Enumeration of Petroleum-Degrading Microorganisms

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1976, p Copyright C) 1976 Americn Society for Microbiology Vol. 31, No. 2 Printed in U.S.A. Enumertion of Petroleum-Degrding Microorgnisms J. D. WALKER' AND R. R. COLWELL* Deprtment of Microbiology, University of Mrylnd, College Prk, Mrylnd 2742 Received for publiction 3 Jnury 1975 A vriety of fctors, including concentrtion of oil, ntibiotics, dyes, nd inoculum shes, ere exmined to determine their effect on the totl counts of microorgnisms on oil-contining medi. The medi found to be best for enumerting petroleum-degrding microorgnisms contined.5% (vol/vol) oil nd.3% phenol red, ith Fungione dded for isolting bcteri nd streptomycin nd dded for isolting yests nd fungi. Wshing the inoculum did not improve recovery of petroleum degrders. Specificlly, silic gel-oil medium nd yest medium re recommended for enumertion of petroleum-degrding bcteri nd yests nd fungi, respectively. It is suggested tht counts of petroleum degrders be expressed s percentge of the totl popultion rther thn totl numbers of petroleum degrders per se. Incubtion temperture nd presence of oil s found to influence the numbers of petroleum-degrding microorgnisms t given smpling site. Becuse of the mrkedly incresed explortion for oil nd relted energy sources, public MATERIALS AND METHOI)S ttention hs been directed to environmentl Smpling nd identifiction of isoltes. Methods for smpling ter nd sediment nd procedures effects of such explortion, prticulrly ith respect to potentil contmintion of the environ- described elsehere (23; J. J. Clomiris, J. D. for identifying the pure cultures isolted hve been ment ith oil. Since the first line of defense, Wlker, nd R. R. Colell, J. Appl. Bcteriol., in so to spek, ginst oil pollution in the environment is the microbil popultion, it becomes Medi. The bsl medi used in the isoltion of press). impertive to kno hether microbil degrders of oil re present in ter nd soil of the isoltion of heterotrophic yests nd fungi hve heterotrophic bcteri nd the yest medium for the re to be impcted. Hence, enumertion of been described elsehere (26). Oil gr (OA) no. 2, petroleum-degrding microorgnisms is importnt both to determine the potentil for re- selective medium used for the isoltion of petroleum-degrding microorgnisms, hs lso been described previously (25). Severl dyes nd grs ere movl of oil vi microbil degrdtion nd to compred in this study. All pltings ere done in ssess the mount of oil pollution tht hs occurred, if the popultions of petroleum-degrd- The five-tube MPN technique s lso used to duplicte. ing microorgnisms prove to be relted to the estimte the number of petroleum-degrding microorgnisms (1); 5 ml of double-strength oil slts concentrtion of polluting oil present. In this study, petroleum-degrding microorgnisms ere isolted from smples of se- sediment nd 1 ml of single-strength oil slts solu- solution (26) s inoculted ith 5 ml of ter or ter nd bottom sediments t sttions locted tion s inoculted ith 1 or.1 ml of ter or beteen 4 south ltitude nd 6 north sediment (Tble 1). Fungione (finl concentrtion, ltitude (32). Severl methods ere used to 1 /g/ml) nd streptomycin nd (finl concentrtion, 5,tg/ml ech) ere dded to medi enumerte the petroleum-degrding bcteri, s pproprite. including the most-probble-number (MPN) Silic gel medium s prepred by using the method (9, 13, 33) nd plting on oil gr (7, procedure of Funk nd Krulich (8), s described 23), gr in inverted petri dishes ith n oilsturted filter on the bottom of the petri dish prepred by dding 2 g of KNO:,, 1 g of MgSO4, 3 mg previously (5). Double-strength slts solution s (16), nd silic gel-oil gr (5, 18, 24). Some of of phenol red indictor, nd 1 ml of