APPLIED
AND
ENVIRONMENTAL MICROBIOLOGY, Oct. 1979, p. 742-746
Vol. 38, No. 4
0099-2240/79/10-0742/05$02.00/0
Microbial Growth on Hydrocarbons: Terminal Branching Inhibits Biodegradation TERI L. SCHAEFFER, SUSAN G. CANTWELL, JEFFREY L. BROWN, DAVID S. WATT, AND R. RAY FALL*
Department of Chemistry, University of Colorado, Boulder, Colorado 80309
Received for publication 18 July 1979
A variety of octane-utilizing bacteria and fungi were screened for growth on terminally branched dimethyloctane derivatives to explore the effects of iso- and anteiso-termini on the biodegradability of such hydrocarbons. Of 27 microbial strains tested, only 9 were found to use any of the branched hydrocarbons tested as a sole carbon source, and then only those hydrocarbons containing at least one iso-terminus were susceptible to degradation. Anteiso- or isopropenyl termini prevented biodegradation. None of the hydrocarbonoclastic yeasts tested was able to utilize branched-hydrocarbon growth substrates. In the case of pseudomonads containing the OCT plasmid, whole-cell oxidation of n-octane was poorly induced by terminally branched dimethyloctanes. In the presence of a gratuitous inducer of the octane-oxidizing enzymes, the iso-branched 2,7-dimethyloctane was slowly oxidized by whole cells, whereas the anteiso-branched 3,6-dimethyloctane was not oxidized at all. This microbial sampling dramatically illustrated the deleterious effect of alkyl branching, especially anteiso-terminal branching, on the biodegradation of hydrocarbons. some
n-Alkanes are readily oxidized by many microorganisms, predominantly via an initial oxidation of a terminal methyl group followed by a ,3-oxidation sequence (14, 19, 22, 28). Alkylbranched alkanes are generally less susceptible to biodegradation (17, 24), and certain branching patterns confer biological recalcitrance, causing these compounds to accumulate in the biosphere (1, 2). If alkyl branches are located near the terminus, it seems likely that decreased biodegradability could be the result of steric inhibition of terminal oxidizing enzymes, although transport defects or failure to induce the appropriate oxidizing system or both may be responsible, too. In the case where an alkyl branch occurs at the /3-position (i.e., anteiso-terminus), the branch will prevent /3-oxidation, requiring an additional strategy such as a-oxidation (3, 15), /3-alkyl group removal (8,27), or w-oxidation (24) to allow oxidation to proceed. We have been searching for microorganisms that are able to carry out the oxidation of /3methyl-substituted alkanes, especially where the pseudomonad type of /3-alkyl group removal occurs (8, 27). Twenty-seven different octane-utilizing strains of bacteria and fungi were obtained (see Table 1) and were screened for growth on 3,6-dimethyloctane (3,6-DMO), a dimethyloctane with /8-methyl substituents (anteiso-) at both ends of the molecule. For comparison, each strain was also screened for growth on 2,7-di-
methyloctane (2,7-DMO), which contains amethyl (iso-) termini, or 2,6-dimethyloctane (2,6-DMO), which contains both iso- and anteiso-termini. Screening with 2,6-dimethyl-2-octene will be described below. Growth tests were carried out by the following general procedure (23). Cultures were grown in minimal media (see Table 1) supplemented with 0.5% (wt/vol) yeast extract (Difco Laboratories), then washed and suspended in the appropriate minimal medium, and used to inoculate minimal media containing 1% (vol/vol) of the appropriate filter-sterilized hydrocarbon. The cultures were transferred to metal tins with tight-fitting lids to maintain a hydrocarbon vapor atmosphere. The tins were opened every 48 h to admit oxygen, the cultures were agitated with a Vortex mixer and