Aromatic and aliphatic hydrocarbon consumption and transformation by the styrene degrading strain Pseudomonas putida CA-3.

Pseudomonas putida CA-3 is capable of consuming a number of aromatic and aliphatic hydrocarbons. With the exception of styrene none of the alkenes tested are capable of supporting the growth of P. putida CA-3 as sole sources of carbon and energy. The highest rate of alkene consumption was observed with styrene as the substrate. A 6.5- and 15.5-fold lower rate of substrate consumption was observed with indene and indole with the concomitant formation of 2-indanone and indigo, respectively. The presence of a sulphur (benzothiopene) or oxygen (benzofuran) in the cyclopentene ring resulted in further decreases in the rate of substrate consumption by whole cells of P. putida CA-3. P. putida CA-3 is incapable of consuming benzene and consumes toluene at a low rate. No detectable products were observed in supernatants of cultures incubated with benzothiopene, benzofuran or toluene. The aliphatic alkenes 1-octene and 1,7-octadiene were both consumed by whole cells of P. putida CA-3 at a rate equivalent to indene consumption. The consumption of (R) styrene oxide was 1.7- and 1.25-fold higher than that of the S isomer and the racemic mix, respectively. The rate of racemic indene oxide, 1,2-epoxyoctane and 1,2-epoxy-7-octene consumption was lower than their equivalent alkene and 55-, 11.8-, and 27.5-fold lower than the rate of racemic styrene oxide consumption. A transposon mutant incapable of growth with styrene or styrene oxide failed to transform indole to indigo. The ratio of styrene utilisation relative to other substrates changes in the mutant strain compared to the wild-type strain, e.g., Indene biotransformation by mutant AF5 is 1.9-fold higher than styrene consumption compared to the wild-type strain CA-3 where the rate of styrene consumption is 6.7-fold higher than indene consumption. This trend is also observed for other alkenes and epoxides.

Comparison of morphological characteristics of Streptomyces natalensis by image analysis and focused beam reflectance measurement.

A morphological interpretation is presented for data collected during growth of a filamentous organism, using a focused beam reflectance measurement (FBRM) system. The morphology of the organism was also obtained using conventional semiautomatic image analysis to support the interpretation of the FBRM data. The model organism employed is the filamentous soil-borne actinomycete Streptomyces natalensis, which produces the antifungal agent pimaricin. The organism was cultivated both in shake flasks and in a bench-scale stirred tank bioreactor. It was found that FBRM could be used to track changes in the morphology of the organism throughout the course of its growth on both scales. These changes were highlighted using both the median chord length and length-weighted mean chord length obtained from the chord length distribution measured with the FBRM probe. The ability of the FBRM probe to respond to changes in both the size and morphology of mycelial aggregates was supported by standard image analysis parameters, including equivalent diameter, convex area, and compactness.

Growth behavior in plant cell cultures based on emissions detected by a multisensor array.

The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line.

Generation of reactive oxygen and antioxidant species by hydrodynamically stressed suspensions of Morinda citrifolia.

The generation of reactive oxygen species (ROS) by plant cell suspension cultures, in response to the imposition of both biotic and abiotic stress, is well-documented. This study investigated the generation of hydrogen peroxide by hydrodynamically stressed cultures of Morinda citrifolia, over a 5-h period post-stress imposition. Suspensions were exposed to repeated passages through a syringe, under laminar flow conditions, corresponding to cumulative energy dissipation levels of approximately 3-6 J kg-1. Extracellular hydrogen peroxide was detected using a luminol-based chemiluminescence assay. The addition of exogenous hydrogen peroxide facilitated the detection of low levels of hydrogen peroxide in the presence of antioxidants. Immediately after shear exposure, there was evidence of significant antioxidative capacity in the sheared cell cultures, which potentially masked any oxidative burst (OB), but which decreased over the following 40 min. This antioxidative capacity was determined to derive from the shearing process. Trials in which ground cellular debris was added to control suspensions suggested that some of the antioxidative capacity observed in stressed suspensions was directly associated with debris generated by the shearing process. Using UV-vis spectrophotometry and HPLC, stress-related increases in the levels of phenolic compounds were detected in suspension filtrates. Under the stress conditions investigated, maximum hydrogen peroxide levels of 11.5 muM were observed, 5 h after shear exposure. This study emphasizes the importance of considering both oxidative and antioxidative capacities as part of a holistic approach to the determination of the OB in hydrodynamically stressed plant cell suspension cultures.

Focussed beam reflectance measurement (FBRM) monitoring of particle size and morphology in suspension cultures of Morinda citrifolia and Centaurea calcitrapa.

Laser light scattering technology, as applied in the Lasentec focussed beam reflectance measurement (FBRM) system, was used to characterise two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa. Shake-flask suspensions were analysed in terms of biomass concentration and aggregate size/shape over the course of typical batch growth cycles. For the heavily aggregated C. calcitrapa, biomass levels [from 10-160 g fresh weight (fw) l(-1))] were linearly correlated with FBRM counts. For M. citrifolia, which grows in unbranched chains of 2-10 elongated cells, linear correlation of biomass concentration with FBRM counts was applicable in the range 0-100 g fw l(-1); at higher levels (100-300 g fw l(-1)), biomass was non-linearly correlated with FBRM counts and length-weighted average FBRM chord length. For both cell systems, particle morphology (size/shape) was quantified using semi-automated digital image analysis. The average aggregate equivalent diameter (C. calcitrapa) and average chain length (M. citrifolia), determined using image analysis, closely tracked the FBRM average chord length. The data clearly demonstrate the potential for applying the FBRM technique for rapid characterisation of plant cell suspension cultures.