Biodegradation of phenol at high initial concentration by Alcaligenes faecalis.

Strain Alcaligenes faecalis was isolated and identified as a member of the genus Alcaligenes by using BIOLOG and 16S rDNA sequence analysis. The phenol biodegradation tests showed that the phenol-degrading potential of A. faecalis related greatly to the different physiological phases of inoculum. The maximum phenol degradation occurred at the late phase of the exponential growth stages, where 1600 mg L(-1) phenol was completely degraded within 76 h. A. faecalis secreted and accumulated a vast quantity of phenol hydroxylase in this physiological phase, which ensured that the cells could quickly utilize phenol as a sole carbon and energy source. In addition, the kinetic behavior of A. faecalis in batch cultures was also investigated over a wide range of initial phenol concentrations (0-1600 mg L(-1)) by using Haldane model. It was clear that the Haldane kinetic model adequately described the dynamic behavior of the phenol biodegradation by the strain of A. faecalis.

Biodegradation of phenol and 4-chlorophenol by the yeast Candida tropicalis.

Biodegradation of phenol and 4-chlorophenol (4-cp) using a pure culture of Candida tropicalis was studied. The results showed that C. tropicalis could degrade 2,000 mg l(-1) phenol alone and 350 mg l(-1) 4-cp alone within 66 and 55 h, respectively. The capacity of the strain to degrade phenol was obviously higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Phenol beyond 800 mg l(-1) could not be degraded in the presence of 350 mg l(-1) 4-cp. Comparatively, low-concentration phenol from 100 to 600 mg l(-1) supplied a sole carbon and energy source for C. tropicalis in the initial phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in the fact that 4-cp biodegradation velocity was higher than that without phenol. And the capacity of C. tropicalis to degrade 4-cp was increased up to 420 mg l(-1) with the presence of 100-160 mg l(-1) phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and mixed substrates in batch cultures. The results illustrated that the models proposed adequately described the dynamic behaviors of biodegradation by C. tropicalis.

Mutation of Candida tropicalis by irradiation with a He-Ne laser to increase its ability to degrade phenol.

Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 x 10(7) cells ml(-1) were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter(-1) phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter(-1). The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did.

Mutant AFM 2 of Alcaligenes faecalis for phenol biodegradation using He-Ne laser irradiation.

He-Ne laser technology was utilized in this study to investigate the response of Alcaligenes faecalis to laser stimulation. The irradiation experiments were conducted by the adjustment of the output power from 5 to 25 mW and the exposure time from 5 to 25 min. The results showed that the survival rate changed regularly with the variety of irradiation dose, and high positive mutation frequency was determined by both the energy density and the output power. The mutant strain AFM 2 was obtained. Phenol biodegradation assay demonstrated that AFM 2 possessed a more prominent phenol-degrading potential than its parent strain, which presumably attributed to the improvements of phenol hydroxylase and catechol 1,2-dioxygenase activities. The phenol of 2000 mgl(-1) was completely degraded by AFM 2 within 85.5h at 30 degrees C. In addition, the cell growth and phenol degradation kinetics of the mutant strain AFM 2 and its parent strain in batch cultures were also investigated at the wide initial phenol concentration ranging from 0 to 2000 mgl(-1) by Haldane model. The results of these experiments further demonstrated that the mutant strain AFM 2 possessed a higher capacity to resist phenol.

[Synergistic effects of biotic and abiotic elicitors on the production of tanshinones in Salvia miltiorrhiza hairy root culture].