monitored for growth, and the tins were resealed. Additional portions of hydrocarbons were added after 6, 12, and 18 days. Growth was compared with controls containing inoculated medium with no hydrocarbon and incubated separately. Growth at 23°C was monitored for up to 22 days with slow-growing cultures. The hydrocarbons 3,6-DMO, 2,7-DMO, and 2,6DMO were each 99+% grade obtained from Chemical Samples Co. 2,6-Dimethyl-2-octene was prepared by Wolff-Kishner reduction (21) of citronellal (Pfaltz & Bauer, Inc.). The product was bulb to bulb distilled at 65 to 72°C (50-mm pressure) and obtained pure by preparative gas 742
VOL. 38, 1979
743
NOTES
chromatography on a 10% Carbowax 20 M column (10-ft [ca. 3-m] column; helium flow rate, 45 ml/min; injector temperature, 175°C; detector temperature, 225°C; retention time, 3 min). Each hydrocarbon was passed over a silicic acid column before use to remove traces of oxidized substances. Of the 27 octane-utilizing strains tested, none grew with 3,6-DMO as a sole carbon source (Table 1). The possibility that the 3,6-DMO contained a growth inhibitor as a trace contaminant was ruled out, since a 1:1 mixture of 3,6-
DMO and n-octane supported the growth of several strains as weli as n-octane alone. These results show that the symmetrical anteiso-substitution pattern present in 3,6-DMO represents a recalcitrant structural feature preventing biodegradation. Nine strains were able to utilize the symmetrically iso-branched 2,7-DMO as a sole carbon source (Table 1), suggesting that an isobranched terminus is more readily degraded than an anteiso-branched terminus. Still, most of the 27 strains tested could not utilize this
TABLE 1. Ability of octane-utilizing microorganisms to grow on branched octane derivatives as a sole carbon source Growth substrate Strain
Bacteria Achromobacter sp. Acinetobacter sp. H01-N Arthrobacter ap. Brevibacterium erythrogenes Corynebacterium hydrocarbonoclastus 15961 Corynebacterium ap. Flavobacterium sp. Mycobacterium convolutum R-22 Mycobacterium rhodochrous OFS Mycobacterium rhodochrous 7E1C Nocardia sp. Nocardia rubra Pseudomonas aeruginosa 17423 Pseudomonas oleovorans 8062 Pseudomonas oleovorans Pseudomonas putida AC4 Pseudomonas Pxy
Fungi Aspergillus versicolor Candida maltosa 28140 Candida petrophilum 20226 Candida sake 14478 Candida tropicalis 750 Cladosporium resinae UD42 Cunninghamella blakes-
Source or reference
Growth 2,6-Dimedium' n-Octane 3,6-DMO 2,7-DMO 2,6-DMO methyl-2octene
Octane enrichment from oil seep W. R. Finnerty (26) Sewage R. M. Atlas (25)
H
2
0
0
0
0
H H HS
1 2 2
0 0 0
0 0 1
0 0 1
0 0 0
ATCCb
H
0
0
0
0
Octane enrichment Octane enrichment J. J. Perry
H H H
0 0 0
2 0 1
1 0 1
0 0 0
W. R. Finnerty
H
W. R. Finnerty
H
2
0
0
0
0
Octane enrichment W. R. Finnerty ATCC
H H H
2 1 2
0 0 0
1 0 1
1 0 1
0 0
ATCC
H
2
0
0
0
M. J. Coon A. Chakrabarty A. Chakrabarty (11)
H H
3 3 2
0 0 0
0 0 0
0 0 0
0 0 0
J. J. Perry ATCC ATCC ATCC ATCC J. J. Cooney (10, 16)
YNBS YNB YNB YNB YNB YNB
2 1 1 1 1 1
0 0 0 0 0 0
2 0 0 0 0 0
1 0 0 0 0 0
0 0 0 0 0 0
Hd
3 1 2
1
0
OM)
0 0 2 YNB ATCC leeana 8688a 0 1 1 0 2 YNBS J. J. Perry EupeniciUum sp. 0 0 0 0 1 YNB ATCC Rhodotorula glutinis 2527 0 0 0 0 1 YNB R. R. Colwell Sporobolomyces sp. a Growth media have been described elsewhere (12, 20). HS is H medium containing 3% (wt/vol) NaCl, and YNBS is also supplemented with this level of NaCl. Growth ratings are as follows: 0, no growth; 1, weak but consistent growth; 2, moderate growth; 3, abundant growth. h ATCC, American Type Culture Collection. 'After prolonged incubation in liquid medium, several positive clones were isolated. These will be described in a separate communication. d The H medium was supplemented with 20 ug of L-methionine per ml.