nondetergent these methods ere evluted ith respect to motor oil (1-2 eight) to 1 liter of distilled ter. enumertion of petroleum-degrding yests, Potssium silicte s prepred by dissolving 1 g fungi, nd bcteri from esturine nd mrine of silic gel (Fisher grde 923, 1 to 2 mesh) in 1 ml of 7% KOH ith heting. The slts solution smples. nd potssium silicte ere dispensed seprtely in ' Present ddress: Environmentl Technology Center, 5-ml liquots to 125-ml Erlenmeyer flsks nd Mrtin Mriett Corp., 145 South Rolling Rd., Bltimore, Md slts solution cooled to mbient temperture,.5 ml utoclved for 15 min t 121 C, 1 tm. After the 198 Donloded from on September 23, 218 by guest

2 VOL. 31, 1976 PETROLEUM-DEGRADING MICROORGANISMS 199 TABLE 1. Procedure folloed in prepring tubes for estimting MPN of petroleum-degrding microorgnisms ph of oil slts Antibiotics Colgte Creek sediment Colgte Creek sediment Colgte Creek sediment Colgte Creek sediment Colgte Creek sediment Colgte Creek sediment Colgte Creek ter Colgte Creek ter Colgte Creek ter Colgte Creek ter Colgte Creek ter Colgte Creek ter Estern By sediment Estern By sediment Estern By sediment Estern By ter Estern By ter Estern By ter Colgte Creek sediment Colgte Creek sediment Colgte Creek sediment Colgte Creek ter Colgte Creek ter Colgte Creek ter Estern By sediment Estern By sediment Estern By sediment Estern By ter Estern By ter Estern By ter of KH2PO4 (1%) nd 1 ml of 2% phosphoric cid ere dded. A 5-ml liquot of potssium silicte (ph 6 to 7, red-ornge) s dded to 5 ml of slts solution, mixed quickly, nd poured into petri dishes. Geltion occurred ithin pproximtely 1 min. Unless stted otherise, ech medium contined.5% (vol/vol) 1-2 W nondetergent motor oil Ẇshing of inoculum. To determine hether nutrient crryover from the inoculum supported microbil groth, ter nd sediment inocul ere shed nd plted on plte count gr, OA no. 2, nd silic gel-oil (SGO) medium s described belo. Fresh sediment (5 g) s dded to 4 ml of mixture of esturine slts solution (26) nd 5 ml of sodium pyrophosphte. The ltter s dded s Dilution of Amt of Rtio: mt of ininocu- lum (ml) oculum-mt oil slts of "' 1" " 1" " 1" 1" 5. 1:1 1. 1:1.1 1:1 5. 1:1 1. 1:1.1 1:1 5. 1:1 1. 1:1.1 1:1 5. 1:1 1. 1:1.1 1:1 5. 1:1 1. 1:1.1 1:1 5. 1:1 1. 1:1.1 1:1 5. 1:1 1. 1:1.1 1:1 5. 1: :1 1:1 1:1 1. 1:1.1 1:1 5. 1:1 1. 1:1.1 1:1 seprte sterile solution to give finl concentrtion of.1% (vol/vol). The sediment suspension s mixed in Wring blender tice, 3 s ech time, fter hich the sediment s lloed to settle. Plte counts ere mde using the superntnt fluid. The superntnt fluid s centrifuged t 36 x g for 15 min, fter hich it s removed from the first centrifugtion nd gin centrifuged t 36 x g for 15 min. The superntnt fluid s removed from the second centrifugtion nd centrifuged t 13, x g for 15 min. The resulting pellet s shed ith esturine slts solution plus.1% sodium pyrophosphte nd centrifuged t 36 x g. The remining superntnt fluid s centrifuged t 13, x g, nd the resulting pellet s shed three times ith esturine slts plus. 1% sodium Donloded from on September 23, 218 by guest

3 2 WALKER AND COLWELL pyrophosphte, folloed ech time by centrifugtion t 13, x g. After the third sh, the pellet s resuspended in 45 ml of esturine slts plus.1% sodium pyrophosphte nd plte counts ere mde. Portions of the ter smples ere plted immeditely, nd the rest of the smple s centrifuged t 36 x g for 15 min. The superntnt fluid s centrifuged t 13, x g for 15 min, nd the resulting pellet s shed three times ith esturine slts plus.1% sodium pyrophosphte, folloed by centrifugtion t 13, x g. After three shings, the pellet s resuspended to the originl volume, using esturine slts plus. 1% sodium pyrophosphte, nd plte counts ere mde. Estimting the efficiency of medi for