Oxygen uptake kinetics Of Pseudomonas sp. strain VB grown under limiting concentration of 2-methoxyethanol

Oxygen uptake and 2-methoxyethanol oxidation kinetics of 2-methoxyethanol degrading


INTRODUCTION
2-methoxyethanol is a member of the organic compounds called glycols.It comprises one unit of ethylene oxide, with a C-O-C ether linkage terminating with an hydroxyl bond (-OH) at one end.The ether linkage is the most common and unifying structural feature which confers on both biological and xenobiotic compounds a high degree of resistance to biological mineralization (Tidswell et al., 1996).Its diverse uses makes the substance ubiquitous as an environmental xenobiotic which has considerable implications within the global biosphere.
The microbial mixed cultural degradation of 2methoxyethanol under anaerobic conditions has been reported (Tanaka et al., 1986;Tanaka and Pfennig, 1988).A novel bacterium Pseudomonas sp.strain VB able to degrade 2-methoxyethanol under aerobic conditions was isolated from anaerobic sewage sludge (Ekhaise, 2002).The bacterium utilizes 2-methoxyethanol as sole source of carbon and energy.
Oxygen metabolism of aerobic microorganisms can be studied using an oxygen electrode.Enzymes are readily studied using a Clarke oxygen electrode provided oxygen is involved in the reaction.Glucose oxidase, D-amino oxidase and catalase are examples of enzymes whose *Corresponding author: Present Address: Department of Microbiology, Faculty of Science, University of Benin, P.M.B. 1154, Benin City, Nigeria.E-mail: fredfldyn@yahoo.com.
properties can be studied in this way (Wilson and Walker, 1994).
The goal of this study was to determine oxygen uptake kinetics of the newly isolated bacterium, Pseudomonas sp.strain VB in relation to substrate concentration.

Chemicals
All the chemicals used were of analytical grade (≥99.9%pure).2methoxyethanol was obtained from Fluka (Buchs, Switzerland).

Organism and culture conditions
An overnight culture of Pseudomonas sp.strain VB was used to inoculate 10 L of mineral salt medium containing 126.7 mmol of 2methoxyethanol.The culture was aerated by bubbling air through it and incubated at 30 o C. The growth was monitored turbidometrically at 436 nm with a spectrophotometer (LKB Biochrom Ultrospec 4050, Cambridge, England).The cells were harvested after 24 h of incubation.

Measurement of oxygen uptake and substrate oxidation kinetics
The determination of oxygen uptake rate was performed polarographically at room temperature using a Clarke-type oxygen electrode (Model 10 Ranke Brothers, Birmingham, Great Britain).An overnight culture of Pseudomonas sp.strain VB was centrifuged, washed twice and re-suspended in 1 ml of fresh phosphate buffer (50 mM, pH 7.2).From this cell suspension, 100 Afr.J. Biotechnol.
µl was added to 3 ml of saturated oxygen-phosphate buffer.After 10 min, the oxygen consumption by the intact cells was measured and this constituted the endogenous respiratory rate.The following limiting concentrations of 2-methoxyethanol 42.2, 84.5 and 105.5 nmol were added to the cultures respectively and after 10 min, oxygen consumption rate was measured.This is the exogenous respiratory rate.The oxygen consumption rate at the expense of the substrate was determined by subtracting the value for endogenous respiratory rate from exogenous respiratory rate.

RESULTS AND DISCUSSION
The stoichiometry of 2-methoxyethanol with Pseudomonas sp.strain VB as shown in Table 1 was calculated to be in the ratio of 1:1.Pseudomonas sp.strain VB utilizes 1 mol oxygen per 1 mol 2methoxyethanol as compared to the theoretically predicted ratio of 4 mol oxygen per 1 mol 2methoxyethanol for the complete oxidation of 2methoxyethanol under aerobic conditions.Total oxidation of 2-methoxyethanol is as follows: The subtrate oxidation curves obeyed Michaelis-Menten kinetics (not shown).The rate of oxygen consumption (velocity rate) was plotted against varying concentrations of 2-methoxyethanol.The double reciprocal according to Lineweaver-Burk was used to determine the K m and V max values of 11.526 mM and 33.856 mM, respectively.
The measurement of the initial rate of an enzymecatalyzed reaction is fundamental to a complete understanding of the mechanism by which the enzyme works (Wilson and Walker, 1994), as well as the estimation of the activity of an enzyme in a biological sample.This is influenced by many factors, among which are substrate concentration, enzyme concentration, pH, temperature and the presence of catalyst.For the majority of enzymes, substrate dependent rate of reaction varies hyperbolically with substrate concentration.At low substrate concentrations in such cases approximately first order kinetics are observed, but at high substrate concentrations saturation zero-order kinetics exists and the initial rate is dependent on substrate concentration as expressed by the Michaelis-Menten reaction.
Stoichiometrically, Pseudomonas sp.strain VB utilizes one mole of oxygen per one mole of 2-methoxyethanol as compared to four moles of oxygen per one mole of 2methoxyethanol required for the total oxidative metabolism (Table 1).This finding therefore supports a hypothesis in which the degradative pathway is not a one-step reaction mechanism and this indicate that intermediate metabolites are involved during the biodegradation of 2-methoxyethanol in Pseudomonas sp.Strain VB under aerobic conditions.
Competition only for oxygen is probably rare under natural conditions since limiting amounts of the electron donors also influence the performance at low oxygen availability (Bodiler, and Laambroek, 1997).From the 2methoxyethanol oxidation kinetics of Pseudomonas sp.strain VB, it is evident that the bacterium uses oxygen as a good electron donor.For single enzymes, the K m and V max values range from 9.5 x 10 -5 to 2.5 x 10 -2 µM (Voet and Voet, 1990).The values obtained in this study are so high that, multiple enzymes may be involved in the degradation processes.Since the magnitude of K m value varies widely with the identity of the enzyme and the substrate, it can be concluded that the bacterium possessed a high substrate utilization capacity with low catalytic efficiency.
1Control was determined under endogenous rate without substrate.