Denis Rho

Analysis of the relative abundance of different types of bacteria capable of toluene degradation in a compost biofilter.

Abstract A microbial community of a compost biofilter treating toluene vapors was investigated using serum-bottle assays and mineral-agar plates. Toluene was not consumed in the absence of oxygen. However, filter-bed extracts exposed to toluene vapor as the only carbon source produced distinct colony types (phenotypic groups) that were counted separately. Strains from each group were isolated and checked for toluene-degradation activity in serum bottles. Only 15% of colonies were true toluene degraders. This population was divided into 11 genotypic groups based on DNA fingerprints. Identification of a member of each group using 16S rRNA gene-sequence comparison showed that they belonged to seven genera: Acinetobacter, Azoarcus, Mycobacterium, Nevskia, Pseudomonas, Pseudonocardia and Rhodococcus. Together, members of the genera Pseudonocardia and Rhodococcus were 34 times more numerous than all the others. We hypothesized that these two organisms are K-strategists (adapted to a resource-restricted and crowded environment) and that the compost biofilter is a K-environment. This would explain why they are not outnumbered by faster growers like Pseudomonas or Acinetobacter species, which would be r-strategists (adapted to a resource-abundant and uncrowded environment).

Oxygen requirement in pullulan fermentation

SummaryOxygen was essential for the biosynthesis of pullulan by Aureobasidium pullulans. In a growth medium, pullulan yield and synthesis rate were proportional to the oxygen availability. However, under controlled oxygen environment in a non-growth medium, the synthesis rate and the yield of pullulan were inversely proportional to the oxygen tension. A relationship between melanin production and oxygen transfer conditions was also observed. The elapsed time prior to the appearance of the pigment was dependent upon the degree of oxygen availability.

Tween 80 enhanced TNT mineralization by Phanerochaete chrysosporium

The effect of a nonionic surfactant (Tween 80) on 2,4,6-trinitrotoluene (TNT) mineralization by the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767, was investigated in a liquid culture at 20, 50, and 100 mg TNT.L-1. The presence of 1% (w/v) Tween 80, at 20 mg.L-1 TNT, added to a 4-d-old culture, allowed the highest TNT mineralization level, that is 29.3% after 24 d, which is two times more than the control culture, without Tween 80 (13.9%). The mineralization of TNT resumed upon additional Tween 80 supplementation, consequently, 39.0% of the TNT was respired on day 68. Orbital agitation of the fungal culture was found detrimental to TNT mineralization, with or without Tween 80 in the culture medium. The surfactant also stimulated the growth of P. chrysosporium without any notable effect on either the glycerol consumption rate or the extracellular LiP and MnP activity levels. Respirometric assays highlighted some differences between the oxygen uptake rate of the fungal culture supplemented with or without Tween 80.

Nutritional and hormonal requirements of Ginkgo biloba embryo-derived callus and suspension cell culture.

Calli were obtained from Ginkgo biloba embryos grown on Murashige and Skoog (MS) medium. The G. biloba cells could grow on either MS or Gamborg B5 mineral salt medium supplemented with sucrose (3% and 2%, respectively) and naphthaleneacetic acid (NAA) and kinetin (K) in concentrations ranging from 0.1 to 2.0 mg·L−1. Best growth and maintenance of callus cultures were achieved using MS medium supplemented with 2 mg·L−1 NAA and 1 mg·L−1 K (N2K1MS). Light was required to maintain healthy growth of the callus tissue.In both MS and B5 based media, sucrose was hydrolyzed extracellularly before being taken up by Ginkgo cell suspension cultures. Specific growth rates of 0.13 d−1 and 0.08 d−1 were obtained in MS medium supplemented with 1 mg·L−1 NAA, 0.1 mg·L−1 K and 30 g·L−1 sucrose (N1K0.1MS) and B5 medium supplemented with the same growth regulator regime and 20 g·L−1 sucrose (N1K0.1B5) respectively. Complete phosphate and ammonium uptake was observed in 11 days when cultured in MS medium and 10 days and 4 days respectively when cultured in B5 medium. During the culture, G. biloba cells consumed only 64% and 29% of the nitrate content of N1K0.1MS and N1K0.1B5 media respectively. Maximum dry biomass concentrations were 13.4 g·L−1 and 7.9 g·L−1, and yields on carbohydrate were 0.39 and 0.45 in N1K0.1MS and N1K0.1B5 media respectively. The better performance of MS cultures came from the higher sucrose and nitrogen salts concentrations of this medium.

Physiological aspects of surface-immobilized Catharanthus roseus cells

SummaryGrowth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O2 consumption and CO2 production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures.