isolting petroleum-degrding microorgnisms. Colonies ppering on ech of the medi used ere counted, nd representtive colonies ere picked from ech of the duplicte pltes prepred for the vrious gr medi nd purified for further study. Pltes prepred by trnsfer from dilutions of the MPN tube cultures ere included in the smpling of colonies for dditionl study. Five drops of the shed cultures, prepred from pure cultures gron for 2 dys t 25 C, s dded to 5 ml of slts solution supplemented ith NO:, nd P4 nd contining.5% motor oil. A petroleum-degrding isolte, for the purposes of this study, s pure culture producing groth in tubes contining oil, but not in tubes contining slts ithout oil dded, the ltter serving s controls. Altertion of motor oil during utoclving. Motor oil (5 g, 5.6 ml) s dded to ech of to 2-liter flsks contining 1, ml of esturine slts. One flsk s utoclved for 15 min t 121 C, 1 tm. After the sterile oil cooled to mbient temperture, the contents of both flsks ere extrcted tice ith 5 ml of benene. The extrcts ere dried over 1 g of N2SO4 nd dded to 25-ml round-bottom flsks, together ith n dditionl 5 ml of benene to sh the N2SO4. The extrcts ere concentrted to constnt eight under N2 t 4 C. Ech extrct s nlyed by gs-liquid chromtogrphy s described elsehere (27). RESULTS AND DISCUSSION Stndrd methods for enumerting petroleum-degrding bcteri, yests, nd fungi re required if dt from different lbortories re to be compred. From the studies of the enumertion of petroleum-degrding bcteri reported here, correltions cn be drn beteen hydrocrbon-degrding ctivity nd numbers of petroleum degrders (r =.86) or the percentge of the totl count tht re petroleum degrders (r =.99) (25). Results of preliminry studies, using OA no. 1 hereby the oil s coted on silic gel s described by Bruh et l. (3), shoed tht the numbers of petroleum-degrding microorgnisms found t n oil-polluted site, nmely, Colgte Creek in Bltimore Hrbor, nd t n oil-free site, Estern By, south of Prson's Islnd in Chespeke By, could be relted to the mount of oil present in the respective environments (5, 22). The OA no. 1 medium used in the erlier study s improved by substituting n oil soniction procedure for silic gel, i.e., OA no. 2. Autoclving the medium ith the oil did not result in significnt loss of the oil, nmely, 2.6% lost during utoclving compred to.14% lost during lbortory evportion to constnt eight. Exmintion of the oil, fter utoclving, by gs-liquid chromtogrphy reveled no significnt differences beteen nonutoclved nd utoclved oil. In this study the concentrtion of oil in the medi used s.5% (vol/vol), the concentrtion yielding highest recovery of bcteri (Fig. 1). To enumerte bcteri, yests, nd fungi, the medi used ere supplemented ith ntibiotics. Addition of ntibiotics resulted in selective inhibition; e.g., nd streptomycin inhibited bcteril groth but did not ffect dversely the counts of yests nd fungi. Similrly, Fungione prevented groth of yests nd fungi but did not inhibit bcteril groth. It should be noted tht ddition of Fungione to OA no. 2 s found to be selective for ctinomycetes (25). Addition of dyes (phenol red,.3%; bromothymol blue,.3%; bromocresol purple,.5%; methyl red,.3%) to the medi provided colored bckground ginst hich colonies could be observed more esily thn hen the stndrd opque medium s used. The dyes did not inhibit groth of bcteri nd, in some cses, cid production s observed round the colonies. Of 1 bcteril strins picked from OA no. 2 pltes, 3 produced cid on the phenol red medium, but only 1 of the ltter utilied oil s sole crbon nd energy source. Hence, there s no correltion obx 1-8 APPL. ENVIRON. MICROBIOL. \. r PERCENT OIL IN MEDIUM FIG. 1. Correltion of log number of bcteri per milliliter ith mount of oil incorported into solid medium. Points represent men of triplicte pltings of duplicte smples of Colgte Creek sediment inoculted cultures on OA no. 2. Donloded from on September 23, 218 by guest

