Low-cost optical cavity based sensor with a large dynamic range.

We have proposed an optical cavity based biosensor using a differential detection method for point-of-care diagnostics. For experimental demonstration of the proposed device using a two-laser system through refractive index measurements, an optical cavity structure is designed, fabricated, and measured. The differential value calculated with intensities of two different wavelengths has a larger responsivity with respect to the refractive index change as compared with the intesity of an individual wavelength. However, the repeatability test shows that the two-laser system has a tight fabrication tolerance due to its small dynamic range. In this paper, we introduce a three-laser system for an optical cavity sensor with a chained differential detection method and present the experimental measurement results showing a large dynamic range and large fabrication tolerance. The measured dynamic range of this system is more than three times greater than that of the two-laser system. The repeatability test using the three-laser system demonstrates a larger fabrication tolerance as a result of the increased dynamic range.

Dynamics of living and dead bacterial cells within a mixed-species biofilm during toluene degradation in a biotrickling filter.

AIMS: To study the accumulation of the bacterial living cells (LC) and dead cells (DC) in a mixed-species biofilm developed in a 3 l biotrickling filter (BTF) challenged with toluene. METHODS AND RESULTS: The bacterial LC and DC within the biofilm developed on polypropylene Pall rings in a toluene-degrading BTF were enumerated as fluoro-microscopic counts during a 62-operating day period using nucleic acid staining and the direct epifluorescence filter technique. The biofilm development could be separated into three distinct phases: (i) cell attachment, (ii) biofilm establishment and (iii) biofilm maturation. The LC were always dominant (>/=72%) in the biofilm during the establishment phase whereas the average LC fraction decreased to 51% of the total cells in the maturation phase. The concentration of LC and DC was observed to level off after 41 days at 1010 cells per ring. The biofilm thickness and the dry weight increased independently of the cell number during the maturation phase. CONCLUSIONS: After the LC reached a maximum concentration in the biofilm, the biofilm proliferation was only characterized by the accumulation of DC and organic matter. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in the present study are of particular relevance for biofilm mathematical modelling and numerical simulations. They will also be useful to estimate the contribution of the living bacteria within the biofilm in bioprocesses.

Population dynamics of free-floating and attached bacteria in a styrene-degrading biotrickling filter analyzed by denaturing gradient gel electrophoresis.

Population dynamics was studied in a 52-l biotrickling filter (BTF) operated for 182 days and used to clean air contaminated with styrene vapors. In the BTF, biomass grew either as free-floating (planktonic) or attached (sessile) microorganisms. PCR-amplified 16S rDNA fragments from planktonic and sessile cells within the bioreactor were analyzed using denaturing gradient gel electrophoresis (DGGE). The results indicated that the complexity of biofilm community was always more pronounced than the complexity of the planktonic cell community. Notably, Rhodococcus erythropolis was identified, based on DNA sequence analysis, as one of the biofilm-specific strains. It was also shown that the inoculum, even when enriched with styrene-degrading bacteria, was not adapted to the growth conditions imposed by the BTF. After a 35-day microbial acclimation period, the DGGE analysis also showed less variation in the banding pattern representing the microbial complexity of the biofilm. In addition, the phylogenic fingerprinting method used demonstrated similar banding profiles in the biofilm along the filter bed.

Transformation of 2,4,6-trinitrotoluene (TNT) by immobilized Phanerochaete chrysosporium under fed-batch and continuous TNT feeding conditions.

The cometabolic transformation of 2,4,6-trinitrotoluene (TNT) by an immobilized Phanerochaete chrysosporium culture was investigated under different TNT and/or glycerol feeding conditions in a 5-L reactor. In the fed-batch feeding mode, as a result of four spiking events at an average feeding rate of 20 mg TNT L(-1) d(-1) and 250 mg glycerol L(-1) d(-1), the initial TNT transformation rate and the glycerol uptake rate of the 7-day-old immobilized cell culture were 2.41 mg L(-1) h(-1) and 16.6 mg L(-1) h(-1), respectively. Thereafter, the TNT fed into the reactor depicted a negative effect on the cell physiology of P. chrysosporium, i.e., both rates decreased constantly. At 32 mg TNT L(-1) d(-1) feeding rate, also in the presence of glycerol (200 mg L(-1) d(-1)), this effect on the fungal cell metabolism was even more significant. When TNT was fed alone at 3.7 mg L(-1) d(-1), it showed an initial 0.75 mg L(-1) h(-1) rate of TNT transformation, i.e., one-third the initial level observed in the presence of glycerol. In contrast, in the continuous feeding mode (dilution rate, D = 0.11 d(-1)), at 5.5 mg TNT L(-1) d(-1) and 220 mg glycerol L(-1) d(-1), the immobilized cell culture exhibited a constant TNT transformation rate for cultivation periods of 50 and 61 days, under uncontrolled and controlled pH conditions, respectively. Thereafter, during the latter experiment, 100% TNT biotransformation was achieved at 1,100 mg L(-1) d(-1) glycerol feeding rate. Immobilized cells (115-day-old), sampled from a continuous TNT feeding experiment, mineralized [(14)C]-TNT to a level of 15.3% following a 41-day incubation period in a microcosm.