744
NOTES
hydrocarbon, further illustrating the deleterious effect of alkyl branching on the microbial assimilation of hydrocarbons (19). That there is also a chain length specificity involved in the biodegradability of such branched hydrocarbons is indicated by the fact that several of the strains tested, including the Acinetobacter species, Mycobacterium rhodochrous 7E1C, and Nocardia rubra, can use pristane (2,6,10,14-tetramethylpentadecane) as a sole carbon source (data not shown), but not 2,7-DMO. Pristane is a highly branched hydrocarbon that also contains isobranched termini like 2,7-DMO, and its degradation via terminal oxidation and,8-oxidation or a combination of f- and w-oxidation has been reported in several microorganisms (24). Thus, an iso-branched terminus is not necessarily a recalcitrant structural feature in the strains mentioned above, but perhaps in the case of 2,7DMO their octane-oxidizing systems cannot initiate oxidation of the branched molecule. Those organisms which could utilize 2,7-DMO but not 3,6-DMO were able in each case to also utilize 2,6-DMO (Table 1). This suggests that oxidation of 2,6-DMO proceeded in these instances via the iso-terminus, although direct experimental evidence is lacking. We were also interested in exploring the possibility that 2,6-dimethyl-2-octene could be degraded via the citronellol pathway (27). If this branched hydrocarbon was hydroxylated at the terminal methyl group of the anteiso-terminus, this would produce citronellol for which there is an established degradative pathway in certain pseudomonads (8, 27; see Fig. 1 of reference 12). Screening with 2,6-dimethyl-2-octene (Table 1) showed that it is also a very recalcitrant structure. Of the strains tested, only a Pseudomonas aeruginosa strain showed any growth, and then only when a few positive clones arose after prolonged exposure to the hydrocarbon. (The properties of these clones will be described in a separate communication; see reference 12.) Strains that were able to utilize 2,6-DMO were in each case unable to use 2,6-dimethyl-2-octene (Table 1), suggesting that the isopropenyl terminus in the latter hydrocarbon is a recalcitrant feature. In the microbial screening, six species of hydrocarbonoclastic yeasts (20) were included. It is worth noting that none of the yeasts grew on any branched hydrocarbon tested. Whether this is a general property of hydrocarbonoclastic yeasts remains to be determined. The interpretation of negative growth tests is very difficult since one must consider several factors, including (i) toxic effects of the added hydrocarbon, (ii) failure of the added hydrocarbon to be transported or diffused into cells effec-
APPL. ENVIRON. MICROBIOL.
tively, (iii) failure of the branched hydrocarbon
to act as an inducer of the alkane-oxidizing system, or (iv) failure of any of the alkane-oxidizing enzymes to utilize branched substrates (2, 5, 13, 28). We began to consider the latter two points with two pseudomonad strains, Pseudomonas oleovorans (also classified as Pseudomonas pu-
tida PpG6 [13]) and Pseudomonasputida AC4, both of which contain the OCT plasmid. OCT is a transmissible plasmid which confers the ability to grow on n-alkanes of 6 to 10 carbons (9, 13), because it codes for soluble and particulate alkane hydroxylase components and an aliphatic alcohol dehydrogenase (4, 6, 13), which initiate a terminal oxidation sequence (i.e., production of n-alkanals) that is completed by chromosomally coded oxidizing activities. The w-hydroxylase component of the alkane hydroxylase complex has been extensively investigated by McKenna and Coon (18), and although it is capable of hydroxylation of some branched alkanes, its specificity for branched alkane substrates has not been thoroughly investigated (M. J. Coon, personal communication). Since P. oleovorans and P. putida AC4 were not able to grow at the expense of any of the dimethyloctanes tested (Table 1), we first determined whether these branched alkanes were able to induce OCT and carried out induction experiments similar to those described by Grund et al. (13). Cells were grown on a pyruvate-salts medium and exposed for 2 h to a 0.1% (vol/vol) concentration of n-octane, 2,7-DMO, or 3,6DMO or to 1 mM dicyclopropyl ketone (DCPK), a gratuitous inducer of OCT (13, 29). Cells were then washed to remove these compounds, and the ability to oxidize n-octane was measured by oxygen uptake with an oxygen electrode (7). Results are shown in Table 2 (experiment 1). Whereas n-octane and the gratuitous inducer DCPK were effective as inducers, the levels of 2,7-DMO or 3,6-DMO tested produced only a low-level induction of octane oxidation. A similar result was reported by Grund et al. (13) with 2-methylhexane, which is not as effective as nheptane in inducing OCT. To test the possibility that strains containing OCT might be able to assimilate branched alkanes in the presence of a gratuitous inducer, growth tests in liquid culture and solid agar were carried out with 1 mM DCPK present. Neither P. oleovorans nor P. putida AC4 grew consistently in either case. That these two strains are capable of oxidation of 2,7-DMO is shown by the results described in Table 2 (experiment 2). Cells cultured with n-octane as a sole carbon source were washed to remove excess n-octane and allowed to deplete residual n-octane for 4 h in minimal
NOTES
VOL. 38, 1979
TABLE 2. Measurement of whole-cell alkaneoxidizing activity in P. oleovorans 8062 and P. putida AC4' Q(02) Substrate P. oleovorans P. putida Expt Inducer 1
None n-Octane DCPK 3,6-DMO 2,7-DMO
n-Octane n-Octane n-Octane n-Octane n-Octane
8062 5 210 240 32 57
AC4 7 173 204 41 50
205 180 DCPK n-Octane 2 0 3,6-DMO DCPK 29 27 2,7-DMO DCPK Each strain was cultured in H medium or, in the case of P. putida AC4, H medium supplemented with 50,g of Lmethionine per ml and treated as described in the text. To prevent further adaptation, cells were washed with buffer containing 200 pg of chloramphenicol per ml (13). For the oxygen consumption measurements a YS1 electrode was used, and basal oxygen uptake was subtracted. Oxygen quotient [Q(02)] values are in units of microliters of 02 consumed per hour per milligram (dry weight) of cells.