4 VOL. 31, 1976 served beteen cid production on phenol red medium nd oil utilition. Nevertheless, the dyes provided useful ddition to the medi hen colonies ere enumerted. From the results of this study, phenol red s the dye of choice becuse it provided the best bckground for enumerting the colonies. Significnt numbers of non-oil-utiliing microorgnisms ere lys encountered on OA no. 2. Becuse OA no. 2 s the first to be developed in this study, it s used s screening medium nd for purposes of comprison in this study. Counts obtined on OA no. 2 prepred ith purified gr (Difco), compred ith counts on OA no. 2 mde up ith shed, purified grs nd contining no oil, shoed little or no difference, except for pltes inoculted ith Colgte Creek sediment, here counts differed by n order of mgnitude (Tble 2). The lo percentge of gr digesting bcteri in Chespeke By (2 to 3%) s not sufficient to ccount for the result observed. It is concluded tht bcteri in the smples could gro in the presence of the impurities in the gr nd/or nutrients in the inocul. The ltter possibility, i.e., crryover of nutrients in the inoculum, s ruled out for sediment nd ter inocul by series of experiments in hich the inocul ere shed crefully prior to ddition to the count pltes. If dsorption to sediment contributed significntly to the decrese in counts fter shing, then the percent decrese ould hve been significntly greter for pltes inoc- TABLE 2. PETROLEUM-DEGRADING MICROORGANISMS 21 ulted ith sediment, result not observed in this study. The totl vible erobic, heterotrophic count s much loer in ll cses hen the inoculum s shed (Tble 3). The portion of the totl popultion tht comprised the petroleum degrders s evluted s follos. If the lo percentge of oil-utiliing bcteri shoing groth on the pltes resulted from utilition of nutrients in the inoculum, then shing the inoculum should increse the percentge of oil utiliers. This s not the cse hen OA no. 2 (Tble 4) or SGO medium (Tble 5) s used, except for those pltes inoculted ith Colgte Creek ter plted on SGO medium. An increse in the percentge of petroleum degrders in the ltter cse s very likely relted to the lo percentge of petroleum degrders before shing (36%), s discussed belo. The higher percentge of petroleum-degrding bcteri, s determined using SGO medium, compred ith OA no. 2 suggested tht impurities in the gr supported groth of non-oil-utiliing bcteri. Silicic cid s introduced s solidifying gent in microbiologicl medi by Winogrdsky (31). A number of reports hve ppered describing the preprtion of silic gel medi (8, 1-12, 15, 17, 19-22). The inorgnic source of nitrogen in our medi, NH,NO:,, provided to sources of nitrogen. Use of NH,NO:, in the preprtion of SGO (24), using the method of Prmer (15) or of Funk nd Krulich (8), results in the production of mmoni during gel- Groth of bcteri on OA no. 2 nd purified gr" Source of inoculum Medium Colgte Creek Estern By Wter Sediment Wter Sediment OA 4.4 x x 1-' 1.5 x 1' 1. x 14 Purified gr 3.7 x x x x 13 Bushnell-Hs gr 3.9 x x x 1' 9.1 x 13 Ion gr no x x x 1' 9.3 x 13 Ion gr no. 2S 1.5 x x x 1' 7.4 x 13 Noble gr 2.7 x x x x 13 Counts re given per milliliter of ter or per grm of sediment. TABLE 3. Effect of shing the inoculum on the totl counts of erobic heterotrophic bcteri Determintion Colgte Creek sedi- Colgted Creek - Estern By sedi- Estern By ter ment ter ment Before shing 7.5 x x x x 13 After shing 2.8 x x x x 12 Numericl decrese 7.2 x x x x 13 Percent decrese Counts re given per milliliter of ter or per grm of sediment smple. Donloded from on September 23, 218 by guest

