  control  chromate  sensitive  strain  were  analyzed  by STEM EDS for their cellular and endospore Cr content under different conditions. Results indicate Cr(VI) tolerant bacteria are  present  both  in  low  and  high  Cr  environments.  Majority  of  isolates  belonged  to  the  B.  cereus  group  indicating  its overall high tolerance to  Cr(VI). Certain strains exhibited high  tolerance and reduction  ability,  indicating their possibleusefulness  in practical  bioremediation  application.  STEM  EDS  analysis  of  Cr(VI)-sensitive  B.  subtilis  PY79  strain  and Cr(VI)-resistant  B. cereus  group strain  NCr1a revealed  significant differences in their response to Cr(VI)  and in  their  Cr cellular and endospore content.
U ovom radu ispitana je tolerantnost  zemljišnih  bakterija na šestovalentni hrom (Cr(VI)). Prvo, ispitan je uticaj visokog nivoa  hroma  antropogenog  i  geogenog  porekla  na  kultivabilnu  bakterijsku  zajednicu  zemljišta.  Dalje,  izolovani  su bakterijski sojevi sa visokom tolerancijom na Cr(VI) iz različitih sredinskih uzoraka   kao što su zemljište, sediment, voda i otpadni materijal. Sojevi su identifikovani i određen je nivo njihove Cr(VI) tolerancije i sposobnost redukcije toksičnog Cr(VI)  u  manje  toksični  Cr(III).  Odabrani  sojevi  Bacillus  cereus  grupe  su  dalje  karakterisani  –  određene  su  njihove morfološke i biohemijske karakteristike, 16S rDNK i  pycA  sekvence, potencijal formiranja biofilma i otpornost na druge teške  metale.  Takođe,  sprovedeno  je  detaljnije  ispitivanje  njihove  tolerancije  i  redukcije  Cr(VI).  Soj  sa  najvišom otpornošću  je  uporedo  sa  kontrolnim  osetljivim  sojem  analiziran  pomoću  STEM  EDS  na  sadržaj  hroma  u  ćelijama  I endosporama u različitim uslovima. Rezultati ukazuju da su bakterije tolerantne na Cr(VI) prisutne i u sredinama sa niskim i  sa  visokim  koncentracijama  hroma.  Većina  izolata  pripadala  je  B.  cereus  grupi  što  ukazuje  na njenu  uopšteno  visoku otpornost na Cr(VI). Pojedini sojevi su pokazali visoku otpornost i sposobnost  redukcije Cr(VI), što ukazuje na mogućnost njihove praktične primene u bioremedijaciji. STEM EDS analiza osetljivog B. subtilis PY79 soja i Cr(VI)- rezistentnog soja B.  cereus  grupe  NCr1a  otkrila  je  značajne  razlike  u  njihovom  odgovoru na  Cr(VI)  i  sadržaju  Cr  u njihovim  ćelijama  i endosporama.

Thesis (M.S.)--University of Cincinnati, 2006.
Title from electronic thesis title page (viewed Apr. 13, 2006). Includes abstract. Keywords: Giardia intestinalis; Cytophaga-Flavobacterium; Cyst degradation; Microbial antagonism. Includes bibliographical references.

Food fermentation is a method widely used in the past to extend the storage life of food. Numerous studies on fermented food have revealed that they not only have biopreservative properties but also health benefits. Lactic acid bacteria are the major group of microorganisms involved in food fermentation and the properties that influence food are primarily due to the compounds released from microorganisms such as organic acids and bacteriocins. Their health benefits are exerted through several mechanisms including inhibiting the growth of pathogenic bacteria and modifying the host immune response. A number of strains that have been investigated show different properties even between the same species thus emphasizing the importance of strain identification. To determine if some traditional fermented Swedish foods contain lactic acid bacteria, bacteria from four fermented Swedish foods (two surströmming and two sausages) were isolated using MRS broth. Bacterial isolates were examined for their colony and cell morphology and Gram staining and were found to be predominantly Gram-positive cocci or rods. 16S rRNA PCR amplifications of selected isolates was performed using universal prokaryotic primers and sequenced. The sequencing results showed that the bacterial isolates from Oskars surströmming Filéer and Gognacs medvurst were Lactobacillus sakei and the isolate from Mannerströms surströmming was Enterococcus sp. This study showed that the traditional Swedish fermented food evaluated did contain lactic acid bacteria.

Bacteriophage (phage), virus of bacteria, has been proposed as a mean to inactivate bacteria that are pathogens of humans. Applied prophylatically to food, phage might decrease the numbers of potential pathogens we ingest. Much active research on using the phages of bacteria to control Gram negative foodborne pathogens are described in the literatures, but comparatively little research describes the phages of Gram positive bacteria and their use as biocontrol agents on food. In this work, previous undescribed phages, able to infect Bacillus cereus and Listeria monocytogenes, were isolated from soil and ruminants faecal material, respectively. As the first step in assessing their potential as biocontrol agents, the isolated phages were purified, concentrated and characterised (albeit to different degrees). The Bacillus phages had a narrow host range while the Listeria phages had a broad host range. Listeria phages also infected L. monocytogenes 2000/47, a strain which recurs in New Zealand clinical cases. Both Bacillus and Listeria phages appeared to be of the Myoviridae family judging by their structure in electron micrographs. The Bacillus FWLBc1 and FWLBc2 phages were lytic phages with a latent period of 106 and 102 min at 37°C, and an average burst size of 322 and 300 phages per infected cell, respectively. Moreover, they both had genomes of approximately 134 kb. All newly isolated and characterized phages were chloroform resistant and survived storage better at 4°C than at room or freezing temperatures. Bacillus phages significantly reduced the bacterial population in mashed potatoes within 24 h at room temperature, when applied at a phage to host ratio of 1000. Listeria phages rapidly inactivated the host population to a low optical density. The findings of this thesis will add to the current knowledge of phages in the context of various environmental conditions for different bacteria and will demonstrate the potential of phages as food safety biocontrol agents.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2006.
Fruit juices were until recently considered to only be susceptible to spoilage by yeasts, mycelial fungi and lactic acid bacteria. Spoilage by these organisms was prevented by the acidic pH of fruit juices and the heat-treatment applied during the hot-fill-hold process. Despite these control measures, an increasing number of spoilage cases of fruit juices, fruit juice products and acidic vegetables due to contamination by thermophilic acidophilic bacteria (TAB) have been reported. The genus Alicyclobacillus, containing TAB were first classified as Bacillus, but were reclassified in 1992. Species of Alicyclobacillus are Gram-positive, rod-shaped, endospore-forming bacteria. The unique characteristic of these organisms is the presence of ω-alicyclic fatty acids, such as ω-cyclohexane and ω-cycloheptane, as the major components of the cellular membrane. This organism has been shown to survive pasteurisation conditions of 95°C for 2 min and grows within a pH range of 2.5 to 6.0 and temperatures between 25° and 60°C. The genus currently consists of 11 species, with A. acidoterrestris, A. acidocaldarius and A. pomorum being the only species associated with the spoilage of fruit juices and fruit juice products. The aim of this study was to evaluate culture-dependent and culture-independent approaches for the detection and isolation of Alicyclobacillus spp. from pasteurised South African fruit juices and concentrates. The culture-dependent approach was evaluated by comparing five different growth media, for growth and recovery of A. acidoterrestris, A. acidocaldarius and A. pomorum at different incubation temperatures, from sterile saline solution (SSS) (0.85% (m/v) NaCl), diluted and undiluted fruit juice concentrates. The five media evaluated included potato dextrose agar (PDA), orange serum agar (OSA), K-agar, yeast extract (YSG)-agar and Bacillus acidocaldarius medium (BAM). The culture-independent approach was used to identify the micro-organisms present in fruit juices and concentrates from different South African manufacturers before and after pasteurisation, using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. Spread plates of PDA at pH 3.7 and incubation temperature of 50°C for 3 days was found to be the best isolation media for species of Alicyclobacillus from fruit juice and fruit juice concentrate. With the inclusion of a heat shock treatment at 80°C for 10 min the growth media of preference for spores of Alicyclobacillus from fruit juice concentrates was OSA at pH 5.5 and an incubation temperature of 50°C for 3 days. The culture-dependent approach could detect cells or endospores at a minimum concentration of 104 cfu.ml-1 in SSS and diluted fruit juices. PCR-based DGGE analysis was more sensitive and detected cells of Alicyclobacillus spp. from fruit juices and concentrates at a minimum concentration of 103 cfu.ml-1. Alicyclobacillus acidoterrestris was found to be present in South African apple juice, pear juice, white grape juice and aloe vera juice. White grape juice was also found to contain A. pomorum. Other organisms present in the orange, apple, mango and pear juices were two uncultured bacteria that were identified as members of the genus Bacillus, and one uncultured bacterium closely related to Alcaligenus faecalis. This study confirmed the presence of TAB in pasteurised South African fruit juices and concentrates and emphasises the need for the rapid and accurate detection of TAB in food products.