745
paper we show that some of this recalcitrance can be overcome by genetic manipulation of pseudomonad strains (12). We thank R. M. Atlas, A. Chakrabarty, R. R. Colwell, M. J. Coon, J. J. Cooney, W. R. Finnerty, and J. J. Perry for supplying microbial cultures. This investigation was supported in part by grant CHE7616788 from the National Science Foundation to D.S.W., Public Health Service grant HL16628 to R.R.F. and grant GM22978 to D.S.W. from the National Institutes of Health, and a joint grant to D.S.W. and R.R.F. from the Council on Research and Creative Work of the University of Colorado.
2
medium. Cell suspensions in miniimal medium were supplemented with n-octane, 2,7-DMO, or 3,6-DMO, and oxygen uptake was measured with an oxygen electrode. Control experiments with no added alkane were also carried out. Whereas rapid oxygen consumption was seen when n-octane was added, addition of 2,7-DMO resulted in a slow 02 uptake, and addition of 3,6DMO produced an 02 uptake curve identical to the control with no added hydrocarbon. These results suggest that cellular oxidation of branched alkanes is severely limited by iso-termini and prevented by anteiso-termini. The exact location of the block, as well as the analysis of the oxidation products (if any) and direct measurements of hydrocarbon uptake, remains to be determined. McKenna and Coon (18) found that in vitro the w-hydroxylase is capable of oxidation of some branched alkanes, but it is not yet known whether 2,7-DMO and 3,6-DMO are substrates for this enzyme. We have shown here that iso- and anteisosubstituted dimethyloctanes are less susceptible or resistant to degradation by microorganisms capable of metabolizing the parent alkane, noctane. These are clear examples of how alkyl branches can confer molecular recalcitrance. Clearly, an understanding of the cause(s) of such recalcitrance will be important to developing approaches to specific bioaccumulation problems associated with alkyl-branched compounds. However, the exact mechanism(s) for the molecular recalcitrance of these alkanes remains to be resolved and may be the result of very different mechanisms depending on the microorganism involved. In an accompanying
LITERATURE CITED 1. Alexander, M. 1973. Nonbiodegradable and other recalcitrant molecules. Biotechnol. Bioeng. 15:611-647. 2. Bartha, R., and R. M. Atlas. 1977. The microbiology of aquatic oil spills. Adv. Appl. Microbiol. 22:225-266. 3. Beam, H. W., and J. J. Perry. 1974. Microbial degradation of cycloparaffinic hydrocarbons via co-metabolism and commensalism. J. Gen. Microbiol. 82:163-169. 4. Benson, S., M. Fennewald, J. Shapiro, and C. Huettner. 1977. Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations. J. Bacteriol. 132:614-621. 5. Benson, S., and J. Shapiro. 1975. Induction of alkane hydroxylase proteins by unoxidized alkane in Pseudomonasputida. J. Bacteriol. 123:759-760. 6. Benson, S., and J. Shapiro. 1976. Plasmid-determined alcohol dehydrogenase activity in alkane-utilizing strains of Pseudomonas putida. J. Bacteriol. 126:794798. 7. Bflliar, R. B., M. Knappenberger, and B. Little 1970. Xanthine oxidase for calibration of the oxygen electrode apparatus. Anal. Biochem. 36:101-104. 8. Cantwell, S. G., E. P. Lau, D. S. Watt, and R. R. Fall. 1978. Biodegradation of acyclic isoprenoids by Pseudomonas species. J. Bacteriol. 135:324-333. 9. Chakrabarty, A. M. 1976. Plasmids in Pseudomonas. Annu. Rev. Genet. 10:7-30. 10. Cofone, L., Jr., J. D. Walker, and J. J. Cooney. 1973. Utilization of hydrocarbons by Cladosporium resinae. J. Gen. Microbiol. 76:243-246. 11. Davey, J. F., and D. T. Gibson. 1974. Bacterial metabolism of para- and meta-xylene: oxidation of a methyl substituent. J. Bacteriol. 119:923-929. 12. Fall, R. R., J. L. Brown, and T. L. Schaeffer. 1979. Enzyme recruitment allows the biodegradation of recalcitrant branched hydrocarbons by Pseudomonas citronellolis. Appl. Environ. Microbiol. 38:715-722. 13. Grund, A., J. Shapiro, M. Fennewald, P. Bacha, J. Leaky, K. Markbreiter, M. Nieder, and M. Toepfer. 1975. Regulation of alkane oxidation in Pseudomonas putida. J. Bacteriol. 123:546-556. 14. Klug, M. J., and A. J. Markovetz. 1971. Utilization of aliphatic hydrocarbons by microorganisms. Adv. Microb. Physiol. 5:1-43. 15. Lough, A. K. 1973. The chemistry and biochemistry of phytenic, pristanic and related acids. Prog. Chem. Fats Other Lipids 14:1-48. 16. Markovetz, A. J., Jr., J. Cazin, and J. E. Allen. 1968. Assimilation of alkanes and alkenes by fungi. Appl. Microbiol. 16:487-489. 17. McKenna, E. J. 1972. Microbial metabolism of normal and branched chain alkanes, p. 73-97. In Degradation of synthetic organic molecules in the biosphere. National Academy of Sciences, Washington, D.C. 18. McKenna, E. J., and M. J. Coon. 1970. Enzymatic woxidation. IV. Purification and properties of the w-hy-
746
19. 20.
