Reduction of perchlorate by an anaerobic enrichment culture

Summary A mixed bacterial culture capable of reducing perchlorate stoichiometrically to chloride under naerobic conditions was enriched from municipal digester sludge. The reduction of 10 mM perchlorate resulted in oxidation of the medium and cessation of perchlorate reduction. The activity was recovered on addition of a reducing agent. Addition of air to the culture during perchlorate reduction immediately terminated the process and aeration for 12 h permanently destroyed the ability of the culture to reduce perchlorate. The culture also reduced nitrite, nitrate, chlorite, chlorate and sulfate. The presence of 10 mM nitrite or chlorite completely inhibited perchlorate reduction, whereas the same concentration of chlorate decreased the reduction rate. Nitrate or sulfate did not affect perchlorate reduction. Chlorate and chlorite, suspected intermediates in the reduction of perchlorate to chloride, were not detected in any cultures during reduction of perchlorate.     

Inhibition of aerobic respiration and dissimilatory perchlorate reduction using cyanide

The effect of low concentrations of cyanide on dissimilatory perchlorate and chlorate reduction and aerobic respiration was examined using pure cultures of Azospira sp. KJ. Cyanide at a concentration of 38 microM inhibited cell growth on perchlorate, chlorate and molecular oxygen, but it did not inhibit the activity of chlorite dismutase. When oxygen accumulation was prevented by adding an oxygen scavenger (Oxyrase or L-cysteine), however, cells completely reduced perchlorate in the presence of cyanide. It was concluded that the inhibition of dissimilative perchlorate reduction by cyanide at this concentration was a consequence of oxygen accumulation, not inhibition of the enzymes used for perchlorate reduction. This finding on the effect of cyanide on respiratory enzymes provides a new method to control and study respiratory enzymes used for perchlorate reduction.     

Peptide biomarkers as evidence of perchlorate biodegradation

Perchlorate is a known health hazard for humans, fish, and other species. Therefore, it is important to assess the response of an ecosystem exposed to perchlorate contamination. The data reported here show that a liquid chromatography-mass spectrometry-based proteomics approach for the detection of perchlorate-reducing enzymes can be used to measure the ability of microorganisms to degrade perchlorate, including determining the current perchlorate degradation status. Signature peptides derived from chlorite dismutase (CD) and perchlorate reductase can be used as biomarkers of perchlorate presence and biodegradation. Four peptides each derived from CD and perchlorate reductase subunit A (PcrA) and seven peptides derived from perchlorate reductase subunit B (PcrB) were identified as signature biomarkers for perchlorate degradation, as these sequences are conserved in the majority of the pure and mixed perchlorate-degrading microbial cultures examined. However, chlorite dismutase signature biomarker peptides from Dechloromonas agitata CKB were found to be different from those in other cultures used and should also be included with selected CD biomarkers. The combination of these peptides derived from the two enzymes represents a promising perchlorate presence/biodegradation biomarker system. The biomarker peptides were detected at perchlorate concentrations as low as 0.1 mM and at different time points both in pure cultures and within perchlorate-reducing environmental enrichment consortia. The peptide biomarkers were also detected in the simultaneous presence of perchlorate and an alternate electron acceptor, nitrate. We believe that this technique can be useful for monitoring bioremediation processes for other anthropogenic environmental contaminants with known metabolic pathways.     

Limitations to Natural Bioremediation of Perchlorate in a Contaminated Site

Perchlorate (ClO₄ ^?) has been detected in many drinking water supplies in the United States, including the Las Vegas Wash and Lake Mead, Nevada. These locations are highly contaminated and contribute perchlorate to Lake Mead and the Colorado River system. Essential elements for perchlorate bioremediation at these locations were examined, including the presence of perchlorate-reducing bacteria (PRB), sufficient electron donors, occurrence of competing electron acceptors, and ability of PRB to utilize a variety of electron donors. Enumeration of PRB was performed anoxically using most probable number (MPN). Values ranged from ≤20 to 230 PRB/100 ml or ≤20 to ≥ 1.6× 10⁵ PRB/g for Lake Mead water samples and Las Vegas Wash sediments, respectively. 16S rRNA sequences revealed that isolates were γ -proteobacteria, Aeromonas, Dechlorosoma, Rahnella and Shewanella. A screening of potential electron donors using BIOLOG^TM demonstrated that all isolates were capable of metabolic versatility. Measurements of total organic carbon (TOC), nitrate and dissolved oxygen (DO) indicated limited presence of electron donor at all sites, whereas the electron acceptors varied throughout the Wash and Lake Mead. The persistence of perchlorate in the sites is attributed to lack of available electron donor and/or the presence of competing electron acceptors. A location has been identified where perchlorate biodegradation could be implemented thereby halting the transport of perchlorate to Lake Mead and the Colorado River.     

Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.     

Limitations to Natural Bioremediation of Perchlorate in a Contaminated Site

Perchlorate (ClO₄^-) has been detected in many drinking water supplies in the United States, including the Las Vegas Wash and Lake Mead, Nevada. These locations are highly contaminated and contribute perchlorate to Lake Mead and the Colorado River system. Essential elements for perchlorate bioremediation at these locations were examined, including the presence of perchlorate-reducing bacteria (PRB), sufficient electron donors, occurrence of competing electron acceptors, and ability of PRB to utilize a variety of electron donors. Enumeration of PRB was performed anoxically using most probable number (MPN). Values ranged from ≤20 to 230 PRB/100 ml or ≤20 to ≥ 1.6× 10⁵ PRB/g for Lake Mead water samples and Las Vegas Wash sediments, respectively. 16S rRNA sequences revealed that isolates were γ -proteobacteria, Aeromonas, Dechlorosoma, Rahnella and Shewanella. A screening of potential electron donors using BIOLOG^TM demonstrated that all isolates were capable of metabolic versatility. Measurements of total organic carbon (TOC), nitrate and dissolved oxygen (DO) indicated limited presence of electron donor at all sites, whereas the electron acceptors varied throughout the Wash and Lake Mead. The persistence of perchlorate in the sites is attributed to lack of available electron donor and/or the presence of competing electron acceptors. A location has been identified where perchlorate biodegradation could be implemented thereby halting the transport of perchlorate to Lake Mead and the Colorado River.     

Kinetics of perchlorate- and chlorate-respiring bacteria

Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated "Dechlorosoma" sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h(-1), respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h(-1), respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h(-1), respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h(-1), respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h(-1)) and exceeded that of strain KJ on perchlorate (0.14 h(-1)). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 +/- 0.07 [n = 7]), chlorate (0.44 +/- 0.05 [n = 7]), and perchlorate (0.50 +/- 0.08 [n = 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.     

Proteomic detection of proteins involved in perchlorate and chlorate metabolism

Mass spectrometry and a time-course cell lysis method were used to study proteins involved in perchlorate and chlorate metabolism in pure bacterial cultures and environmental samples. The bacterial cultures used included Dechlorosoma sp. KJ, Dechloromonas hortensis, Pseudomonas chloritidismutans ASK-1, and Pseudomonas stutzeri. The environmental samples included an anaerobic sludge enrichment culture from a sewage treatment plant, a sample of a biomass-covered activated carbon matrix from a bioreactor used for treating perchlorate-contaminated drinking water, and a waste water effluent sample from a paper mill. The approach focused on detection of perchlorate (and chlorate) reductase and chlorite dismutase proteins, which are the two central enzymes in the perchlorate (or chlorate) reduction pathways. In addition, acetate-metabolizing enzymes in pure bacterial samples and housekeeping proteins from perchlorate (or chlorate)-reducing microorganisms in environmental samples were also identified.     

Characterization of DitA3, the [Fe3S4] ferredoxin of an aromatic ring-hydroxylating dioxygenase from a diterpenoid-degrading microorganism

DitA3, a small soluble ferredoxin, is a component of a ring-hydroxylating dioxygenase involved in the microbial degradation of the diterpenoid, dehydroabietic acid. The anaerobic purification of a heterologously expressed his-tagged DitA3 yielded 20 mg of apparently homogeneous recombinant protein, rcDitA3, per liter of cell culture. Each mole of purified rcDitA3 contained 2.9 equivalents of iron and 4.2 equivalents of sulfur, indicating the presence of a single [Fe(3)S(4)] cluster. This conclusion was corroborated by UV-Visible absorption (epsilon(412)=13.4 mM(-1) cm(-1)) and EPR (g(x,y)=2.00 and g(z)=2.02) spectroscopies. The reduction potential of rcDitA3, determined using a highly oriented parallel graphite (HOPG) electrode, was -177.0+/-0.5 mV vs. the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, pH 7.0, 22 degrees C). This potential is similar to those of small, soluble Rieske-type ferredoxin components of aromatic-ring dihydroxylating dioxygenases. In contrast to these Rieske-type ferredoxins, DitA3 appears to exist as a dimer in solution. The dimeric ferredoxin may be more stable or may increase the catalytic efficiency of the dioxygenase by delivering the two reducing equivalents required for turnover of the oxygenase.     

Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (alpha beta)(2) dimer

We describe and compare the main kinetic characteristics of the (alpha beta)(2) form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high (K(0.5)=4 microM) and low (K(0.5)=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate (K(0.5)=0.5 mM) and a lower hydrolysis rate (V(M)=42 U/mg). Stimulation of ATPase activity by K(+) (K(0.5)=0.63 mM), Na(+) (K(0.5)=11 mM) and Mg(2+) (K(0.5)=0.60 mM) all showed V(M)'s of approximately 600 U/mg and negative cooperativity. K(+) (K(0.5)=0.69 mM) and Mg(2+) (K(0.5)=0.57 mM) also stimulated PNPPase activity of the (alpha beta)(2) form. Ouabain (K(0.5)=0.01 microM and K(0.5)=0.1 mM) and orthovanadate (K(0.5)=0.06 microM) completely inhibited the ATPase activity of the (alpha beta)(2) form. The kinetic characteristics obtained constitute reference values for diprotomeric (alpha beta)(2)-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.     

Characterization of a new feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase AroF of Escherichia coli

Tyrosine feedback-inhibits the 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase isoenzyme AroF of Escherichia coli. Here we show that an Asn-8 to Lys-8 substitution in AroF leads to a tyrosine-insensitive DAHP synthase. This mutant enzyme exhibited similar activities (v=30-40 U mg(-1)) and substrate affinities (K(m)(erythrose-4-phosphate)=0.5 mM, positive cooperativity with respect to phospho(enol)pyruvate) as the wild-type AroF, but showed decreased thermostability. An engineered AroF enzyme lacking the seven N-terminal residues also was tyrosine-resistant. These results strongly suggest that the N-terminus of AroF is involved in the molecular interactions occurring in the feedback-inhibition mechanism.     

Transformation of (per)chlorate into chloride by a newly isolated bacterium: reduction and dismutation

 Bacterial strain GR-1 was isolated from activated sludge for its ability to oxidize acetate with perchlorate as electron acceptor. Sequencing of 16S rDNA revealed the isolate to belong to the β subgroup of Proteobacteria. When strain GR-1 was grown on acetate and perchlorate, the release of chloride was proportional to the disappearance of perchlorate, showing that this compound was completely reduced. In addition to perchlorate, strain GR-1 used chlorate, oxygen, nitrate and Mn(IV) as electron acceptor. The oxidation of acetate is coupled to the reduction of perchlorate and chlorate, whereas chlorite reduction is not affected by the addition of acetate. Strain GR-1 disproportionates chlorite into molecular oxygen and chloride. As a consequence, the strain oxidizes acetate by simultaneously reducing perchlorate to chlorite and molecular oxygen to water. Comparison of growth yields with oxygen, chlorate and perchlorate and calculated ΔG ^0′ values confirms this finding. Received: 26 June 1995/Received revision: 11 October 1995/Accepted: 16 October 1995     

Environmental factors that control microbial perchlorate reduction

As part of a study to elucidate the environmental parameters that control microbial perchlorate respiration, we investigated the reduction of perchlorate by the dissimilatory perchlorate reducer Dechlorosoma suillum under a diverse set of environmental conditions. Our results demonstrated that perchlorate reduction by D. suillum only occurred under anaerobic conditions in the presence of perchlorate and was dependent on the presence of molybdenum. Perchlorate reduction was dependent on the presence of the enzyme chlorite dismutase, which was induced during metabolism of perchlorate. Anaerobic conditions alone were not enough to induce expression of this enzyme. Dissolved oxygen concentrations less than 2 mg liter(-1) were enough to inhibit perchlorate reduction by D. suillum. Similarly to oxygen, nitrate also regulated chlorite dismutase expression and repressed perchlorate reduction by D. suillum. Perchlorate-grown cultures of D. suillum preferentially reduced nitrate in media with equimolar amounts of perchlorate and nitrate. In contrast, an extended (40 h) lag phase was observed if a similar nitrate-perchlorate medium was inoculated with a nitrate-grown culture. Perchlorate reduction commenced only when nitrate was completely removed in either of these experiments. In contrast to D. suillum, nitrate had no inhibitory effects on perchlorate reduction by the perchlorate reducer Dechloromonas agitata strain CKB. Nitrate was reduced to nitrite concomitant with perchlorate reduction to chloride. These studies demonstrate that microbial respiration of perchlorate is significantly affected by environmental conditions and perchlorate reduction is directly dependent on bioavailable molybdenum and the presence or absence of competing electron acceptors. A microbial treatment strategy can achieve and maintain perchlorate concentrations below the recommended regulatory level, but only in environments in which the variables described above can be controlled.     