As very little information was available on mercury sensitivity testing, the first aim was to determine the most suitable agar and concentration of mercuric chloride to use in this project. The primary objective of the study was to determine whether mercury released from amalgam fillings could increase the prevalence of mercury-resistant bacteria in the oral flora of children. This was achieved through cross-sectional and longitudinal studies. The second aim was to determine whether changes in mercury resistance correlated with changes in the incidence of antibiotic resistance. The final aim was to determine whether individual mercury-resistant isolates contained the merA gene. In the cross-sectional study, saliva and plaque samples were collected from patients with and without amalgam fillings. No significant differences in the proportion of mercury-resistant bacteria were detected between the two groups. One hundred and thirty nine mercury-resistant bacteria were isolated and 41% (with amalgam) and 33% (without amalgam) of these were also resistant to one or more antibiotics. Resistance to tetracycline was most common. Sixteen patients were enrolled into the longitudinal study. The proportions of mercury-and antibiotic-resistant bacteria were determined on 3 separate occasions (2 pre-amalgam and 1 post-amalgam). There was not a statistically significant change in the incidence of mercury- or antibiotic-resistant bacteria during the month after the installation of the amalgam fillings. However, a linear association between the number of surfaces and proportion of mercury-resistant bacteria was observed. Eighty eight mercury-resistant bacteria were isolated and 27% (pre-amalgam) and 40% (post- amalgam) of these were resistant to one or more antibiotics. Resistance to erythromycin was most common. One hundred and thirty two mercury-resistant bacteria were screened for the merA gene using PCR and 2 sets of primers. Sixty three percent of the streptococci were found to contain the merA gene. Coagulase-negative staphlyococci, Rothia dentocariosa and Neisseria species contained the merA gene, while Staphylococcus aureus and Pseudomonas stutzeri did not. All sequenced amplicons were found to be up to 95% identical to the Bacillus cereus RC607 merA gene. The results of this study have failed to demonstrate any definitive link between the presence of mercury amalgam in teeth and the presence, or proportion, of mercury- or antibiotic-resistant bacteria in the oral cavity of children.

In order to characterise thermostable hydantoin-hydrolysing enzymes from bacteria, locally-isolated thermophilic organisms were screened for the ability to convert hydantoin to N-carbamylglycine at 55°C using the hydantoinase enzyme. Cell disruption of a selected strain, RU-20-15, was conducted by French pressing to release enzyme from within the cell. In all of the experiments conducted, the amounts of product were low. In view of the low yields of products formed by the thermophiles, a previously-isolated Gram negative strain, RU-KM3L was selected from a number of mesophiles by screening for hydantoinase and carbamylase activity over a 40-55°C temperature range. Hydantoin conversion at 40°C using crude extract from pressed cells of this organism was similar to conversion at 50°C, and therefore subsequent assays were conducted at the higher temperature. The growth kinetics of RU-KM3L cells were studied and the enzyme activities of the extracts were compared in complete and chemically-defined media. The results suggested that the optimal time to harvest cells was at early stationary phase, when using complete medium for culture of cells; the specific activity of enzyme extracts produced by culture in complete medium was higher than that obtained in chemically-defined medium. 5-methylhydantoin was shown to be the preferred substrate for both the hydantoinase and carbamylase enzymes in the crude extract of RU-KM3L. The substrate specificity of the hydantoinase and carbamylase enzymes of the crude RU-KM3L extract was observed to be altered in the presence of increasing amounts of hydantoin, 5,5-dihydrouracil (DHU) and 5-thiouracil (TU) as inducers, showing selectivity for 5-methylhydantoin over hydantoin at inducer concentrations of 0.1 to 1%. A limiting effect on the hydrolysis of 5-methylhydantoin was observed when DHU and 5,5-dimethylhydantoin (DMH) were used as inducers, while the limiting effect on hydantoin specificity was observed when DHU and TU were used as inducers. The limiting effect was observed to be dependent upon the concentration of inducer, and was not observed when hydantoin was used as an inducer. The optimal time for assay of the hydantoinase enzyme in crude extract preparations at 50°C was observed to be 3h. Alkaline conditions were shown to be optimal for both the hydantoinase and carbamylase enzymes of RU-KM3L. Assay for enzyme activities of RU-KM3L extract in the presence of metal ions showed Mn²⁺ ions (and to a lesser extent, Co²⁺) to activate both the hydantoinase and carbamylase activities. Cu²⁺ ions were observed to inhibit the hydantoinase enzyme. In order to determine the location of the enzymes within the cell, cell debris from disrupted cells of RU-KM3L was removed by centrifugation. A decrease in enzyme activity in the supernatant was observed, and suggested association of the enzymes with the cell membrane. Ammonium sulfate fractionation experiments conducted on the crude extract provided further evidence for this result. Sonication of the crude enzyme extract was the only successful method for the releasing of membrane-associated enzyme. Of a number of strategies investigated, the use of sucrose at 50% (w/v) concentration was shown to preserve the hydantoinase and carbamylase enzyme activities during lyophilisation. Furthermore, assay for these enzyme activities showed the activities to be higher after lyophilisation in the presence of sucrose. However, sucrose did not increase the thermostability of lyophilised crude enzyme extracts. Water-miscible organic solvents at 1% concentration were shown to be inhibitory to the hydantoinase and carbamylase enzymes of RU-KM3L, and the inhibition was also observed to increase with increasing concentrations of these solvents. Hydantoinase activity in the presence of water-immiscible organic solvents was shown to increase with an increase in the hydrophobicity of these solvents, but the activity observed was not significantly higher than activity in the absence of solvent when hydantoin and 5-methylhydantoin were used as substrates. The possibility of reversing the hydantoinase enzyme reaction by water-immiscible organic solvents was investigated, and the results obtained suggested that the reaction could be reversed. It was thought that the partitioning of substrates or products into hydrophobic organic solvents could influence the reaction equilibrium, but the partitioning observed was not sufficient to affect reaction rates. Peptide synthesis was shown to have occurred in small amounts when the hydantoinase reaction was carried out in the presence of water-immiscible organic solvents. In conclusion, the hydantoin-hydrolyzing enzyme activity of a crude extract preparation from the bacterial strain RU-KM3L was characterised at elevated temperatures, and in the presence of watermiscible and -immiscible organic solvents.

A novel screening method for detecting lignin-degradation activity on agar plates was developed using nitrated lignin. Using this method, ten lignindegrading bacteria have been isolated from environmental sources, including seven mesophilic soil bacteria and three thermotolerant strains from composted wheat straw. All of the isolates have demonstrated activity towards lignin degradation in the assays, the most active strain being a thermotolerant Sphingobacterium strain from the Bacteroidetes family. The ability of each strain to degrade a variety of aromatic carbon sources and size-fractionated Kraft lignin has been examined by laboratory-scale growth experiments and gel filtration chromatography respectively, and the bioconversion of different lignin-containing feedstocks by three of the most active strains has been examined in a series of laboratory-scale fermentation experiments. Purification of extracellular lignin-degrading enzymes from the culture supernatant of Sphingobacterium sp. has highlighted several different enzyme activities and possible lignin-degrading enzymes.