5 22 WALKER AND COLWELL TABLE 4. Effect of shing the inoculum on the counts ofpetroleum degrders (PD) on OA no. 2 Determintion Colgte Creek Colgte Creek Estern By Estern By sediment ter Sediment ter Before shing 4.8 x x x x 1 After shing 1.5 x x 12 <1' 1. x 1 Numericl decrese 1. x 1X 4.3 x 13 >1.9 x x 1O Percent decrese >9 8 PD before shing 4. x x 1" Type colonies PD before shing 1. 1 Percent PD before shing 77 2 PD fter shing 4. x 12 Numericl decrese PD NDI 3.6 x 13 ND ND Type colonies PD fter shing 1 Numericl decrese colonies PD ND ND ND Percent PD fter shing 47 Percent decrese PD ND 3 ND ND Counts re given per milliliter of ter or per grm of sediment. b Increse. Indictes distinct colonil type nd representtive colony. d ND, Not determined. TABLE 5. Effect of shing the inoculum on the counts ofpetroleum degrders (PD) on SGO medium Determintion Colgte Creek Colgte Creek Estern By Estern By sediment ter sediment ter Before shing 1. x x x x 1 After shing 1. x 1' 1. x x 1' 1. x 1" Numericl decrese 9. x 1' 4.5 x x 1' 1.5 x 1 Percent decrese PD before shing 9. x 1' 2. x x 1' 1.2 x 1 Type colonies PD before shing Percent PD before shing PD fter shing <11 5. x 1' 1. x 11 1 Numericl decrese PD NDb 1.5 x x 11 ND Type colonies PD fter shing 3 1 Numericl decrese colonies PD ND 2C ND Percent PD fter shing 5 32 Percent decrese PD ND 14' 8 ND Counts re given per milliliter of ter or per grm of sediment smple. b ND, Not determined. " Increse. tion nd should be voided. Therefore, groth of bcteri on OA no. 2 contining NH,NO:I or KNO:, s nitrogen sources s compred. Resonbly similr counts ere obtined using these compounds s nitrogen source for bcteri, yests, nd filmentous fungi. Numbers of petroleum-degrders ere compred using the MPN technique (Tble 6), bsl gr, OA no. 2, nd SGO (Tble 7). As might hve been expected, the highest totl counts ere observed on the bsl gr generlly used for totl erobic heterotrophic bcteril counts. Similr results ere observed ith the MPN method nd SGO, s reported previously by Seki (18). The SGO medium hs APPL. ENVIRON. MICROBIOL. lso been used to enumerte petroleum-degrding mrine bcteri using the membrne filter method (3). Comprison of the MPN method ith results obtined using yest gr, OA no. 2, nd SGO medium for yests nd fungi provided similr results. Highest counts ere obtined on yest gr, medium used for enumertion of heterotrophic yests nd fungi. Loer counts ere obtined on OA no. 2, nd the loest counts ere those here the MPN method nd SGO ere used, the ltter supporting slightly higher counts of filmentous fungi (Tble 8). To estimte the percentge of petroleum degrders, i.e., tht portion of the totl number Donloded from on September 23, 218 by guest

6 VOL. 31, 1976 PETROLEUM-DEGRADING MICROORGANISMS 23 TABLE 6. Groth of microorgnisms on motor oil using the MPN technique No. of positive tubes for: Dilution Bcteri Fungi nd yests 1:1 1:1 1:1 1:1 1:1 1:1 Colgte Creek sediment Colgte Creek sediment Colgte Creek ter Colgte Creek ter Estern By sediment Estern By ter medium rtio. TABLE 7. Totl counts of bcteri on different medi Medium MPN Bsl gr OA no. 2 SGO Colgte Creek sediment 9.2 x x x x 12 Colgte Creek ter 1.6 x x x x 11 Estern By sediment 8. x 1 9. x x x 11 Estern By ter.5 x 1 6. x x x 1 Counts re given per milliliter of ter or per grm of sediment. TABLE 8. Counts of petroleum-degrding yests nd fungi on different medi Medium MPN Yest gr OA no. 2 SGO Colgte Creek sediment 2.5 x 1 1. x x x 1 Colgte Creek ter (ml) 2/1 12/1 1/1 1/1 Estern By sediment <1" <1 <1 <1 Estern By ter (ml) <1/1 59/1 2/1 <1/1 Counts re given per milliliter of ter or per grm of sediment. of colonies from plte of given medium, tht degrded petroleum, the MPN dilution tubes ere plted nd the percentge of petroleum degrders s clculted, s described