Effect of nitrate, acetate, and hydrogen on native perchlorate-reducing microbial communities and their activity in vadose soil

The effect of nitrate, acetate, and hydrogen on native perchlorate-reducing bacteria (PRB) was examined by conducting microcosm tests using vadose soil collected from a perchlorate-contaminated site. The rate of perchlorate reduction was enhanced by hydrogen amendment and inhibited by acetate amendment, compared with unamendment. Nitrate was reduced before perchlorate in all amendments. In hydrogen-amended and unamended soils, nitrate delayed perchlorate reduction, suggesting that the PRB preferentially use nitrate as an electron acceptor. In contrast, nitrate eliminated the inhibitory effect of acetate amendment on perchlorate reduction and increased the rate and the extent, possibly because the preceding nitrate reduction/denitrification decreased the acetate concentration that was inhibitory to the native PRB. In hydrogen-amended and unamended soils, perchlorate reductase gene (pcrA) copies, representing PRB densities, increased with either perchlorate or nitrate reduction, suggesting that either perchlorate or nitrate stimulates the growth of the PRB. In contrast, in acetate-amended soil pcrA increased only when perchlorate was depleted: a large portion of the PRB may have not utilized nitrate in this amendment. Nitrate addition did not alter the distribution of the dominant pcrA clones in hydrogen-amended soil, likely because of the functional redundancy of PRB as nitrate-reducers/denitrifiers, whereas acetate selected different pcrA clones from those with hydrogen amendment.     

Whole cell immobilized amperometric biosensor based on Saccharomyces cerevisiae for selective determination of vitamin B1 (thiamine)

A new amperometric whole cell biosensor based on Saccharomyces cerevisiae immobilized in gelatin was developed for selective determination of vitamin B1 (thiamine). The biosensor was constructed by using gelatin and crosslinking agent glutaraldehyde to immobilize S. cerevisiae cells on the Teflon membrane of dissolved oxygen (DO) probe used as the basic electrode system combined with a digital oxygen meter. The cells were induced by vitamin B1 in the culture medium, and the cells used it as a carbon source in the absence of glucose. So, when the vitamin B1 solution is injected into the whole cell biosensor system, an increase in respiration activity of the cells results from the metabolic activity and causes a decrease in the DO concentration of interval surface of DO probe related to vitamin B1 concentration. The response time of the biosensor is 3 min, and the optimal working conditions of the biosensor were carried out as pH 7.0, 50mM Tris-HCl, and 30 degrees C. A linear relationship was obtained between the DO concentration decrease and vitamin B1 concentration between 5.0 x 10(-3) and 10(-1) microM. In the application studies of the biosensor, sensitive determination of vitamin B1 in the vitamin tablets was investigated.     

A simple and rapid method for monitoring dissolved oxygen in water with a submersible microbial fuel cell (SBMFC)

A submersible microbial fuel cell (SBMFC) was developed as a biosensor for in situ and real time monitoring of dissolved oxygen (DO) in environmental waters. Domestic wastewater was utilized as a sole fuel for powering the sensor. The sensor performance was firstly examined with tap water at varying DO levels. With an external resistance of 1000?, the current density produced by the sensor (5.6±0.5-462.2±0.5mA/m(2)) increased linearly with DO level up to 8.8±0.3mg/L (regression coefficient, R(2)=0.9912), while the maximum response time for each measurement was less than 4min. The current density showed different response to DO levels when different external resistances were applied, but a linear relationship was always observed. Investigation of the sensor performance at different substrate concentrations indicates that the organic matter contained in the domestic wastewater was sufficient to power the sensing activities. The sensor ability was further explored under different environmental conditions (e.g. pH, temperature, conductivity, and alternative electron acceptor), and the results indicated that a calibration would be required before field application. Lastly, the sensor was tested with different environmental waters and the results showed no significant difference (p>0.05) with that measured by DO meter. The simple, compact SBMFC sensor showed promising potential for direct, inexpensive and rapid DO monitoring in various environmental waters.     