Oil spills are a universal threat impacting local, national and world communities alike. Bioremediation that is natural, efficient, economical and safe is the best solution for protecting the environment from oil related damages. In this study, motor oil degrading bacteria were isolated from oil-contaminated soil samples from a suburban Atlanta, Georgia community. Mineral salt broth containing 1 Ow-40 motor oil as the sole carbon source was used to isolate motor oil degrading bacteria. Motor oil tolerant and metabolizing bacteria were identified using morphological and biochemical tests. Two bacterial isolates were then tested for their tolerance varying concentrations of diesel and kerosene oils for comparison with motor oil consumption. Observed results suggest that the isolated bacteria from oil contaminated soil possess abilities to metabolize motor oil, kerosene and diesel. Knowledge of the tolerance ranges of the isolated bacteria can indicate their potential to be of use in the remediation of terrestrial petroleum oil spills in a manner that is natural, economical, quick and efficient.

Includes bibliographical references.
Bacterial strains were isolated by enrichment cultures from oil-contaminated soil samples. In the present study, several strains, capable of growing on crude oil, were isolated. Isolates were screened for their inherent abilities to produce bioemulsifiers when they were grown on hydrocarbon substrates.

The sulphidogenic activity of sulphate-reducing bacteria (SRB) is of great economic importance to many industrial sectors, since it is strongly implicated in a range of problems, including the souring of oil reservoirs and the anaerobic corrosion of mild steel. Despite increasing reports of SRB activity throughout oil fields, little attention has yet been given to further characterisation and identification of those SRB species involved. In an attempt to understand the role of these organisms in the oil field environments this work is to isolate, characterise, and identify SRB present in a range of samples from different oil fields. Using selective enrichment cultivation methods, a number of nutritional types of mesophilic and thermophilic SRB cultures have been obtained from a range of oil field samples. This suggests that SRB are widespread throughout oil fields and may be important contributors in the biogenic production of H₂S in such environments. The successful isolation of 7 SRB pure cultures (6 mesophiles and 1 thermophile) was achieved. Using both classical methods and nucleic acid analyses, 3 types of SRB strains have been identified: 5 mesophiles (strains FM3, EF2, FM2, GF2, and MM6) are members of Desulfomicrobium; mesophilic strain EM2 is a Desulfovibrio; thermophile NM2 is an archaebacterium, phylogenetically closely related to Archaeoglobus fulgidus. Such strains may be the causative micro-organisms in the generation of H₂S in oil fields. 16S rDNA comparative analysis was applied to directly identify SRB present in 4 thermophilic enrichment cultures from 3 different oil field samples (sandstone core, drilling mud, and production water). The results show that thermophilic SRB, Desulfotomaculum-related species, were commonly distributed in all oil field samples. Micro-organisms related to Gram-positive, thermophilic, non-sulphate-reducing, anaerobic bacteria were also found in cultures from sandstone core and production water. These organisms may play an important ecological role alongside SRB in oil field environments.