bove. The highest percentge of petroleum-degrding bcteri s obtined using the SGO medium (Tble 9). Silic gel is less likely to contin the impurities present in gr. In generl, yest gr gve the highest estimte of petroleumdegrding yest nd fungi, lthough OA no. 2 nd the SGO medium ere reltively efficient (Tble 1). Results from bttery of txonomic tests ere condensed to provide generic description of the bcteril popultions occurring on SGO medium, bsl gr, nd OA no. 2 (Tbles 11 nd 12). A petroleum-degrding Pseudomons sp. s predominnt in the popultion ppering on the SGO medium. Other fctors possibly ffecting the petroleum-degrding microorgnisms re incubtion temperture nd mount of oil present in the sediment or ter under exmintion. The temperture of incubtion ill influence the number of petroleum degrders recovered. Furthermore, there my be sesonl effect, s shon for petroleum degrders enumerted from SGO medium (Clomiris et l., J. Appl. Bcteriol., in press) (Fig. 2). At lest to tempertures for incubtion should be used for enumertion of petroleum-degrding bcteri on monthly bsis, nd temperture dpttion index (13) might prove useful. Expressing results s the percentge of totl popultions verged for given temperture is method for normliing the dt nd is recommended for environmentl sites not smpled on regulr bsis. Correltion of numbers of petroleum-degrding microorgnisms ith mount of oil present in the given environment cn be esily ccomplished by extrcting the ter or sediment smple ith n orgnic solvent nd compring the results ith the number of petroleum degrders estimted for the smple. Using this pproch, the totl number of petroleum-degrding bcteri in Colgte Creek sediment Donloded from on September 23, 218 by guest

7 24 WALKER AND COLWELL TABLE 9. Efficiency of vrious medi for enumerting petroleum-degrding bcteri % Petro- Medium leum degrders Colgte Creek sediment MPN 39. Colgte Creek sediment Bsl gr 2.6 Colgte Creek sediment OA no Colgte Creek sediment SGO 69.6 Colgte Creek ter MPN 1.8 Colgte Creek ter Bsl gr Colgte Creek ter OA no Colgte Creek ter SGO 1. Estern By sediment MPN Estern By sediment Bsl gr Estern By sediment OA no. 2 Estern By sediment SGO 1. Estern By ter MPN Estern By ter Bsl gr Estern By ter OA no. 2 Estern By ter SGO 1. TABLE 11. TABLE 1. Efficiency of vrious medi for enumerting petroleum-degrding yests nd fungi % Petro- Medium leum degrders Colgte Creek sediment MPN 33.3 Colgte Creek sediment Yest gr 75. Colgte Creek sediment OA no Colgte Creek sediment SGO 7. Colgte Creek ter MPN 5. Colgte Creek ter Yest gr 1. Colgte Creek ter OA no Colgte Creek ter SGO 1. Estern By sediment MPN ND Estern By sediment Yest gr 1. Estern By sediment OA no Estern By sediment SGO 1. Estern By ter MPN ND Estern By ter Yest gr 5. Estern By ter OA no Estern By ter SGO 5. ND, Not determined becuse of lck of groth in ny of the tubes (see Tble 11). Number nd percentge of bcteril generic groups isolted from Estern By on SGO, bsl (B), nd OA no. 2 Wter Sediment Genus or generic SGO B OA SGO B OA group _ No. % No. % No. % No. % No. % No. % Acinetobcter Actinomycete Aeromons Bcillus Coryneform Enterobctericee Flvobcterium Lctobcillus Lucibcterium 1 4. MCFFb Pseudomons Vibrio Unidentified Common nme for bcteri in the gener Arthrobcter, Brevibcterium, Cellulomons, Corynebcterium, Erysipelothrix, Jenseni, Kurthi, Listeri, Microbcterium, Mycobcterium, Nocrdi, nd Propionobcterium. b MCFF, Morxell, Cytophg, Flvobcterium, nd Flexibcterium. s compred ith the mount of beneneextrctble mteril in the sediment. No correltion s observed. A positive correltion s observed, hoever, hen the percentge of petroleum-degrding bcteri in the totl vible, heterotrophic count s compred ith percentge of benene-extrctble mteril (Fig. 3). Thus, it is