21. 22.
23. 24.
NOTES
droxylase of Pseudomonas oleovorans. J. Biol. Chem. 245:3882-3889. McKenna, E. J., and R. E. Kallio. 1965. The biology of hydrocarbons. Annu. Rev. Microbiol. 19:183-208. Meyer, S. A., K. Anderson, R. E. Brown, M. T. Smith, D. Yarrow, G. Mitchell, and D. G. Ahearn. 1975. Physiological and DNA characterization of Candida maltosa, a hydrocarbon-utilizing yeast. Arch. Microbiol. 104:225-231. Minlon, IL 1946. A simple modification of the WolffKishner reduction. J. Am. Chem. Soc. 68:2487-2488. Nieder, M., and J. Shapiro. 1975. Physiological function of the Pseudomonasputida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids. J. Bacteriol. 122:93-98. Palleroni, N. J., and M. Doudoroff. 1972. Some properties and taxonomic subdivisions of the genus Pseudomonas. Annu. Rev. Phytopathol. 60:215-231. Pirnik, M. P. 1977. Microbial oxidation of methyl
APPL. ENVIRON. MICROBIOL. branched alkanes. Crit. Rev. Microbiol. 5:413-422. 25. Pirnik, M. P., R. M. Atlas, and R. Bartha. 1974. Hydrocarbon metabolism by Brevibacterium erythrogenes: normal and branched alkanes. J. Bacteriol. 119: 868-878. 26. Scott, C. C. L., R. A. Makula, and W. R. Finnerty. 1976. Isolation and characterization of membranes from a hydrocarbon-oxidizing Acinetobacter sp. J. Bacteriol. 127:469-480. 27. Seubert, W., and E. Fass. 1964. Untersuchungen uber den bakteriellen Abbau von Isoprenoiden. V. Der Mechanismus des Isoprenoid-Abbaues. Biochem. Z. 341: 3544. 28. Van der Linden, A. C., and G. J. E. Thijsse. 1965. The mechanisms of microbial oxidations of petroleum hydrocarbons. Adv. Enzymol. 27:469-546. 29. van Eyk, J., and T. J. Bartels. 1968. Paraffin oxidation in Pseudomonas aeruginosa. I. Induction of paraffin oxidation. J. Bacteriol. 96:706-712.
AND
ENVIRONMENTAL MICROBIOLOGY, Oct. 1979, p. 742-746
Vol. 38, No. 4
0099-2240/79/10-0742/05$02.00/0
Microbial Growth on Hydrocarbons: Terminal Branching Inhibits Biodegradation TERI L. SCHAEFFER, SUSAN G. CANTWELL, JEFFREY L. BROWN, DAVID S. WATT, AND R. RAY FALL*
Department of Chemistry, University of Colorado, Boulder, Colorado 80309
Received for publication 18 July 1979
A variety of octane-utilizing bacteria and fungi were screened for growth on terminally branched dimethyloctane derivatives to explore the effects of iso- and anteiso-termini on the biodegradability of such hydrocarbons. Of 27 microbial strains tested, only 9 were found to use any of the branched hydrocarbons tested as a sole carbon source, and then only those hydrocarbons containing at least one iso-terminus were susceptible to degradation. Anteiso- or isopropenyl termini prevented biodegradation. None of the hydrocarbonoclastic yeasts tested was able to utilize branched-hydrocarbon growth substrates. In the case of pseudomonads containing the OCT plasmid, whole-cell oxidation of n-octane was poorly induced by terminally branched dimethyloctanes. In the presence of a gratuitous inducer of the octane-oxidizing enzymes, the iso-branched 2,7-dimethyloctane was slowly oxidized by whole cells, whereas the anteiso-branched 3,6-dimethyloctane was not oxidized at all. This microbial sampling dramatically illustrated the deleterious effect of alkyl branching, especially anteiso-terminal branching, on the biodegradation of hydrocarbons. some
n-Alkanes are readily oxidized by many microorganisms, predominantly via an initial oxidation of a terminal methyl group followed by a ,3-oxidation sequence (14, 19, 22, 28). Alkylbranched alkanes are generally less susceptible to biodegradation (17, 24), and certain branching patterns confer biological recalcitrance, causing these compounds to accumulate in the biosphere (1, 2). If alkyl branches are located near the terminus, it seems likely that decreased biodegradability could be the result of steric inhibition of terminal oxidizing enzymes, although transport defects or failure to induce the appropriate oxidizing system or both may be responsible, too. In the case where an alkyl branch occurs at