Luminescence is the emission of light by an object. Living organisms including certain bacteria are capable of luminescence. Bacteria are the most abundant luminescent organisms in nature. Bacterial luminescence has been studied most extensively in several marine bacteria. Bacterial luminescence is due to the action of the enzyme called luciferase. The luminescent bacteria exist in nature either as free living bacteria or in symbiotic association ship with certain marine organisms. Research on luminescent bacteria has always been a fascinating one. In the present study, twenty free living luminescent bacteria were isolated from Bay of Bengal, India using soft agar overlay method in sea water complex agar (SWCA). All the 20 strains were characterized for certain biochemical tests and they were tentatively identified that they are all Photobacterium spp. The effect of salinity, pH glycerol concentration and heavy metals on the growth and luminescence of these 20 strains was also studied. In this part of experiment, visual scoring was done to categorize the luminescence. In case of salinity, it has been found that up to 6% of NaCl the intense of luminescence was good and thereafter it declined. Further, in some strains it was completely ceased beyond 9% of salinity. Luminescence was not greatly affected by pH in liquid medium however; the same was affected in solid medium. The intensity of luminescence has increased with increasing concentrations of glycerol ranging from 0.3 to 1.2%. All the 20 luminescent bacteria were characterized for their tolerance to heavy metals and antibiotics. Copper and zinc at 1 mg/ml concentration have inhibited the growth and luminescence of the all strains. Surprisingly, mercury at the same concentration has inhibited only two strains (AMET1913 and AMET1920). However, at 2 mg/ml concentration mercury has inhibited the growth and luminescence of all the 20 strains. Selected six luminescent bacterial strains were also characterized for their antibiotic susceptibility against six different antibiotics. It has been found that most of the strains were sensitive to all the six antibiotics tested. Since, the bioluminescence is regulated by quorum sensing, the effect of culture filtrate extracted with dichloromethane was also tested for its effect on luminescence. These DCM extracts haven‟t influenced the luminescence much.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: Wine quality is greatly influenced by the number of microorganisms, which occur throughout the winemaking process. Yeasts are responsible for the alcoholic fermentation, the lactic acid bacteria (LAB) are responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) are responsible for converting ethanol to acetic acid. These microorganisms are present on the grapes and in the cellar and these consequently serve as gateways to the fermentation tanks where they will affect the wine quality. However, these microorganisms can be seen either as beneficial or as wine spoilage microorganisms, depending on the conditions that prevail throughout the winemaking process. It is thus very important to prevent any process that could lead to the lowering of the wine quality. In this regard, some of the factors that should always be evaluated include the quality of the grapes, winemaking techniques and quality control. One of the measures that have been implemented during winemaking to ensure the microbial stability is the use of chemical preservatives. Sulphur dioxide (502) has been, and is, used widely as primary preservative in winemaking. However, an ever-increasing consumer resistance against the use of chemical preservatives has developed as it poses possible health risks and decreases the sensorial quality of wine. An alternative approach to chemical preservation that has triggered numerous new investigations, is biological preservation or biopreservation. This is the use of the natural microbial flora and/or their antimicrobial products, such as bacteriocins, to inhibit or destroy the other sensitive microorganisms that are unwanted in the same environment. Evidence in the wine industry has shown that bacterial spoilage still is a very common problem in many wineries. This bacterial spoilage can lead to, amongst other, two main problems, which are of great concern to winemakers. This include high levels of volatile acidity, resulting in the wine having a vinegary off-flavour, and sluggish/stuck fermentation, which is the result of compounds such as acetic- and other fatty acids that causes inhibition of the yeast's growth. With acetic acid being the common link in both cases, it became evident that investigations should be performed on the main producer of acetic acid, namely AAB. As a result, AAB turned out to be one of the main spoilage microorganisms associated with winemaking. Most of the research on biopreservation in the food and beverage industry has been performed on the Gram-positive LAB. The fact that their spectrum of inhibition currently excludes most Gram-negative bacteria, specifically AAB, indicated that AAB should be screened in search of possible antimicrobial compounds that could be applied to control their cell numbers during winemaking. No evidence of antimicrobial action amongst AAB could be found in literature, therefore this work was considered novel. The main objectives of this study were to screen wine isolates of AAB for the production of antimicrobial compounds. This was followed by the isolation and preliminary characterisation of the antimicrobial substances produced. Various attempts to optimise the production of the antimicrobial compounds and isolation procedures, were also included. This study forms part of a larger research programme that has been initiated at the Institute for Wine Biotechnology at Stellenbosch University on the biopreservation in wine. Our results indicated that possible antimicrobial compounds of proteinaceous nature, produced by AAB isolated from wine, do exist. It was found that two different species of AAB, namely Acetobacter aeeti and Gluconobacter frateurii, produced antimicrobial compounds that inhibited other species of AAB. Preliminary results indicated that these compounds are heat sensitive and stable in a wide pH range. It was also shown that after the action of proteolytic enzymes, such as proteinase K and a-chemotrypsin, all inhibitory activity was lost. This study also revealed the existence of the species Gluconobacter frateurii, which have not yet been associated with the winemaking environment. This study made a valuable contribution to the limited amount of information and understanding of AAB, not only in the wine environment, but also elsewhere. The results and findings of this research would serve as platform for further projects. This might soon lead to the development of antimicrobial substances or tailored wine-yeasts with antimicrobial abilities, which can be applied during winemaking to assist the winemaker in combatting high cell numbers and subsequent spoilage by AAB.
AFRIKAANSE OPSOMMING: Wynkwaliteit word beïnvloed deur 'n verskeidenheid van mikroorganismes wat regdeur die wynrnaakproses teenwoordig is. Die giste is vir die alkoholiese fermentasie, die melksuurbakterieë (MSB) vir die appelmelksuurgisting, terwyl die asynsuurbakterieë (ASB) vir die omskakeling van etanol na asynsuur verantwoordelik is. AI hierdie mikroorganismes is teenwoordig op die druiwe en in die kelder, en dit dien gevolglik as 'n weg waardeur hulle in die fermentasietenke kan kom om sodoende die wynkwaliteit te beïnvloed. Hierdie mikroorganismes kan egter gesien word as óf voordelig óf as wynbederfmikroorganismes, afhangende van die heersende kondisies gedurende die wynrnaakproses. Dit is daarom baie belangrik om enige proses te voorkom wat tot 'n verlaging in wynkwaliteit kan lei. Wat laasgenoemde aanbetref, is daar sekere faktore wat altyd geëvalueer moet word, naamlik die druifkwaliteit, wynrnaaktegnieke en kwaliteitsbeheer. Een van die maatreëls wat geïmplementeer is om mikrobiologiese stabiliteit tydens die wynrnaakproses te handhaaf, is die gebruik van chemiese preserveermiddels. Swaweidioksied (S02) word algemeen gebruik as primêre preserveermiddel tydens wynrnaak. Daar is egter 'n toenemende verbruikersweerstand teen die gebruik van chemiese preserveermiddels, aangesien dit moontlike gesondheidsrisiko's kan inhou, asook tot 'n verlaging in sensoriese kwaliteit van die wyn kan lei. 'n Alternatiewe benadering vir chemiese preservering, wat reeds tot verskeie nuwe ondersoeke gelei het, is biologiese preservering of biopreservering. Dit is die gebruik van die natuurlike mikroflora en/of hulle antimikrobiese produkte, soos bv. bakteriosiene, om die sensitiewe mikroorganismes wat in dieselfe omgewing voorkom, se groei te inhibeer óf om hulle dood te maak. Aanduidings vanuit die wynbedryf dui daarop dat bakteriese bederf steeds 'n algemene probleem is wat in baie kelders ondervind word. Hierdie bakteriese bederf kan onder andere twee hoofprobleme veroorsaak, wat 'n groot bekommernis vir verskeie wynmakers is. Dié probleme sluit in hoë vlakke van vlugtige suurheid, wat gevolglik die wyn 'n asyn-afgeur gee, en slepende/gestaakte fermentasies, wat die gevolg is van komponente soos asynsuur en ander vetsure, wat die gis se groei inhibeer. Die feit dat asynsuur die gemeenskaplike faktor in beide gevalle was, het daarop gedui dat 'n ondersoek rakende die hoofproduseerder van asynsuur, naamlik ASB, benodig word. ASB word gevolglik as een van die hoofbederforganismes wat met die wynrnaakproses geassosieer word, beskou. Die meeste navorsing oor biopreservering in die voedsel -en drank bedryf is op die Gram-positiewe MSB gedoen. Die spektrum van inhibisie van die bakteriosiene van MSB sluit egter die meeste Gram-negatiewe bakterieë uit, veral ASB, en dit dui daarop dat ASB gesif moet word in 'n soektog na antimikrobiese substanse wat moontlik gebruik kan word om hul getalle tydens die wynrnaakproses te beheer. Geen bewyse kon tot dusver uit die literatuur gekry word met betrekking tot antimikrobiese aktiwiteit teen ASB nie, daarom word hierdie navorsing dus as nuut beskou. Hierdie studie se hoofdoelwittewas om die wyn-isolate van ASB vir die produksie van antimikrobiese peptiede te sif. Dit is gevolg deur die isolasie en voorlopige karakterisering van die geproduseerde antimikrobiese komponente. Daar is ook verskeie pogings aangewend om die produksie van die antimikrobiese substanse, asook die isolasieprosedures, te optimiseer. Hierdie studie vorm deel van 'n groter navorsingsprogram oor biopreservering van wyn wat deur die Instituut vir Wynbiotegnologie by die Universiteit van Stellenbosch geïnisieer is. Die resultate het daarop gedui dat antimikrobiese substanse van proteïenagtige aard, afkomstig vanaf wyn-isolate van ASB, wel bestaan. Daar is gevind dat twee veskillende spesies, naamlik Aeefobaefer aeefi en Glueonobaefer frafeurii, antimikrobiese peptiede produseer, wat ander spesies van ASB kan inhibeer. Voorlopige resultate het getoon dat hierdie substanse hitte-sensitief is en ook stabiel is oor 'n wye pH-reeks. Daar was ook aanduidings dat, ná die aksie van proteolitiese ensieme, soos bv. proteïnase K en a-chemotripsien, al die inhibitoriese aktiwiteit verlore gegaan het. Hierdie studie het ook die voorkoms van die spesies Glueonobaefer frafeurii aangedui, wat nog nie tot dusver met die wynrnaakomgewing geassosieer is nie. Hierdie studie maak 'n waardevolle bydrae tot die beperkte hoeveelheid inligting oor en begrip van ASB, nie net in die wynomgewing nie, maar ook in die algemeen in die natuur. Die bevindinge en resultate van hierdie navorsing sal as basis dien vir verdere projekte wat sal volg. Dit kan moontlik binnekort lei tot die ontwikkeling van antimikrobiese substanse, en ook pasgemaakte wyngiste met antimikrobiese vermoëns, wat tydens die wynrnaakproses gebruik kan word om sodoende die wynmaker in staat te stelom die hoë bakteriese getalle en die gevolglike bederf deur ASB, te beheer.