cler tht normliing the dt provides better estimte. The benene-extrctble mteril in Colgte Creek sediment hs been identified s petroleum by computeried lo-resolution mss spectrometry (29). No correltion s observed beteen the mount of benene-extrctble mteril nd the totl APPL. ENVIRON. MICROBIOL. number of petroleum-degrding bcteri in Colgte Creek ter (Fig. 4, nor s such correltion observed for petroleum-degrding fungi in Colgte Creek sediment (Fig. 5) or petroleum-degrding yests nd fungi in Colgte Creek ter (Fig. 6). It is concluded tht there is "threshold" concentrtion of oil in the environment or percentge of petroleum-degrding microorgnisms in the microbil popultion of the environment belo hich there is little correltion beteen the to. High numbers of petroleum-degrding bcteri did not correlte ith mount of benene-extrctble mteril from oil-contminted Colgte Creek Donloded from on September 23, 218 by guest

8 VOL. 31, 1976 PETROLEUM-DEGRADING MICROORGANISMS 25 TABLE 12. Number nd percentge of bcteril generic groups isolted from Colgte Creek on SGO, bsl (B), nd OA no. 2 Wter Sediment Genus or generic SGO B OA SGO B OA group No. % No. % No. % No. % No. % No. % Acinetobcter Actinomycete Aeromons Bcillus Coryneform Enterobctericee Flvobcterium Lucibcterium Lctobcillus MCFF Pseudomons Vibrio Unidentified Cl, -J m LI 4 See footnotes to Tble 11. 1' I lo, D 1' -J I- i. Z / MAY g NOVEMBER INCUBATION TEMPERATURE ( C) FIG. 2. Petroleum-degrding bcteri enumerted t 5, 15, nd 25 C for Colgte Creek sediment smpled in November 1973 nd My SGO medium (see Mterils nd Methods) s used in the experiments from hich dt shon in Fig. 2-4 ere tken. S ter (Fig. 4). Similrly, lo numbers or percentges of petroleum-degrding fungi did not correlte ith high mounts of benene-extrctble mteril in sediment (Fig. 5). These dt re importnt if the number of petroleumdegrding microorgnisms in smple is to be correlted ith the mount of petroleum pollution. Other fctors, such s concentrtion of ' C', -J "2 1' co CD 1 H i D BENZENE-EXTRACTABLE MATERIAL (MG) FIG. 3. Correltion of number of petroleum-degrding bcteri per milliliter of sediment (O) nd percentge ofpetroleum-degrding bcteri () ith mount of benene-extrctble mteril in Colgte Creek sediment. 1 I 8!8 <t F- L) m CD, 6 C) LI) 4 LWI -J CL 2 L cr L IHC orgnic nd inorgnic nutrients, extent of gring on the bcteri by higher trophic levels, etc., cn ffect the number of petroleum-degrding microorgnisms, but these fctors cn usully be normlied by clculting the percentge of petroleum degrders comprising the totl popultion nd compring the vlues thus u Donloded from on September 23, 218 by guest

9 26 WALKER AND COLWELL APPL. ENVIRON. MICROBIOL _ cr LUJ 1 8 e 2 9: LUJ O~~~~~~~~~~ m _L lo,. 6 t < L' 6 H.~~~~~~~~~~~~~~~~~~~ (D L'2 O LU o IL Z!O, oi2~~~~~~ C O~~~~~~~ ~~~~~~~~~~~I Ii o o~~~~~~ H 2 L o ~~~~~~~ BENZENE-EXTRACTABLE MATERIAL (MG) FIG. 4. Correltion of number of petroleum-degrding bcteri per milliliter of ter (@) nd percentge of petroleum-degrding bcteri () ith mount of benene-extrctble mteril in Colgte Creek ter. C') (9 cr LUI lo ()lo, LUJ -J BENZENE-EXTRACTABLE MATERIAL (MG) FIG. 5. Correltion of number of petroleum-degrding fungi per milliliter of sediment (A) nd percentge of petroleum-degrding fungi (A) ith mount of benene-extrctble mteril in Colgte Creek sediment. Medium used s yest gr (see Mterils nd Methods). A, : -i LL -8 F- ZU6 (9 L- Z 63 '2 (I) Z) LUJ (9 LUJ -LJ 9: CL.