the /3-position (i.e., anteiso-terminus), the branch will prevent /3-oxidation, requiring an additional strategy such as a-oxidation (3, 15), /3-alkyl group removal (8,27), or w-oxidation (24) to allow oxidation to proceed. We have been searching for microorganisms that are able to carry out the oxidation of /3methyl-substituted alkanes, especially where the pseudomonad type of /3-alkyl group removal occurs (8, 27). Twenty-seven different octane-utilizing strains of bacteria and fungi were obtained (see Table 1) and were screened for growth on 3,6-dimethyloctane (3,6-DMO), a dimethyloctane with /8-methyl substituents (anteiso-) at both ends of the molecule. For comparison, each strain was also screened for growth on 2,7-di-
methyloctane (2,7-DMO), which contains amethyl (iso-) termini, or 2,6-dimethyloctane (2,6-DMO), which contains both iso- and anteiso-termini. Screening with 2,6-dimethyl-2-octene will be described below. Growth tests were carried out by the following general procedure (23). Cultures were grown in minimal media (see Table 1) supplemented with 0.5% (wt/vol) yeast extract (Difco Laboratories), then washed and suspended in the appropriate minimal medium, and used to inoculate minimal media containing 1% (vol/vol) of the appropriate filter-sterilized hydrocarbon. The cultures were transferred to metal tins with tight-fitting lids to maintain a hydrocarbon vapor atmosphere. The tins were opened every 48 h to admit oxygen, the cultures were agitated with a Vortex mixer and monitored for growth, and the tins were resealed. Additional portions of hydrocarbons were added after 6, 12, and 18 days. Growth was compared with controls containing inoculated medium with no hydrocarbon and incubated separately. Growth at 23°C was monitored for up to 22 days with slow-growing cultures. The hydrocarbons 3,6-DMO, 2,7-DMO, and 2,6DMO were each 99+% grade obtained from Chemical Samples Co. 2,6-Dimethyl-2-octene was prepared by Wolff-Kishner reduction (21) of citronellal (Pfaltz & Bauer, Inc.). The product was bulb to bulb distilled at 65 to 72°C (50-mm pressure) and obtained pure by preparative gas 742
VOL. 38, 1979
743
NOTES
chromatography on a 10% Carbowax 20 M column (10-ft [ca. 3-m] column; helium flow rate, 45 ml/min; injector temperature, 175°C; detector temperature, 225°C; retention time, 3 min). Each hydrocarbon was passed over a silicic acid column before use to remove traces of oxidized substances. Of the 27 octane-utilizing strains tested, none grew with 3,6-DMO as a sole carbon source (Table 1). The possibility that the 3,6-DMO contained a growth inhibitor as a trace contaminant was ruled out, since a 1:1 mixture of 3,6-
DMO and n-octane supported the growth of several strains as weli as n-octane alone. These results show that the symmetrical anteiso-substitution pattern present in 3,6-DMO represents a recalcitrant structural feature preventing biodegradation. Nine strains were able to utilize the symmetrically iso-branched 2,7-DMO as a sole carbon source (Table 1), suggesting that an isobranched terminus is more readily degraded than an anteiso-branched terminus. Still, most of the 27 strains tested could not utilize this
TABLE 1. Ability of octane-utilizing microorganisms to grow on branched octane derivatives as a sole carbon source Growth substrate Strain
Bacteria Achromobacter sp. Acinetobacter sp. H01-N Arthrobacter ap. Brevibacterium erythrogenes Corynebacterium hydrocarbonoclastus 15961 Corynebacterium ap. Flavobacterium sp. Mycobacterium convolutum R-22 Mycobacterium rhodochrous OFS Mycobacterium rhodochrous 7E1C Nocardia sp. Nocardia rubra Pseudomonas aeruginosa 17423 Pseudomonas oleovorans 8062 Pseudomonas oleovorans Pseudomonas putida AC4 Pseudomonas Pxy
Fungi Aspergillus versicolor Candida maltosa 28140 Candida petrophilum 20226 Candida sake 14478 Candida tropicalis 750 Cladosporium resinae UD42 Cunninghamella blakes-
Source or reference
Growth 2,6-Dimedium' n-Octane 3,6-DMO 2,7-DMO 2,6-DMO methyl-2octene