The microbial ecology of 45 high temperature (> 50 ° C) petroleum reservoirs was investigated by isolating and characterizing bacteria that were present in their produced fluids. Initial work was aimed at selecting a suitable high temperature petroleum reservoir for the study of natural microbial populations. Experimental work then focussed on establishing the physico-chemical conditions that prevail in the selected reservoir and on developing media and enrichment conditions for the isolation of microorganisms indigenous to the reservoir. The ability of reservoir bacteria to grow and survive under the physical and chemical conditions found in the selected reservoir was used to assess the likelihood of an indigenous origin for these bacteria. The petroleum reservoir selected for study was the Alton petroleum reservoir (SW Queensland, Australia). It was established that most of the physico-chemical conditions in the Alton reservoir had remained unchanged since oil recovery began. The stability of redox conditions (90 mV) in the reservoir over its operating life was identified as an important factor in the coexistence of strict aerobic and strict anaerobic bacterial populations within the reservoir. An important change that has occurred in the Alton reservoir over its operating life because of oil recovery was an increase in water pH from 6.41 to 8.42 as a result of carbon dioxide loss (1.36 atm to 0.0134 atm) from the reservoir. Development of novel enrichment procedures that simulated Alton reservoir conditions led to the isolation of previously unreported aerobic and anaerobic populations of thermophilic bacteria. The aerobic bacteria isolated were identified as either endosporeforming heterotrophic bacteria from the genus Bacillus or nonspore-forming heterotrophic bacteria resembling members of the genus Thermoleophilum. All aerobes grew on carbon sources such as acetate and n-heptadecane that are normal constituents of the reservoir. The anaerobic bacteria isolated were characterized as sheathed fermentative bacteria from the order Thermotogales or non-sheathed fermentative bacteria. In parallel studies, the natural microbial populations in other reservoirs were investigated and I concluded that fermentative microorganisms were common inhabitants of high temperature petroleum reservoirs. The isolation of fermentative bacteria from these high temperature petroleum reservoirs established that fermentative bacteria are a fourth major microbial group, together with hydrocarbon-oxidizers, sulphate-reducers and methanogens, to be reported in petroleum reservoirs. The fermentative bacteria use organic nutrients and carbohydrates, but not contemporary crude oil as the principal nutrient source within reservoir waters. The thermophilic bacteria isolated from Alton petroleum reservoir demonstrated growth characteristics such as temperature (optima 50-70 ° C and range 37-85 ° C), pH (optima 6.0-9.0 and range 5.0-9.0 and salinity (optima 0-15 g per litre and range 0-30 g per litre), that were consistent with conditions encountered in the Alton reservoir (temperature 75 � C, pH 8.5 and TDS 2.7 g per litre). The isolated bacteria also demonstrated a number of characteristics that might enable them to survive adverse conditions that could be encountered in a petroleum reservoir environment. The characteristics that contribute to aerobic bacteria surviving in and overcoming periods of oxygen limitation include well-documented processes such as sporulation, by Bacillus spp., and microaerophily. The characteristics that contribute to fermentative bacteria surviving were: (1) a natural tolerance to reservoir physico-chemical fluctuations, (2) an ability to remain viable when metabolic activity was suppressed to very low rates by the growth-limiting conditions imposed, and (3) possible formation of viable ultramicrobacteria (UMB). Formation of UMB (bacteria smaller than 0.3 |im) by thermophilic bacteria has not been reported previously. The recovery of thermophilic UMB by filtration from the Alton reservoir water indicates that these bacteria occur in natural habitats. This study found the formation of thermophilic UMB and their survival characteristics differed considerably from that reported for the mesophilic, marine bacterium Vibrio sp. DWI. Unlike mesophilic marine bacteria, thermophilic bacteria did not always respond to nutrient deprivation by forming UMB and that these UMB did not show any increased ability to survive in the face of adverse conditions. Although the formation of UMB as part of routine cell growth and division was not demonstrated directly in this study, circumstantial evidence suggests that they form part of a natural life cycle. The exact conditions that result in UMB formation and their role in survival remain unresolved. The capacity of nonspore-forming indigenous populations from Alton to survive sudden shifts in environmental conditions that might result from common oilfield operations was poor. Such operations were demonstrated to be inhibitory or lethal to Alton reservoir bacteria. It also was concluded that such oilfield operations suppress indigenous microbiota. However, the impacts of most oilfield operations within a reservoir are likely to be confined to the immediate area surrounding injection and producing wells. Minimizing the localized effects of oilfield practices on indigenous reservoir populations will lead to the better management of undesirable microbial activity in reservoirs such as H2S formation (souring) and facilitate development of better microbially mediated oil recovery process. This study showed that selected reservoir isolates possess characteristics which are suitable for in situ biotechnological applications such as microbially enhanced oil recovery (MEOR). Characteristics favourable for enhanced oil recovery include a capability for UMB formation, which would enable better dispersion, and resistance to high concentrations of reservoir components such as calcium, magnesium, strontium, heavy metals and hydrocarbons.

博士
國立成功大學
化學工程學系碩博士班
96
Approaches for rapid screening of lipase-producing bacteria were first developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of thirty-three pure bacteria possessing lipase activity. Among them, sixteen strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). Among the 33 strains, the isolate having best transesterification activity was obtained by strategy C (SSC) and was identified as Burkholderia sp. C20. Literature shows that the Burkholderia lipase could be used to produce biodiesel. Thus, in this study, Burkholderia sp. C20 was used as a target lipase producing strain for biodiesel production. Next, an experimental design tool (i.e., response surface methodology; RSM) was used to improve lipase production from Burkholderia sp. C20. The factors affecting the lipase production of Burkholderia sp. C20 were screened by two-level factorial design and the optimal conditions for lipase production were determined by response surface methodology (RSM). Preliminary batch tests were employed to obtain the favorable conditions for lipase activity analysis and found that the optimal temperature and pH for lipase activity assay was 55oC and 9.0, respectively. Comparison of cell growth and lipase activity profiles shows that the fermentation time of 67 h was suitable for harvesting lipase products in the batch culture of Burkholderia sp. C20. A two-level design of 25-1 experiments was applied to identify the most significant influential factors out of five factors; namely, temperature, pH, and concentrations of olive oil, CASO and NaCl. After analysis with two-level design, concentrations of CASO and NaCl were selected for RSM analysis, predicting an optimal composition of 0.12% and 0.16% for CASO and NaCl, respectively. Using the optimal conditions, lipase production by Burkholderia sp. C20 was enhanced nearly 5 fold (from 0.8 to 3.9 U/ml). However, it is insufficient to provide enough amount of lipase from flask cultivation for biodiesel production. Therefore, a 5-L fermentor was used for scale-up production of lipase using the Burkholderia sp. C20 strain. The target factors affecting fermentative lipase production were aeration rate, agitation rate, and incubation time. By adjusting the aeration rate from 0.5 to 2 vvm, the overall lipase productivity increased from 0.057 to 0.076 U/ml-h. In addition, lipase production by fermentation could be further improved by optimizing agitation speed at 100 rpm, giving an overall lipase productivity of 0.09 U/ml-h. The formation of lipase from Burkholderia sp. C20 was found to be a mixed growth-associated event with a yield coefficient of 524 U/g-dry-cell-weight. It was also found that pH controlled at 7.0 was preferable to the cell growth while pH controlled at 6.0 was adaptable to lipase production. A stepwise addition of olive oil was found to be able to enhance lipase production in flask experiments and the results were also confirmed in fermentor tests. To enhance the recovery, efficiency and reusability of the lipase for further applications in biodiesel synthesis, the lipase originating from Burkholderia sp. C20 was immobilized onto cellulose nitrate (CN) membrane and celite via filtration and covalent bonding, respectively. The optimal condition for lipase immobilized onto celite matrix was as follows: crude lipase concentration, 70 g/L; pH 7.0; incubation time, 3 h. Under the optimal conditions, the maximum immobilized lipase activity was about 273.5 U/g celite while the lipase loading amount was around 62.9 mg per gram celite. The optimal lipolysis condition of CN-lipase and celite-lipase was pH 9.0, 55℃ and pH 10.0, 55℃, respectively. Kinetic analysis shows that the dependence of lipolytic activity of free and immobilized lipase on oil substrate can be described by Michaelis–Menten model with good agreement. The estimated kinetic constants for free lipase were vmax = 133.33 U/mg protein and Km = 0.09 mM; for CN-lipase were vmax = 31.25 U/mg protein and Km = 9.44 mM; for celite-lipase were vmax = 11.29 U/mg protein and Km = 12.06 mM, respectively. As a result, the employment of lipase immobilization would lead to a decrease in vmax and an increase in Km. Finally, biodiesel was produced by the immobilized lipase from Burkholderia sp. C20. Celite-lipase was used to produce biodiesel. The optimal condition of biodiesel production by celite-lipase was 0.6 g celite-lipase and a methanol-to-olive oil molar ratio of 3. Under that optimal condition, the maximum conversion of olive oil to biodiesel was 43%.

