Philip 1. Kumler and Peter J. DeJong Saginaw Valley College University Center, Michigan 48710
Microbial Oxidation of Alkenes An integrated organic- biology experiment
Instructors in organic chemistry lahoratory sequences are faced with classes that have a high percentage of hiological science majors. Partly as a result of this type of distribution, a number of experiments have appeared recently which have been devised for these situations (1-6). During the second semester of the organic lahoratory sequence a t Saginaw Valley College all students are required to carry out one experiment which involves hoth chemistry and hiology. For the past two years we have offered the students three options for this experiment: (1) The synthesis of a known allelopath (juglone) and a study of the effect of it on the sprouting behavior on various seeds (51, or (2) preparation of an immobilized enzyme and a study of its pH-activity profile (6). or (3) a study of the microbial epoxidation of alkenes. It is the last option which we wish to describe here. The described experiment: (1) combines organic, hiological, and quantitative techniques, (2) is capable of completion in 2-3, 3-hr lahoratory periods, (3) does not require elaborate apparatus, and (4) introduces students to the techniques used in carrying out chemical conversions employing microorganisms. The experiment involves the conversion of an appropriate terminal alkene to a 1,2-epoxyalkane by Pseudomonas oleouorans
This particular organism has previously been shown to he an efficient epoxidizing agent for terminal alkenes (7, 8). Discussion of the Integrated Experiment
In our particular situation we have chosen to have the students work in pairs with one of the students, a hiology major, or someone with previous laboratory experience in hiology. Even so, many of the hiology majors have not as yet been exposed to pure culture techniques so we supply them with a cell suspension of resting cells of Pseudomonas oleouorans.' Some instructors may choose to have the students prepare their own cell suspension. The conditions of temperature, pH, etc. for maximum production of epoxyalkanes from 1-alkenes have been previously described (7) and are suggested to the students. The cells are suspended in a buffered solution, the alkene is added, and the flask is incubated with shaking for an appropriate period of time. At the end of the incuhation period the epoxyalkane and unreacted alkene are extracted with hexane containing an appropriate alcohol (1- or 2-octanol) as internal standard for quantitative determination of the epoxide/alcohol ratio by vapor phase chromatography which allows quantitative determination of the amount of epoxide present. There are a number of ways that factorial design (9) can he introduced into the experiment if desired. Various mouns of students can studv the effect of chain length by selecting different alkenes iwe have successfully carried out the ex~erimentusina 1-hexene, 1-octene, and l-decene) or thk effect of var;ing incuhation times on the epoxidation of a particular alkene. We have carried out the experiment successfully using either of the above experimental designs, or they can be combined.
After incubating the alkene with the organism for an appropriate time period, the epoxide/octanol ratio can he conveniently determined by quantitative vpc. In our particular instance this ratio was determined using a vpc equipped with a thermal conductivity detector, which necessitated the use of rather large sample sizes (50-75~1)for the quantitative analysis. I€ desired and available, the vpc analysis could he carried out using a flame ionization detector which would allow the use of much smaller sample sizes due to the increased sensitivity. If it is desired to have the studehts report their results in terms of actual epoxide concentration, then it is necessary to determine the-relative response of the particular detector used to hoth epoxide and internal standard. In most cases 2-octanol serves satisfactorily as an internal standard hut if the retention time of the 2-octanol is similar to that of the epoxide then 1-octanol may he conveniently used instead. - . ---.. ~ ~ ~ Even though we have observed considerahle variation in the amounts of enoxide ~ r o d u c e dhv different cultures of the organism it would he possible for the students in a large class to do a statistical analysis of the experimental data. We feel that the rather large observed variation is due to differences in the ages of cultures used by the various groups of students and is dependent on the growth stage at which the cells are harvested; similar