OBJECTIVE: To investigate the synergistic effects of a biotic elicitor yeast extract and different abiotic elicitors (Ag+, Co2+ and alpha-amino isobutyric acid) on the production of tanshinones in Salvia miltiorrhiza hairy root. METHOD: Different elicitors and their combinations were added to S. miltiorrhiza hairy root culture and the contents of three major tanshinones (crypotanshinone, tanshinone I and tanshinone II A) were analyzed by HPLC. RESULT: The combinations of yeast extract with different abiotic elicitors had notable synergistic effects on the tanshinone I and tanshinone II A production but not on crypotanshinone. The combination of yeast extract and Ag+ (300 micromol x L(-1)) yielded the highest tanshinone I content, which was nearly 14-fold of the control, and the synergistic elicitation coefficient was 3.0. The combination of yeast extract and Co2+ (100 micromol L(-1)) led to the highest tanshinone IIA content, which was about 14.5-fold of the control, and the synergistic elicitation coefficient was 2.1. Only yeast extract combined with alpha-amino isobutyric acid (200 micromol x L(-1)) increased the crypotanshinone content more effectively than single elicitors. The highest crypotanshinone content was 1.28 mg x g(-1), about 30-fold of the control with a synergistic elicitation coefficient of 1.3. CONCLUSION: The elicitation by the combination of a biotic elicitor and an abiotic elicitor can generate a synergistic effect, which is more effective than single elicitors to promote secondary metabolite production in plant tissue cultures.

Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation.

In Salvia miltiorrhiza hairy root cultures, the desired secondary metabolites diterpenoid tanshinones are normally produced at low yields and stored within the roots. To enhance tanshinone production and the secondary product recovery, we employed three means, elicitation with a yeast elicitor (YE), in situ adsorption of tanshinones with a hydrophobic polymeric resin (X-5) and semi-continuous mode of operation. YE treatment stimulated the tanshinone biosynthesis, increasing the total tanshinone (TT) content of root by about two-fold, from 0.46 to 1.37 mg/g dry weight (dw) (TT content=total content of three major tanshinones, cryptotanshinone, tanshinone I and tanshinone IIA). The addition of X-5 resins to the culture only increased the tanshinone yield slightly, but recovered more than 80% of tanshinones from the roots. With the application of a semi-continuous culture process involving repeated medium renewal, elicitor addition and resin replacement, starting at the late exponential growth phase, the root biomass was increased to 30.5g dw/l (versus 8-10g dw/l in batch mode) and the volumetric tanshinone yield to 87.5mg/l (about 15-fold increase), with 76.5% adsorbed to the resin. The volumetric productivity of total tanshinone reached 1.46 mg/lday, more than 7.4 times that of the batch culture. The results demonstrate that the integration of multiple elicitation, in situ adsorption and semi-continuous operation can synergistically enhance tanshinone production in S. miltiorrhiza hairy root cultures.

Induction of HL-60 apoptosis by ethyl acetate extract of Cordyceps sinensis fungal mycelium.

The cultivated mycelium of a Cordyceps sinensis (Cs) fungus was sequentially extracted by petroleum ether, ethyl acetate (EtOAc), ethanol and water. The EtOAc extract showed the most potent cytotoxic effect against the proliferation of human premyelocytic leukemia cell HL-60, with an ED50 < or = 25 microg/ml for 2-day treatment. The EtOAc extract induced the characteristic apoptotic symptoms in the HL-60 cells, DNA fragmentation and chromatin condensation, occurring within 6-8 h of treatment at a dose of 200 microg/ml. The activation of caspase-3 and the specific proteolytic cleavage of poly ADP-ribose polymerase were detected during the course of apoptosis induction. These results suggest that the Cs mycelium extract inhibited the cancer cell proliferation by inducing cell apoptosis.

A structured kinetic model for suspension cultures of Taxus chinensis var. mairei induced by an oligosaccharide from Fusarium oxysporum.

A structured kinetic model was established to describe the process of Taxol formation in suspension cultures of Taxus chinesis var. mairei induced by an oligosaccharide from Fusarium oxysporum. In this model, the role of intracellular starch as a storage carbon source had to be taken into account. Substrate uptake, culture growth, cell respiration, and secondary metabolites, predicted by the model, agreed with those obtained experimentally. The effective factors of oligosaccharide elicitation, eta(e,j), defined as the ratio of the parameter values in the system with oligosaccharide to those in control, reflected the effects of the oligosaccharide on cell growth and Taxol production.