碩士
國立中央大學
環境工程研究所
98
The objective of this study was to isolate the indigenous dioxin-degrading bacteria of Taiwan. Also, the characteristics of these bacteria were investigated after isolation followed by culture enrichment and strains acclimation. Three soil samples were gathered from the downstream of the announced dioxin and contamination site. The analytical results of PCDDs/PCDFs and their isomer concentration in all samples confirmed the dioxin contamination of the sampling site with the highest total toxic equivalence of 2450 ng-TEQ/kg of the contaminants. Four bacteria strains which can survive in dioxin media were isolated after sub-culture inoculation. The four strains were identified as Achromobacter xylosoxidans, Ochrobactrum anthropi, Ralstonia mannitolilytica and Agromyces sp.. It was found that all of the strains are gram’s negative in the morphology. In addition, Ochrobactrum anthropi and Agromyces sp. demonstrated the largest configuration with length 2 μm and width 1 μm. Particularly, Agromyces sp. had closest relationship to the PCP-degrading bacterium Mycobacterium chlorophenolicum PCP-1 according to phylogenetic analysis (bootstrap value = 95). The microbial growth kinetics showed that Achromobacter xylosoxidans and Ochrobactrum anthropi had the maximum specific growth rate of 0.115 h-1. Ralstonia mannitolilytica had the smallest half-saturation constants of 43.2 mg/L, meaning least dependence of substrate. Ochrobactrum anthropi has the greatest substrate inhibition coefficient of 0.948 mg/L. However, the microbial growth tests revealed that adding extra carbon source of glucose (1000 mg/L) to the culture medium containing high dioxin concentration (10 μg/kg 2,3,7,8-TCDD) would decrease the growth inhibition. Achromobacter xylosoxidans, Ochrobactrum anthropi, Agromyces sp. could degrade 2,3,7,8-TCDD at lag phase, whereas Ralstonia mannitolilytica decomposed it primarily at log phase. It was also observed that Agromyces sp. had the maximum 2,3,7,8-TCDD degradation efficiency (97%). The results of proteome analysis suggested that two primary proteome molecular weight of 40 and 50kDa for Achromobacter xylosoxidans and Ochrobactrum anthropi had changed during degradation of 2,3,7,8-TCDD. By contrast, 120 kDa proteome of Ralstonia mannitolilytica as well as 20 and 120 kDa proteome of Agromyces sp. would increase when 2,3,7,8-TCDD was degraded.

碩士
國立中山大學
生物科學系研究所
96
The purpose of this study is to isolate and identify the crude oil-degrading bacteria from oil polluted soil. Their physiological characteristics and oil-degrading capability were also studied. Eight polluted soil samples were taken from the Kaohsiung Refingery Factory of the Chinese Petroleum Corporation (CPC). The microbiota of the Kaohsiung refinery soil sample P37-2 (#6) could degrade crude oil from 2000 ppm to 572 ppm in 10 days. Bacteria in polluted soil samples were selected and isolated by minimal medium with 2000 ppm crude oil as the sole carbon source. Biochemical test, PCR-DGGE, and 16S DNA sequencing were used to identify and characterize the bacteria isolates. Three strains were identified as Pseudomonas aeruginosa (NSYSU-1-1), Acinetobacter sp. (NSYSU-4-1), and Pseudomonas sp. (NSYSU-7-1). These three strains and microbiota #6 were tested for their capability of degrading the total petroleum hydrocarbons (TPH). We found that microbiota #6 performed better than the other three bacterial strains in degrading the crude oil. In this study, we also found temperature was not the major factor of influcing the biodegradation; however, high oxygen concentration and providing nitrogen soure couled improve the biodegradation rate. Although both NSYSU-1-1 and NSYSU-7-1 are Pseudomonas strains, they performed different on degrading the oil. All strains tested could degrade the crude oil to a concentration below 1000 ppm to meet the government emission standard. The bacterial strains and techniques developed in this study provide a choice for future bioremediation of crude oil pollution.

碩士
國立中山大學
生物科學系研究所
101
A combination of PCP, mercury and dioxin polluted site in Southern Taiwan causes great damage to the natural environment and endangers the health of nearby residents. The goal of this study is to isolate mercury resistant microorganisms from the polluted site and develop an in situ bioremediation technique. Because the concentration of mercury pollution in the polluted site is about 50 ppm, the medium used to isolate bacteria was designed to contain 60 ppm mercury ion. After mercury-resistant bacteria were isolated, PCR specific primers were applied to detect whether the bacteria contain merA gene or not. Identification of bacteria were based on their 16S rDNA sequences. In this study, three bacteria were isolated, designated as B37, A45 and A46. Species identification showed that B37 was closely related to Enterobacter cloacae while A45 and A46 were associated with Pseudomonas sp.. All three bacterial strains were also confirmed to grow well in 100 ppm Hg2+ media as well as under seawater salinity (3.5%). RT-PCR and real time PCR analysis revealed that the expression of merA gene could be induced by 10 ppm mercury ion in all 3 bacterial strains. Mercury ion removal batch studies exhibited that A45 strain in NB medium, combination of 3 strains in PMM medium, and B37 strain in LB mediums possessed the best treatment effect. After 12 days of incubation, 54.4%, 89.0% and 88.6% of mercury ions could be removed respectively. This study shows that these three bacterial strains are useful candidates in future bioremediation.

碩士
國立臺灣大學
海洋研究所
94
Agar, a complex polysacchrides extracted from red algae, consists of polygalactan. Polygalactan is composed of above 70% agarose and 30% agaropectin, and agarose consists of a linear chain of alternating residues of 3,6-anhydro-L-galactose and D-galactose. Only a relatively few bacterial species are capable of degrading this refractory material. Agarolytic bacteria are ubiquitous in coastal and estuarine habitats. In this study, there were 110 agarolytic marine bacteria isolated from northeast coast and An-Ping Harbor. Phenotypic and phylogenetic data of the isolates revealed that three isolates, TMA1, SA1, and AW18, could be novel species. These three agarolytic isolates were Gram-negative and straight or curved rods. They required NaCl for growth and presented catalase. They could reduce nitrate to nitrite but not further to N2. Other characteristics of the three isolates are listed below. TMA1 was non-motile and non-flagellated. It grew aerobically and was incapable of anaerobic growth by fermenting glucose or other carbohydrates. Cellular fatty acids were predominated by C16:0 (17.5%), C17:1ω8c (12.8%), C17:0 (11.1%), C15:0 iso 2-OH/C16:1ω7c (8.6%) and C13:0 (7.3%). The DNA G+C content was 41.0 mol%. The strain showed highest 16S rRNA gene sequence similarities to JAMB-A33 (98.2%), Thalassomonas ganghwensis (95.2%) and Thalassomonas viridans (94.7%). No other known bacteria shared more than 94% sequence similarity with strain TMA1. SA1 was non-motile and non-flagellated, but some cells were flagellated. It grew aerobically and was capable of anaerobic growth by reducing nitrite to nitrate. Cellular fatty acids were predominated by C15:0 2-OH/C16:1ω7c (28.6%), C17:1ω8 c (22.8%), C16:0 (14.5%), C18:1ω7 c (11.0%), and C17:0 (6.4%). The DNA G+C content was 55.6 mol%. The 16S rRNA gene sequence was similar to Teredinibacter turnerae (92.7%), Pseudomonas argentinensis (92.3%), and Cellvibrio japonicus (92.1%), and no other known bacteria shared more than 92% sequence similarity with strain SA1. AW18 could motion by a single polar flagellum. It was capable of anaerobic growth by fermenting glucose or other carbohydrates. The major fatty acids present at levels greater than 5% included C15:0 iso 2-OH/C16:1ω7c (35.62%), C18:1ω7c (27.03%), C16:0 (16.26%), C14:0 3-OH/C16:1 iso I (6.90%). The DNA G+C content was 52.9 mol%. The strain showed highest 16S rRNA gene sequence similarities to Agarivorans albus (93.9%), and no known bacteria shared more than 92% sequence similarity with strain AW18. Phylogenetic and phenotypic characteristics accumulated in this study support that TMA1, SA1, and AW18 are novel species in