variations have been previously observed (7) We have generally found (within the range Cs-CIa chain lengths) that epoxide production is most efficient with the longer chain length alkenes. When the effect of time is studied it is generally observed that epoxide production reaches a maximum (usually in 1-3 hr) and then actually declines. Such observations have been utilized to suggest that epoxide is metabolized further (7). A typical result of the effect of time on the epoxidation of 1-octene is as follows: after 1-hr incubation the epoxide/2-octanol ratio was 0.46 (corresponding to an epoxide concentration of 0.12 mg/ml) while after 3-hr incuhation the ratio was 0.42 (0.10 mgepoxide/ml). Although we have chosen to supply the students with samples of authentic epoxides corresponding to the alkenez some instructors may prefer to have the students synthesize the epoxide corresponding to the particular alkene by use of m-chloroperbenzoic acid (10). This experiment has been very well received by the students and has led to a number of discussions concerning such features as the problems involved in quantitative vpc analysis, use of microorganisms for hoth lahoratory and commercial scale organic syntheses, and the potential use of "oil-eating" microorganisms in cases of oil spills, etc. ~~~~
Experimental Details
Growth and Harvesting of the Organism The stock culture of Pseudornonas oleouorans' was maintained on a nutrient agar slant to which several drops of sterile octane ' A pure culture of Pseudarnonns oleouorans was obtained from Dr. Robert D. Schwartz at the Essa Research and Engineering
Company. Epoxides with even chain lengths from Cs-C12 are available from Chemical Samples Co., Columbus, Ohio. Volume 52, Number 7. July 1975 / 475
had been added (71. Octane was sterilized by passing it through a membrane filter with 0.45rrm pore in a Nalgene filter unit. To prepare cell suspensions of the organism, cells were washed from the slant with sterile water and transferred to 100 ml of a salts medium in a 500-ml Erlenmeyer flask to which 1 ml of sterile octane had been added.3 The salts medium is composed of NHI.H~POI, 109; K ~ H P O I ,5g; Na2S04, 0.5 g; CaCL, 0.05 g; t a p water, 50 ml; and distilled water, 950ml. The culture was incubated a t 30°C for 18 hr on a rotary shaker and was supplied to the students.
Microbial Epoxidation of the Alkene Each student team was supplied with 100 ml of the above suspension. The cells were centrifuged, washed once with 0.05 M phosphate buffer a t p H 7.8, recentrifuged, and suspended in 20 ml of 0.05 M phosphate buffer at p H 7.8. The cell suspension was divided equally among three 250-ml Erlenmeyer flasks and 0.1 ml of the appropriate alkene (I-hexene, l-octene, or l-decene) was added to each flask. The incubation was carried out in the stoppered flasks by shaking far periods of 1, 2, and 3 hr, respectively (if the effect of chain length is being studied 2 hr is a n apprapri-
ate incubation time). Each of the samples is extracted with 5 ml of hexane which contains 0.25 mg of 2-actanol per ml of hexane; the extraction can be conveniently carried out by shaking for 10 min. After centrifugation to break the resultant emulsion the hexane layer is analyzed by quantitative vpc.
Quantitative VPC Analysis
The response ratio (epoxide/internal standard) can he determined by injecting an accurately weighed mixture of epoxide/intemal standard and measuring the actual peak areas of both epoaide and internal standard; in our particular usage we have found it more convenient to determine the response ratio in advance and supply it to the students before the vpe analysis. Our rationale for supplying this information was t o minimize the amount of time any one particular group required use of the vpc, but the pedagogical advantages of having the students determine this ratio are obvious. Experimental samples (50-75 al) were injected and the eporide/internal standard ratio was determined. When corrected for the relative response ratio this information can be used to determine the actual concentration of e p o ~ i d e . ~ Literature Cited
SIt is s common microbiological practice to flame the mouths of tubes and flasks when making culture transfers. It is obvious that thin must be avoided when working with cultures containing volatile hvdrocarbons. 'Typical oprrating parameters for the vpv ~VarianAerovaph. Mrdel'OO, at ?1WC ~ ~ o t h e r m aun l r a 2J.n X 38-in. Corbownx 20hl column i:iuY/r un Chrun~oaorh30) with a helium flou, rate oi RO ml/min; typical retention times (in mi") are: hexane (3.5). l-oetene (4.5), 1.2-epoxyoctane (11.0). 2-octanol(l4.0).