Kinetics behaviors of Na,K-ATPase: comparison of solubilized and DPPC:DPPE-liposome reconstituted enzyme

We describe and compare the main kinetic characteristics of rabbit kidney Na,K-ATPase incorporated inside-out in DPPC:DPPE-liposomes with the C(12)E(8) solubilized and purified form. In proteoliposomes, we observed that the ATP hydrolysis of the enzyme is favored and also its affinity for Na(+)-binding sites increases, keeping the negative cooperativity with two classes of hydrolysis sites: one of high affinity (K(0.5)=6 microM and 4 microM for reconstituted enzyme and purified form, respectively) and another of low affinity (K(0.5)=0.4 mM and 1.4 mM for reconstituted enzyme and purified form, respectively). Our data showed a biphasic curve for ATP hydrolysis, suggesting the presence of (alphabeta)(2) oligomer in reconstituted Na,K-ATPase similar to the solubilized enzyme. The Mg(2+) concentration dependence in the proteoliposomes stimulated the Na,K-ATPase activity up to 476 U/mg with a K(0.5) value of 0.4 mM. The Na(+) ions also presented a single saturation curve with V(M)=551 U/mg and K(0.5)=0.2 mM with cooperative effects. The activity was also stimulated by K(+) ions through a single curve of saturation sites (K(0.5)=2.8 mM), with cooperative effects and V(M)=641 U/mg. The lipid microenvironment close to the proteic structure and the K(+) internal to the liposome has a key role in enzyme regulation, affecting its kinetic parameters while it can also modulate the enzyme's affinity for substrate and ions.     

Experimental characterization of poly(hydroxyalkyl-L-glutamine) conformations in aqueous calcium chloride and sodium perchlorate solutions

Poly(hydroxyalkyl-L -glutamine) (alkyl = ethyl, propyl, butyl) solutions have been studied by CD as functions of temperature and activity of calcium chloride and sodium perchlorate. Helical content is altered by changes in salt activity and temperature. The helicity of poly(hydroxybutyl-L -glutamine) and poly(hydroxypropyl-L -glutamine) falls to zero in a monotonic fashion with increasing calcium chloride activity. A nonzero helicity reappears at activities in excess of 5–50 mol kg^?1. Poly(hydroxypropyl-L -glutamine) is much more sensitive to calcium chloride than is poly(hydroxybutyl-L -glutamine), and both polypeptides are more sensitive to calcium chloride than are typical proteins. Markedly different behavior is observed with sodium perchlorate. This salt acts as a helix stabilizer at low activities but becomes a destabilizer at activities higher than 0.3–1.0 mol kg^?1. In this respect the effect of sodium perchlorate on nonionic poly(hydroxyalkyl-L -glutamines) resembles that seen with cationic poly(L -lysine) and poly(L -arginine). Helix stabilization at low sodium perchlorate activity is moderate for poly(hydroxybutyl-L -glutamine) and large for poly(hydroxypropyl-L -glutamine) and poly(hydroxyethyl-L -glutamine).     

Regulation of vinblastine biosynthesis in cell suspension cultures of catharanthus roseus

Various elicitors of hydroxylase, peroxidase, acetyltransferase and inhibitors of oxygenase were added to a Catharanthus roseus cell culture medium to investigate the regulatory effects on tabersonine, vindoline and vinblastine biosynthesis. Hydrogen peroxide was found to be the most effective agent for enhancing the biosynthesis of tabersonine. By adding 20???g/L hydrogen peroxide, the tabersonine concentration reached 9.02?mg/g dry weight (DW) after culturing cell suspensions for 7?days. With the addition of 30???g/L acetyl CoA, the most vindoline (final cell content of 0.33?mg/g DW) was produced. By effective inhibition of lochnericine biosynthesis with the addition of 0.5???mol/L benzotriazole, the cell content of vindoline was increased to 0.42?mg/g DW. An orthogonal experiment consisting of multiple regulation factors was carried out to optimize vinblastine biosynthesis. It was shown that optimal vinblastine biosynthesis was achieved by addition of 5?mg/L acetyl CoA, 20???g/L hydrogen peroxide, 0.5???mol/L benzotriazole, 100?mg/L tryptophan, 100?mg/L loganin and 30?mg/L cerium chloride. Under these conditions, the cell content of vinblastine reached 0.81?mg/g DW. Simultaneous changes in cell content and enzyme activities of Cytochrome P-450 monooxygenases, Deacetylvindoline-O-acetyltransferase and Peroxidase enzyme indicated that these enzymes were closely linked to vinblastine biosynthesis.