碩士
國立中興大學
土壤環境科學系
94
The products of petroleum industry are considered as the most frequent organic pollutants of soil and ground water. Because diesel fuel is one of the fractional distillation products, it’s of great urgency to straighten diesel-contaminated site. The past decade, bioremediation techniques have been developed and improved to clean up soils polluted with hazardous chemicals. The aim of this work is to isolate diesel- degrading bacteria from diesel-contaminated soil, and to investigate the diesel-emulsification and diesel-degradation ability of these strains in the aquatic environment. Originally, isolated several bacteria using the selective medium which have diesel as the unique C source. Followly, identified these initial-screening strains by 16S rDNA sequencing, and investigated their diesel-emulsification and diesel-degrading abilities. The initial-screening strains formed colony on the medium in 3 days, and could survive under the high-concertration-diesel condition. The gene identification results showed that these strains belong to Pseudomonas aeruginosa, Pseudomonas stutzeri, Acidovorax temperans, Acinetobacter radioresistens, Sphingomonas paucimobilis and Acinetobacter lwoffii. In the diesel-emulsification experiment, the emulsion index of uncultured control was 1%. Among 19 strains, strain c, e, G had no diesel emulsification ability because of no variation with control in three days. The others could emulsify diesel and strain 1-2 (Pseudomonas aeruginosa) had highest emulsion index in the first day up to 30%. Compared with uncultured control, every strain had ability to utilize diesel in the diesel-degrading experiment, and strain 5 (Pseudomonas stutzeri) had best diesel- degrading ability. Strain 5 degraded 40% diesel after 15 days of incubated. Strain 3-1 (Acidovorax temperans) degraded 15% diesel after 15 days of incubated. Strain V (Acinetobacter lwoffii) degraded 14% diesel after 19 days of incubated. Strain W (Acinetobacter radioresistens) degraded 18% diesel after 12 days of incubated. And strain G (Sphingomonas paucimobilis) degraded 12% diesel after 19 days of incubated. Hence, the isolated strains 3-1, V, W, 5 and G of this work can utilize diesel, and strain 5 had best ability to degrade diesel.

碩士
大仁科技大學
生物科技研究所
99
This research begins by screening the environment for native bacterial strains in the soil by testing for protease activity. The bacterium is the Gram-positive and aerobic, which is classified to bacillus. Identification of the strain was by way of the 16S rDNA. Sequence alignment of the Bacillus strain was analyzed by gene bank, that showed the similarities up to 98%. This strain named Bacillus subtilis PTD25. The strain can grow between 15- 45℃ temperatures and pH 5- 9. The strain exhibited an optimum grow at pH 7.5 and 45℃. In the medium, casein, gelatin and milk can increase protease secretory volume. Adjunction of the casein can induce the most protease production at 30℃. After incubation at 40℃, the concentrated supernatant was crude enzyme. The first of all used phosphate buffer at pH 7.0 to dialysis. It used anion exchange chromatography column for purification. Salt gradients was analyzed to found activity of protease. Used SDS-polyacrylamide gel electrophoresis and zymography to confirm the molecular weight and purification of protein. The results shown its apparent molecular mass was approximately 28 kDa. Further to promote purity of enzyme for proceed the research of particularity.

碩士
國立中興大學
環境工程學系所
100
The objective of this study was to isolate and identify oil-degrading bacteria from different wastewater environments. First of all, a selective media was used as the screening tool for lipase-secreting bacteria, and a total of 32 strains were isolated. The lipase activity of these strains was identified by detecting the clear zone surrounding the bacterial colony when incubated on the specific media plate. After two separated screening processes, 15 strains were selected for detailed study. The strain which have the strongest ability of lipase production was numbered as strain S-6, followed by strain H-4 and G-3. Based on the 16S rDNA sequence analysis results, these strains were identified as Serratia marcescens, Aeromonas hydrophila and Aeromonas veronii, respectively. In this study, the growth characteristics and lipase activity of the isolated strains, were compared with Azospirillum brasilense 14325 (purchased from Bioresource Culture Research Center), one of the lipolytic strains reported in the reference. The size of clear zone produced by Serratia marcescens on the solid medium was about 0.6 cm. But the clear zone was formed slowly by Azospirillum brasilense 14325, and had only pinpoint-like shape of secretion observed. Llipase activity of Serratia marcescens and Azospirillum brasilense 14235 when cultured in liquid medium were (71 U) and (0 U), respectively. Further examination of the lipase activity of Serratia marcescens under different growth conditions showed that the best lipase activity (506 U) could be obtained when this strain was incubated under a concentration of 6% Tween 80, pH8 and 30oC. Finally, when the use of 6% Tween 80 was replaced by 6% salad oil, the oil decomposing rate were 7.43% (3 days), 14.56% (5 days), 29.41% (7 days). We speculated that Serratia marcescens strain may have good degradation effects when applied to the processing of waste edible oil.

Dissertação de mestrado em Biotechnology
The overgrowth of filamentous bacteria leads to worldwide operational problems in activated sludge processes in wastewater treatment plants (WWTP), the most recurring being filamentous bulking and/or filamentous foaming, both resulting in effluents with poor quality. Current control measures of filamentous bacteria are not always effective and are not sustainable and economically viable in the long run. An alternative solution can be the use of bacteriophages to control the overgrowth of filamentous bacteria. The present thesis envisaged the isolation and characterization of a lytic phage which specifically infects Sphaerotilus natans, a filamentous bacterium which causes filamentous bulking, and the evaluation of the effectiveness of the isolated phage in controlling the population of S. natans in batch assays. The growth kinetics parameters of the strain used in this thesis, S. natans DSM 6575T were calculated. Specific growth rate and the generation time of this strain were found to be 0.152 ± 0.002 h-1 and 4.560 h, respectively. The principal objective of the thesis was achieved: a lytic phage with S. natans as a host was isolated from the mixed liquor of a bench scale reactor (4 L) that accurately mimic the secondary treatment of WWTP. Phage infection on pure culture of the host strain resulted in small, uniform and clear plaques, with 1.3 ± 0.3 mm in diameter. S. natans phage belongs to the Podoviridae family, possess an icosahedral head with a size of 60 nm, along with a short tail of 20 nm length by 10 nm width. This newly isolated S. natans phage is the first ever, among those that have S. natans as host, to be classified in this family. S. natans phage did not show a lytic effect against any of the other bacteria tested. S. natans phage proved to be stable after 24 h within the temperatures of 4 °C, 21 °C, 28 °C and 40 °C and within the pH range of 5 to 11. S. natans phage possesses a latent period of 2 h and a burst size of 331.4 PFU per infected cell. The use of S. natans phage at MOI 1 proved to be the most suitable for the purpose of its application, since after 48 h results in the reduction of bacteria concentration in 0.88 logs. Results obtained indicate a possible lytic nature of the phage, a narrow host range and thermal and pH stability within the range values reported in activated sludge processes. In conclusion, the characterization performed on S. natans phage provided the results needed for the future proof of concept regarding the biocontrol of the overgrowth of S. natans by this phage, in activated sludge wastewater treatment.
O crescimento excessivo de bactérias filamentosas induz problemas operacionais nos processos de tratamento de águas residuais por lamas ativadas em todo o mundo, sendo o bulking e o foaming filamentoso os mais recorrentes e resultando em efluentes de baixa qualidade. As atuais medidas de controlo de bactérias filamentosas nem sempre são eficazes e não são sustentáveis e economicamente viáveis a longo prazo. Uma alternativa poderá ser o uso de bacteriófagos para controlar o crescimento excessivo de bactérias filamentosas. A presente tese previa o isolamento e a caracterização de um fago lítico que infetasse especificamente Sphaerotilus natans, uma bactéria filamentosa que causa bulking filamentoso e a avaliação da eficácia da ação do fago isolado no controlo da população de S. natans, em ensaios em batch. Os parâmetros cinéticos de crescimento da estirpe utilizada nesta tese, S. natans DSM 6575T foram calculados, tendo-se obtido para a taxa específica de crescimento e para o tempo de geração, 0.152 ± 0.002 h-1 e 4.560 h, respetivamente. O objetivo principal da tese foi alcançado: um fago lítico tendo S. natans como hospedeiro foi isolado do licor misto de um reator de bancada (4 L) que mimetiza o tratamento secundário de uma ETAR. A infeção fágica na cultura pura do hospedeiro resultou em placas pequenas, uniformes e transparentes, com 1.3 ± 0.3 mm de diâmetro. O fago S. natans pertence à família Podoviridae, possui uma cabeça icosaédrica com 60 nm e uma cauda curta de 20 nm de comprimento por 10 nm de largura. Este fago recém-isolado é o primeiro dos que infetam esta filamentosa a ser classificado nesta família. O fago S. natans não apresentou um efeito lítico contra nenhuma de uma série de outras bactérias testadas. Além disso, é estável após 24 h nas temperaturas de 4 °C, 21 °C, 28 °C e 40 °C e na gama de pH de 5 a 11. Possui um período de latência de 2 h e um burst size de 331.4 unidades formadoras de placas (PFU) por célula infetada. O uso do fago S. natans a uma multiplicidade de infeção (MOI) de 1 demonstrou ser o mais adequado para o propósito de sua aplicação, uma vez que após 48 h resultou na redução da concentração de bactérias em 0.88 logs. Os resultados obtidos evidenciam a possível natureza lítica do fago, a estreita gama de hospedeiros e a estabilidade térmica e de pH dentro da gama de valores relatados em processos de lamas ativadas. Em conclusão, a caracterização do fago de S. natans forneceu os resultados necessários para a futura prova de conceito relativamente ao biocontrolo do crescimento exagerado de S. natans por este fago em ETAR por lamas ativadas.
This study was supported by the project “Innovative strategies to control filamentous overgrowth in activated sludge wastewater treatment” (INOVCONTROLFIL – reference: PTDC/AAG-TEC/3331/2014). In addition, by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.