Octane enrichment from oil seep W. R. Finnerty (26) Sewage R. M. Atlas (25)
H
2
0
0
0
0
H H HS
1 2 2
0 0 0
0 0 1
0 0 1
0 0 0
ATCCb
H
0
0
0
0
Octane enrichment Octane enrichment J. J. Perry
H H H
0 0 0
2 0 1
1 0 1
0 0 0
W. R. Finnerty
H
W. R. Finnerty
H
2
0
0
0
0
Octane enrichment W. R. Finnerty ATCC
H H H
2 1 2
0 0 0
1 0 1
1 0 1
0 0
ATCC
H
2
0
0
0
M. J. Coon A. Chakrabarty A. Chakrabarty (11)
H H
3 3 2
0 0 0
0 0 0
0 0 0
0 0 0
J. J. Perry ATCC ATCC ATCC ATCC J. J. Cooney (10, 16)
YNBS YNB YNB YNB YNB YNB
2 1 1 1 1 1
0 0 0 0 0 0
2 0 0 0 0 0
1 0 0 0 0 0
0 0 0 0 0 0
Hd
3 1 2
1
0
OM)
0 0 2 YNB ATCC leeana 8688a 0 1 1 0 2 YNBS J. J. Perry EupeniciUum sp. 0 0 0 0 1 YNB ATCC Rhodotorula glutinis 2527 0 0 0 0 1 YNB R. R. Colwell Sporobolomyces sp. a Growth media have been described elsewhere (12, 20). HS is H medium containing 3% (wt/vol) NaCl, and YNBS is also supplemented with this level of NaCl. Growth ratings are as follows: 0, no growth; 1, weak but consistent growth; 2, moderate growth; 3, abundant growth. h ATCC, American Type Culture Collection. 'After prolonged incubation in liquid medium, several positive clones were isolated. These will be described in a separate communication. d The H medium was supplemented with 20 ug of L-methionine per ml.
744
NOTES
hydrocarbon, further illustrating the deleterious effect of alkyl branching on the microbial assimilation of hydrocarbons (19). That there is also a chain length specificity involved in the biodegradability of such branched hydrocarbons is indicated by the fact that several of the strains tested, including the Acinetobacter species, Mycobacterium rhodochrous 7E1C, and Nocardia rubra, can use pristane (2,6,10,14-tetramethylpentadecane) as a sole carbon source (data not shown), but not 2,7-DMO. Pristane is a highly branched hydrocarbon that also contains isobranched termini like 2,7-DMO, and its degradation via terminal oxidation and,8-oxidation or a combination of f- and w-oxidation has been reported in several microorganisms (24). Thus, an iso-branched terminus is not necessarily a recalcitrant structural feature in the strains mentioned above, but perhaps in the case of 2,7DMO their octane-oxidizing systems cannot initiate oxidation of the branched molecule. Those organisms which could utilize 2,7-DMO but not 3,6-DMO were able in each case to also utilize 2,6-DMO (Table 1). This suggests that oxidation of 2,6-DMO proceeded in these instances via the iso-terminus, although direct experimental evidence is lacking. We were also interested in exploring the possibility that 2,6-dimethyl-2-octene could be degraded via the citronellol pathway (27). If this branched hydrocarbon was hydroxylated at the terminal methyl group of the anteiso-terminus, this would produce citronellol for which there is an established degradative pathway in certain pseudomonads (8, 27; see Fig. 1 of reference 12). Screening with 2,6-dimethyl-2-octene (Table 1) showed that it is also a very recalcitrant structure. Of the strains tested, only a Pseudomonas aeruginosa strain showed any growth, and then only when a few positive clones arose after prolonged exposure to the hydrocarbon. (The properties of these clones will be described in a separate communication; see reference 12.) Strains that were able to utilize 2,6-DMO were in each case unable to use 2,6-dimethyl-2-octene (Table 1), suggesting that the isopropenyl terminus in the latter hydrocarbon is a recalcitrant feature. In the microbial screening, six species of hydrocarbonoclastic yeasts (20) were included. It is worth noting that none of the yeasts grew on any branched hydrocarbon tested. Whether this is a general property of hydrocarbonoclastic yeasts remains to be determined. The interpretation of negative growth tests is very difficult since one must consider several factors, including (i) toxic effects of the added hydrocarbon, (ii) failure of the added hydrocarbon to be transported or diffused into cells effec-
APPL. ENVIRON. MICROBIOL.
