Enrichment culture techniques

Microbial Involvement through Enrichment Culturing Techniques...175... 

Microbial Transformation of Organic Chemicals in Natural Environments Fate of Chemicals and Substantiation of Microbial Involvement through Enrichment Culturing Techniques... 

In 1977, a Pseudomonad was isolated from soil by enrichment culture technique with linalool as the sole source of carbon and energy [36]. The bacterial strain was later identified as Pseudomonas incognita and given the name linalool strain . It was also capable of growing on geraniol, nerol and limonene. The biotransformation of geraniol by this... 

Limonene (92) is the most widely distributed terpene in nature after a-pinene [68]. The (+)-isomer is present in Citrus peel oils at a concentration of over 90% a low concentration of the (-)-isomer is found in oils from the Mentha species and conifers [26]. The first data on the microbial transformation of limonene date back to the sixties. A soil Pseudomonad was isolated by enrichment culture technique on limonene as the sole source of carbon [69]. This Pseudomonad was also capable of growing on a-pinene, / -pinene, 1-p-menthene and p-cymene. The optimal level of limonene for growth was 0.3-0.6% (v/v) although no toxicity was observed at 2% levels. Fermentation of limonene by this bacterium in a mineral-salts medium resulted in the formation of a large number of neutral and acidic products. Dihydrocarvone, carvone, carveol, 8-p-menthene-1,2-cw-diol, 8-p-menthen-1 -ol-2-one, 8-p-menthene-1,2-trans-diol and 1 -p-menthene-6,9-diol were among the neutral products isolated and identified. The acidic compounds isolated and identified were perillic acid, /Msopropenyl pimelic acid, 2-hydroxy-8-p-menthen-7-oic acid and... 

More recently the biotransformation of limonene by another Pseudomonad strain, P. gladioli was reported [76,77]. P. gladioli was isolated by an enrichment culture technique from pine bark and sap using a mineral salts broth with limonene as the sole source of carbon. Fermentations were performed during 4-10 days in shake flasks at 25°C using a pH 6.5 mineral salts medium and 1.0% (+)-limonene. Major conversion products were identified as (+)-a-terpineol and (+)-perillic acid. This was the first time that the microbial conversion of limonene to (+)-a-terpineol was reported, see pathway 4. The conversion of limonene to a-terpineol was achieved with an enzyme, a-terpineol dehydratase (a TD), by the same group [78]. The enzyme, purified more than tenfold after cell-disruption of Pseudomonas gladioli, stereospecifically converted (4 )-(+)-limonene to (4/ )-(+)-a-terpineol or (4S)-(+)-limonene to (4S)-(+)-a-terpineol. a-Terpineol is widely distributed in nature and is one of the most commonly used perfume chemicals [27]. 

By enrichment culture technique, a bacterium was isolated from local sewage sludge, utilising caryophyllene as the sole source of carbon and energy [103]. Fermentation of / -pinene by this culture in a mineral salt medium (Seuberf s medium) at 30°C with agitation and aeration for four days yielded a few neutral and acidic transformation products. The metabolites isolated and identified were camphor (2), bomeol (1),... 

Two bacterial strains, one from soil and the other from infected local beer, which utilised calarene as the sole source of carbon and energy have been isolated by enrichment culture techniques [149]. Both these bacteria were adapted to grow on valencene as the sole carbon source. Fermentations of valencene (5) by these bacteria of the genus Enterobacter in a mineral salts medium yielded several neutral metabolic products dihydro alpha-agarofuran (200) (7.5%), nootkatone (6) (12%), another ketone (201) (18%) and a-cyperone (202) (8%), Fig. (40). 

Conventional approaches to microbiological examination of specimens require that they be cultured to assess the total numbers of specific groups of microorganisms or to determine the presence or absence of particular named species. The majority of samples taken for examination contain mixtures of different species, so simple plating onto an agar surface may fail to detect an organism that is present at < 2% of the total viable population. Various enrichment culture techniques may therefore be deployed to detect trace numbers of particular pathogens, prior to confirmatory identification. 

ON PLATES OR ON VIALS WITH SPECIAL CULTURE CONDITIONS ENRICHMENT CULTURE TECHNIQUES... 

A new NAD -dependent opine dehydrogenase was purified to homogeneity from Arthrobacter sp. strain 1C isolated from soil by an enrichment culture technique with a synthetic substrate N-[1-DL-(carboxyl)ethyl]-L-phenylalanine [15]. We purified and characterized an NAD+-dependent secondary amine dicarboxylic acid dehydrogenase, and named it opine dehydrogenase (ODH). The enzyme catalyzed a reversible oxidation-reduction reaction of opine-type secondary amine dicarboxylic acids (Scheme 1). 

One example of the use of enrichment culture techniques was the isolation of a microbial culture that could degrade a chemical warfare agent. The chemical agent mustard gas—or HD... 

Enrichment culture techniques can also be used for bioremediation to detoxify xenobiotic pollutants such as polycyclic aromatic hydrocarbons (PAHs), heterocyclic polyaromatics, and halogenated aromatics in soils and sediments through microbial degradation. An effective way to do this is by isolating microbes through enrichment cultures with the substrate one wants to detoxify as a limiting compound. Once this is proven in the laboratory, it can be taken full-scale to the field. 

Enrichment culture techniques rely on creating a condition in which the survival and growth of bacterial cultures, with whatever... 

In addition to selecting naturally occurring microbial cultures that possess a desired metabolic trait, it is also possible to use enrichment culture techniques to develop microbial cultures with unique biochemical traits. The substrate range of enzymes catalyzing a certain reaction can be expanded through the use of enrichment culture techniques. 

This process of evolving new biochemical traits in the laboratory can also be accelerated by the use of directed evolution. In directed evolution, the genes that encode a biochemical trait of interest are subjected to specific mutagenesis, and then enrichment culture techniques are used to isolate derivatives that contain the desired improvements. 

The problem of pyrimidine degradation by microorganisms has been studied by enrichment culture techniques, whereby bacterial strains are grown which utilize pyrimidines as their source of nitrogen and carbon. Studies have been carried out with intact cells as w ell as with purified enzymes obtained from bacteria. Wang and Lampen demonstrated that pyrimidines were converted to barbituric acid by cell-free extracts... 

A thermophilic strain capable of degrading PA-12 was isolated from 100 soil samples using an enrichment culture technique at 60 °C. At this temperature, the strain not only grew on PA-12, but also reduced the molecular weight of the polymer. The strain was identified as a neighbouring species to Bacillus pallidus it has an optimum growth temperature of around 60 °C and was also found to degrade PA-6 as well as PA-12, but not PA-66 [52]. 

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