碩士
弘光科技大學
環境工程研究所
106
Acetaminophen is one of a common antipyretic and analgesic in worldwide. The amount of pollution will damage the environment and people health, if wastes or wastewater contain acetaminophen that are improperly managed. The aims in present study are to select the acetaminophen-utilizing microbes from the acetaminophen-acclimated sludge. And to investigate the acetaminophen removal of mixed strains in synthetic wastewater and purification strains in synthetic wastewater and domestic sewage. The results suggested that the acetaminophen utilizing mixed strains was successfully obtained from the acclimated sludge that can survived and growth in 200 - 1000 mg / L acetaminophen. The acetaminophen and sCOD removal efficiency of mixed strains were above 99 and 96%, respectively. The Klebsiella sp., Bacillus sp. and Deftia sp. were selected from acetaminophen utilizing mixed strains by R2A and TSA. All of the acetaminophen utilizing single strains can survived and growth in 200 - 1000 mg / L acetaminophen. The acetaminophen removal efficiency was above 99 % in 20 – 30 hours in all of the acetaminophen utilizing single strains. In contrast to acetaminophen removal efficiency, sCOD removal efficiency of acetaminophen utilizing single strains was inadequate in 20 – 30 hours, but which was significantly increased when the experiment time were extended from 30 hours to 36 hours.

碩士
嘉南藥理大學
化粧品應用與管理系
105
Lactic acid bacteria can produce antibacterial substances and inbihit the growth of pathogens. Recent studies showed that lactic acid bacteria may alleviate vaginitis. In this study, vaginal swabs were obtained from 23 females in Taiwan, aged between 22 and 30 years old. Seven strains of lactic acid bacteria were isolated by selective culture. Identification of these strains by API 50CHL kit and polymerase chain reaction followed by 16S ribosomal RNA gene sequence analysis indicated that all strains isolated belong to Enterococcus. The results revealed that strains isolated in this study showed no antimicrobial activity against Candida albicans, a major pathogen in vaginitis. Due to the complexity in vaginal environment and various causes of vaginitis, further research will be needed to investigate the potential of these strains used in cosmetics.

碩士
明志科技大學
環境與資源工程研究所
101
During the last several decades, the world is confronted with environmental degradation and fossil fuel depletion, the search for renewable fuels which sustainable development, energy conservation, efficiency and environmental preservation, has become highly pronounced in the present context. Second-generation bioethanol is made from non-edible source—lignocellulose biomass, which can be hydrolyzed to pentoses (xylose) and hexoses (glucose). The biocatalyst used is almost exclusively Saccharomyces cerevisiae. But it lacks the pentose metabolism resulting in the pentose and hexose fermentation processes occurring separately.The four xylose biotransformation bacteria have successfully been isolated from the soils of the Sansing onions field. They have been identified as four strains of the Arthrobacter species—IL01, IL02, IL03 and IL04. They are all gram-negative, 1.2-1.5 μm in size with membrane ruffles, short rods and cocci, without flagellum and pilus. They are all the alkaliphiles and mesophilic bacteria that grow best with pH 7-10 at 30oC-40oC. The μmax and the ks values of Arthrobacter species IL01 for xylose fermentation respectively are 0.2601 hr-1 and 17.223 g/L. The four isolated strains can consume the xylose and glucose simultaneously and show great potential for improving the co-fermentation process in the lignocellulosic ethanol industry. Xylitol has been qualitatively evaluated by the LCMSMS shows it may be the intermediate product in xylose fermentation pathway.

碩士
嘉南藥理科技大學
環境工程與科學系暨研究所
95
Methane is an important greenhouse gas, and the concentrations of methane increase at the rate of 0.5-0.8 % year-1 recently. Most of methane is produced biologically by a group of strictly anaerobic bacteria in highly reduced environments. Landfill is one of the important biogenic methane sources. Methanotrophs are important regulators of methane fluxes from the landfill to atmosphere. There is very limited information presently available on the community structure of methanotrophs in landfills in Taiwan. The study is an investigation on the community structure of methanotrophs as revealed by molecular method in Nan-Sa-Lun landfills in Tainan. The methanotrophic strains will be isolated by enrichment culture and thereby subsequent loss of a variety of other methanotroph. Furthermore, methanotrophic isolates will be sampled by PCR amplification and cloning of particulate methane dehydrogenase (mxaF) gene and 16S rDNA from the total soil population. The isolated will be selected from different cover-age (0-2, 3-5 and 5-7 year-old) landfill soils. The number of methanotrophs in the surface layer of landfill soils will also determined by most-probable number (MPN) method. The 7 test strains (SA2、SA4、SA6、SA9、SA14、SA15 and SD1) obtained from in the 0-2 year-old, one strain (SC2) from 5-7 year-old landfill. SA6, SA9 and SA15 are Gram-positive strains；SA2, SA4, SA14, SC2 and SD1 are Gram-negative strains. All test strains could be detected by oxidase test. Only SA6 and SA14 could be detected by catalase test. Phylogenic analysis of the test strains were identified by 2 primers specific for the 16S rDNA and mxaF gene by NCBI database and MEGA software. Database searches indicated that SA2, SA4 andSC2 are monophyly from the phylogenic tree constructed by 16 S rDNA and would not hybridize to the Type I and Type II methanotrophs. SA6, SA9, SA14, SA15 and SD1 are most closely related to a group Methanobacterium sp.(Type II methanotroph) by phylogenic analysis of partial DNA sequence of mxaF. Changes in various factors of methane concentration, soil water content and incubation temperature will also be estimated based on incubation experiments. The maximum oxidation rates for methane were found in 30℃, 5 % CH4 and moisture content of 20% . Inoculation of methanotrophic isolates would also promote the methane oxidation rates more than 2-5 days.