tively, (iii) failure of the branched hydrocarbon
to act as an inducer of the alkane-oxidizing system, or (iv) failure of any of the alkane-oxidizing enzymes to utilize branched substrates (2, 5, 13, 28). We began to consider the latter two points with two pseudomonad strains, Pseudomonas oleovorans (also classified as Pseudomonas pu-
tida PpG6 [13]) and Pseudomonasputida AC4, both of which contain the OCT plasmid. OCT is a transmissible plasmid which confers the ability to grow on n-alkanes of 6 to 10 carbons (9, 13), because it codes for soluble and particulate alkane hydroxylase components and an aliphatic alcohol dehydrogenase (4, 6, 13), which initiate a terminal oxidation sequence (i.e., production of n-alkanals) that is completed by chromosomally coded oxidizing activities. The w-hydroxylase component of the alkane hydroxylase complex has been extensively investigated by McKenna and Coon (18), and although it is capable of hydroxylation of some branched alkanes, its specificity for branched alkane substrates has not been thoroughly investigated (M. J. Coon, personal communication). Since P. oleovorans and P. putida AC4 were not able to grow at the expense of any of the dimethyloctanes tested (Table 1), we first determined whether these branched alkanes were able to induce OCT and carried out induction experiments similar to those described by Grund et al. (13). Cells were grown on a pyruvate-salts medium and exposed for 2 h to a 0.1% (vol/vol) concentration of n-octane, 2,7-DMO, or 3,6DMO or to 1 mM dicyclopropyl ketone (DCPK), a gratuitous inducer of OCT (13, 29). Cells were then washed to remove these compounds, and the ability to oxidize n-octane was measured by oxygen uptake with an oxygen electrode (7). Results are shown in Table 2 (experiment 1). Whereas n-octane and the gratuitous inducer DCPK were effective as inducers, the levels of 2,7-DMO or 3,6-DMO tested produced only a low-level induction of octane oxidation. A similar result was reported by Grund et al. (13) with 2-methylhexane, which is not as effective as nheptane in inducing OCT. To test the possibility that strains containing OCT might be able to assimilate branched alkanes in the presence of a gratuitous inducer, growth tests in liquid culture and solid agar were carried out with 1 mM DCPK present. Neither P. oleovorans nor P. putida AC4 grew consistently in either case. That these two strains are capable of oxidation of 2,7-DMO is shown by the results described in Table 2 (experiment 2). Cells cultured with n-octane as a sole carbon source were washed to remove excess n-octane and allowed to deplete residual n-octane for 4 h in minimal
NOTES
VOL. 38, 1979
TABLE 2. Measurement of whole-cell alkaneoxidizing activity in P. oleovorans 8062 and P. putida AC4' Q(02) Substrate P. oleovorans P. putida Expt Inducer 1
None n-Octane DCPK 3,6-DMO 2,7-DMO
n-Octane n-Octane n-Octane n-Octane n-Octane
8062 5 210 240 32 57
AC4 7 173 204 41 50
205 180 DCPK n-Octane 2 0 3,6-DMO DCPK 29 27 2,7-DMO DCPK Each strain was cultured in H medium or, in the case of P. putida AC4, H medium supplemented with 50,g of Lmethionine per ml and treated as described in the text. To prevent further adaptation, cells were washed with buffer containing 200 pg of chloramphenicol per ml (13). For the oxygen consumption measurements a YS1 electrode was used, and basal oxygen uptake was subtracted. Oxygen quotient [Q(02)] values are in units of microliters of 02 consumed per hour per milligram (dry weight) of cells.
745
paper we show that some of this recalcitrance can be overcome by genetic manipulation of pseudomonad strains (12). We thank R. M. Atlas, A. Chakrabarty, R. R. Colwell, M. J. Coon, J. J. Cooney, W. R. Finnerty, and J. J. Perry for supplying microbial cultures. This investigation was supported in part by grant CHE7616788 from the National Science Foundation to D.S.W., Public Health Service grant HL16628 to R.R.F. and grant GM22978 to D.S.W. from the National Institutes of Health, and a joint grant to D.S.W. and R.R.F. from the Council on Research and Creative Work of the University of Colorado.
2
medium. Cell suspensions in miniimal medium were supplemented with n-octane, 2,7-DMO, or 3,6-DMO, and oxygen uptake was measured with an oxygen electrode. Control experiments with no added alkane were also carried out. Whereas rapid oxygen consumption was seen when n-octane was added, addition of 2,7-DMO resulted in a slow 02 uptake, and addition of 3,6DMO produced an 02 uptake curve identical to the control with no added hydrocarbon. These results suggest that cellular oxidation of branched alkanes is severely limited by iso-termini and prevented by anteiso-termini. The exact location of the block, as well as the analysis of the oxidation products (if any) and direct measurements of hydrocarbon uptake, remains to be determined. McKenna and Coon (18) found that in vitro the w-hydroxylase is capable of oxidation of some branched alkanes, but it is not yet known whether 2,7-DMO and 3,6-DMO are substrates for this enzyme. We have shown here that iso- and anteisosubstituted dimethyloctanes are less susceptible or resistant to degradation by microorganisms capable of metabolizing the parent alkane, noctane. These are clear examples of how alkyl branches can confer molecular recalcitrance. Clearly, an understanding of the cause(s) of such recalcitrance will be important to developing approaches to specific bioaccumulation problems associated with alkyl-branched compounds. However, the exact mechanism(s) for the molecular recalcitrance of these alkanes remains to be resolved and may be the result of very different mechanisms depending on the microorganism involved. In an accompanying
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