Solid-phase extraction and fluorimetric detection of benzophenanthridine alkaloids from Sanguinaria canadensis cell cultures

Abstract A solid-phase extraction (SPE) method using Sep-Pak C 18 reversed-phase cartridges was developed for the prepurification of benzophenanthridine alkaloids produced by Sanguinaria canadensis cell cultures. Efficient recoveries (>90%) of chelirubine, sanguinarine and chelerythrine were obtained from synthetic solutions using the SPE method. Up to 20 mg of alkaloids were extracted from spiked biomass and culture-medium samples on a 400-mg adsorbent cartridge. This extraction method was more reproducible and as selective as a conventional liquid—liquid extraction technique. Fluorimetric detection was used for the identification and quantification of benzophenanthridine alkaloids after high-performance liquid chromatographic separation. This detection method was compared to conventional UV absorption at 280 nm. The detection limits of both detectors were in the 100-pg range for chelirubine and sanguinarine and in the 1-ng range for chelerythrine. The main advantage of fluorimetry over UV spectrometry was its improved selectivity for the benzophenanthridine alkaloids.

Growth characteristics of Sanguinaria canadensis L. Cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids

SummarySanguinaria canadensis L. plants were harvested from a local forest and calli were initiated from leaf explants. The production of benzophenanthridine alkaloids (i.e. sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S. canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes. Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%,g g−1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g−1). Sanguinarine, chelirubine and chererythrine were the only known alkaloids detected in cultured S. canadensis cells. Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion. The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor. The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days. The maximum sugar and nitrate consumption rates were 1.8 g l−1 per day and 2.3 mm per day respectively. The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O2 l−1 h−1 and 0.95 mmol CO2 l−1 h−1 respectively. The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g−1 dw) by day 15.

Microcalorimetry as a diagnostic and analytical tool for the assessment of biodegradation of 2,4-D in a liquid medium and in soil

The biodegradation of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) byPseudomonas cepacia was assessed by microcalorimetry in a liquid medium and in sterilized soil at 25°C under aerobic conditions. It was found that thermograms of the rate of heat evolved versus time (dQ/dt versust) can be used as a diagnostic tool to identify the timet1 required for the primary biodegradation of 2,4-D and the timetf required for the completion of the biodegradation activity in a liquid medium as well as in soil. Microcalorimetry can also be used as an analytical tool to monitor the progress of 2,4-D consumption during the biodegradation process in a liquid medium and to measure the importance of the soil sorption/desorption of intermediate metabolites. A new concept called “bioeffort” was defined as the product of the biodegradation time (t) and the biomass concentration (X) at that time. This concept was used to predict either the biomass concentration required or the duration of the primary biodegradation of 2,4-D in soil from the data obtained from a liquid medium.

Assessment of the Aerobic Biodegradation Potential of RDX, TNT, GAP, and NC

Laboratory studies were conducted to evaluate the biodegradation potential of four energetic compounds — RDX, TNT, GAP, and NC — under aerobic conditions. Soil samples collected from three sites were screened for microorganisms with the ability to degrade energetic compounds. Two bacteria were enriched and purified from RDX-contaminated soil by their ability to use RDX as a sole source of nitrogen. Using [14C]-labelled RDX, the ability of these isolates to mineralize RDX in liquid culture was verified. Bioaugmentation of RDX-contaminated soil with these isolates enhanced the rate and extent of RDX biodegradation. In contrast, although TNT is more recalcitrant and toxic, [14C]TNT mineralization activity was detected in TNT-contaminated soil. This activity was concentration dependent: in soil, as the concentration of TNT increased, mineralization decreased and was abrogated at 100 ppm TNT. Attempts to enrich and isolate from this soil organisms with the ability to mineralize [14C]TNT have as yet been unsuccessful. Although it remains unknown whether any of the soils we are working with are contaminated with GAP or NC, preliminary studies revealed that some of the soils harboured [14CJGAP and [ 14C]NC mineralization activities. Data obtained to date firmly establish the biodegradation potential of RDX under aerobic conditions, and suggest that biodegradation of TNT, GAP, and NC is also possible.

Effect of Fungal Deterioration on Physical and Mechanical Properties of Hemp and Flax Natural Fiber Composites

The development and application of bio-sourced composites have been gaining wide attention, yet their deterioration due to the growth of ubiquitous microorganisms during storage/manufacturing/in-service phases is still not fully understood for optimum material selection and design purposes. In this study, samples of non-woven flax fibers, hemp fibers, and mats made of co-mingled randomly-oriented flax or hemp fiber (50%) and polypropylene fiber (50%) were subjected to 28 days of exposure to (i) no water-no fungi, (ii) water only and (iii) water along with the Chaetomium globosum fungus. Biocomposite samples were measured for weight loss over time, to observe the rate of fungal growth and the respiration of cellulose components in the fibers. Tensile testing was conducted to measure mechanical properties of the composite samples under different configurations. Scanning electron microscopy was employed to visualize fungal hyphal growth on the natural fibers, as well as to observe the fracture planes and failure modes of the biocomposite samples. Results showed that fungal growth significantly affects the dry mass as well as the tensile elastic modulus of the tested natural fiber mats and composites, and the effect depends on both the type and the length scale of fibers, as well as the exposure condition and time.