An in situ respirometric technique to measure pollution-induced microbial community tolerance in soils contaminated with 2,4, 6-trinitrotoluene.

Long-term exposure to 2,4,6-trinitrotoluene (TNT) can induce changes in the structure and activities of soil microbial communities. Such changes may be associated with an elevated microbial tolerance. An in situ respirometry technique based on the analysis of the substrate-induced respiration response to freshly added TNT was used to examine soil microbial tolerance to TNT at the community level. The specific growth rate derived by fitting an exponential equation to respiration data was taken as the measurement endpoint. Microbial tolerance was evaluated using a tolerance index defined as the ratio of the specific growth rate at a spiking dose of 2000 microg TNT/g soil to that of the control with no spiked TNT. Three soils with long-term exposure histories (TNT level in soil: 1.5, 32, and 620 microg TNT/g, respectively) exhibited significantly higher microbial community tolerance to TNT than two uncontaminated control soils. A soil containing 29,000 microg TNT/g exhibited the highest tolerance. Findings from this study support the hypothesis that pollution-induced community tolerance can be used as a means of identifying those compounds that have exerted selective pressure on the community.

Acute angle-closure glaucoma after albuterol nebulizer treatment.

PURPOSE: To report acute angle-closure glaucoma associated with albuterol nebulizer treatment. METHODS: Case report and review of the relevant literature. RESULTS: In a 75-year-old woman with asthma, acute angle-closure glaucoma in the right eye was probably caused by local absorption of albuterol after nebulizer administration. CONCLUSION: As albuterol is widely used in the management of patients with asthma, increased awareness by health care professionals of the potential for acute angle-closure glaucoma secondary to albuterol may decrease the incidence of this complication.

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.

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

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).

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

Sanguinaria 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 chelerythrine 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.

Effect of culture process on alkaloid production by Catharanthus roseus cells. II. Immobilized cultures.

Two processes for the production of indole alkaloids 2 l surface-immobilized bioreactor cultures of Catharanthus roseus cells using Zenk's Alkaloid Production Medium (APM) were evaluated. The 1-stage process consisted of inoculating APM containing bioreactors and incubating for 15 d. The 2-stage process involved inoculating growth medium-containing bioreactors, growing the immobilized cultures for a certain period of time and subsequently replacing this medium with APM. The production stage which lasted for 15 d. High production in 2-stage cultures required the replacement of the growth regulator 2,4-dichlorophenoxyacetic acid by indole-3-acetic acid in the growth medium and a growth stage of 6 d (late exponential phase) before production initiation. Growth, main nutrient consumption and alkaloid production were monitored. Both culture regimes resulted in similar biomass production, dw (10-13 g l-1). The 2-stage cultures yielded biomass richer in organic nutrients (200-300%) and with higher respiratory activity (approximately 250%), indicated by their lower biomass-to-carbohydrate yields (31% and 26%), as compared to 1-stage cultures (41%). Two-stage cultures produced more known products (10 as compared to 6) at yields (5 to 4800 micrograms g-1) 3 to 5 times higher than 1-stage cultures. More alkaloids were alkaloids released in the medium of 2-stage cultures, under non-lysing conditions, (20 to 4700 micrograms l-1) than in 1-stage cultures (20 to 460 micrograms l-1). These results were compared to those obtained from shake flask cultures performed at the same time, with the same C. roseus cell line and under similar regimes and reported previously. Suspension and immobilized cultures performed according to the 1-stage regime showed similar total production. However, release of known alkaloids was 2 to 3 times higher in immobilized than in suspension cultures. Total alkaloid production of 2-stage suspension cultures was 3.8-fold higher than 2-stage immobilized cultures. Two stage immobilized cultures released 4 more known alkaloids than the 2-stage suspensions. Lower oxygen availability in the 2 l immobilized cultures may explain lower specific growth rates (0.15-0.22 d-1) and total alkaloid production levels, compared to 200 ml suspension cultures (0.2-0.4 d-1) reported in our previous paper.

Stimulated indole alkaloid release from Catharanthus roseus immobilized cultures. Initial studies.

Vacuolar sequestration of valuable secondary metabolites remains the major limitation to the use of immobilization technology for large scale plant-cell-based bioprocesses, which otherwise may be a more efficient culture system than suspension for this biomass. In this initial study, the release of indole alkaloids produced by immobilized Catharanthus roseus cells cultured in Zenk's Alkaloid Production Medium was evaluated. Unstimulated alkaloid release in immobilized cultures reached levels of 10 to 50% of total production or 3 to 100% of known alkaloid content (30 to 4700 micrograms l-1), which was higher than that found for suspension cultures of the cell line used (10 to 25% of total production) without apparent cell lysis. Modifications of the medium pH value of immobilized cultures were explored in order to improve this release. Periodical additions of acid (HCl 0.1 N) or base (KOH 0.1 N) solutions (2% v/v) to different cultures resulted in rapid (less than 3 h) and transient variations in extracellular pH value from 5.5 to 4.3, and 5.8 to 8.5, respectively. In both cases, these variations provoked significant increase in total alkaloid (from approximately 5-10 mg l-1 to 15 mg l-1), ajmalicine (from 0 to approximately 0.29 mg l-1) and serpentine (from 0 to approximately 0.20 mg l-1) release, without apparent cell lysis or decrease in the culture viability. This product release was estimated to represent 100% of alkaloids produced.

Growth and stoichiometry of a Catharanthus roseus cell suspension culture grown under nitrogen-limiting conditions.

The uptake of carbohydrate and nitrate by Catharanthus roseus cell suspension cultures was studied in relation to biomass production in shake flasks. Biomass production was similar when using either 6, 12, 18, or 24 mM nitrate as the nitrogen source and 20 g L(-1) sucrose as the carbon source. In all cases, maximum biomass production was reached when carbohydrates were entirely consumer by the cells. Apparent biomass yields, Y(X/S) and Y(X/N) were 0.49 g biomass g(-1) glucose equivalent and 0.23 g biomass mmol(-1) nitrate, respectively. The determination of the cellular carbon-to-nitrogen ration (C/N ration) resulted in the identification of three district growth phases: an active growth phase, and accumulation phase, and a biomass decline phase (endogenous metabolism). The onset of the last two phases was correlated with nitrate and sugar of the last two phases was correlated with nitrate and sugar exhaustion, respectively. Balanced stoichiometric equations describing the active growth and accumulation phases were proposed based on elemental composition and ash content of the biomass. The stoichiometric equation related to the accumulation phase predicts that the available sugars are stored as starch- and lipid-like materials.

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.

Induction of cellulose in Schizophyllum commune: thiocellobiose as a new inducer.

Several mono-, di, tetra-, and polysaccharides were screened for their ability to induced cellulase production by the tetrapolar hymenomycete Schizophyllum commune. Out of 21 carbohydrates screened, 4 (thiocellobiose, carboxymethylcellulose, cellobiose, and xylan) induced all three enzymes tested (carboxymethylcellulase, beta-glucosidase, and xylanase). The inducing effect increased with rising concentrations of the inducers up to a certain value, beyond which there was either a leveling off or a decrease of the enzymatic activities. The most powerful inducer, thiocellobiose, showed the highest activity at 0.5 mM. Cellobiose, carboxymethylcellulose, and xylan showed their highest activities at 1 mM and 1%, respectively. Surprisingly, sophorose did not enhance enzyme production. The enzymatic activities were monitored over a period of 24 h. Thiocelloboise elicited a response immediately after incubation, but with all other inducers there was a latency period before their effect could be measured. High-performance liquid chromatography showed no hydrolysis of thiocellobiose when incubated in the presence of S. commune extracellular enzymes.