- ~~~~~~ 5 L~~~~~~~~~~~~~ _ (9 Z) 8 U- U (9 'C 4 D(9 LU H. LC <D - BENZENE -EXTRACTABLE MATER IAL (MG ) FIG. 6. Correltion of number of petroleum-degrdingyests (U) nd fungi (A)perlOO ml of ter nd percentge of petroleum-degrding yests (E) nd fungi (A) ith mount of benenie-extrctble mteril in Colgte Creek ter. Medium used s yest gr (see Mterils nd Methods). obtined. The concentrtion of orgnic nd inorgnic nutrients present in smple ill clerly influence the ctivity of petroleum-degrding microorgnisms (28). In summry, enumertion of petroleum-degrding microorgnisms requires tht ll pure cultures tht re isolted be exmined for bility to utilie oil to obtin vlid estimte of the number of petroleum-degrding microorgnisms comprising the popultion. Petroleumdegrding microorgnisms should be expressed s percentge of the totl popultion henever possible. It is recommended tht SGO medium be used to isolte petroleum-degrding bcteri nd tht the yest medium described here be used to isolte petroleum-degrding yests nd fungi. The temperture of incubtion should be considered crefully, nd duplicte sets of count pltes should be incubted t to or more tempertures. If correltions re to be mde beteen presence of petroleum degrders nd concentrtion of oil in the smple, percentge of petroleum degrders in the totl microbil popultion of the smple should be used, not the totl numbers, nd the mount of oil in the orgnic extrctble mteril should be determined. c A. Donloded from on September 23, 218 by guest

10 VOL. 31, 1976 ACKNOWLEDGMENTS We cknoledge the excellent technicl ssistnce of H. F. Austin, J. J. Clomiris, nd P. A. Seesmn. This ork s supported by contrct no. N14-67-A beteen the Office of Nvl Reserch nd the University of Mrylnd. LITERATURE CITED PETROLEUM-DEGRADING MICROORGANISMS Americn Public Helth Assocition Stndrd methods for the exmintion of ter nd steter, p Americn Public Helth Assocition, Inc., Ne York. 2. Atlst, R. M., nd R. Brth Abundnce, distribution nd oil-biodegrdtion potentil of microorgnisms in Rritn By. Environ. Pollut. 4: Bruh, J. N., Y. Alroy, nd R. I. Mteles Incorportion of liquid hydrocrbons into gr medi. Appl. Microbiol. 15: Colell, R. R., J. F. Crney, T. Kneko, J. D. Nelson, nd J. D. Wlker Microbil ctivities in the esturine ecosystem, p In T. Mseg (ed.), First Interntionl Congress of the Interntionl Assocition of Microbiologicl Societies. Science Council of Jpn, Tokyo. 5. Colell, R. R., R. K. Siemore, J. F. Crney, R. Y. Morit, J. D. Nelson, J. H. Pickr, J. R. Schr, S. D. VnVlkenberg, J. D. Wlker, nd R. T. Wright Mrine nd esturine microbiology lbortory mnul. University Prk Press, Bltimore. 6. Colell, R. R., J. D. Wlker, nd J. D. Nelson Microbil ecology nd the problem of petroleum degrdtion in Chespeke By, p In D. G. Ahern nd S. P. Meyers (ed.), The microbil degrdtion of oil pollutnts. Publ. no. LSU-SG-73-1, Louisin Stte University, Bton Rouge. 7. Ehlers, S. E., nd H. G. Hedrick Detection nd identifiction of bcteri utiliing petroleum ste in dilution ters. Dev. Ind. Microbiol. 13: Funk, H. B., nd T. A. Krulich Preprtion of cler silic gels tht cn be streked. J. Bcteriol. 88: Gunkel, W., nd H. H. Trekel On the method of quntittive determintion of oil-decomposing bcteri in oil-polluted sediments nd soils, oil-ter mixtures, oils nd trry substnces. Helgol. Wiss. Meeresunters. 16: Hnks, J. H., nd R. L. Weintrub The preprtion of silicic cid jellies for bcteriologicl medi. J. Bcteriol. 32: Ingelmn, B., nd H. Lurell The preprtion of silicic cid jellies for the cultivtion of microorgnisms. J. Bcteriol. 53: Kingsbury, J. M., nd E. S. Brghoorn Silic gel s microbiologicl medium: potentilities nd ne method of preprtion. Appl. Microbiol. 2: Mulkins-Phillips, G. J., nd J. E. Stert Distribution of hydrocrbon-utiliing bcteri in Northestern Atlntic ters nd costl sediments. Cn. J. Microbiol. 2: Nedell, D. B., nd G. D. Floodgte The sesonl selection by temperture of heterotrophic bcteri in n intertidl sediment. Mr. Biol. 11: Prmer, D The influence of physicl nd chemicl fctors on the preprtion of silic gel medi. Appl. Microbiol. 5: Robertson, B., S. Arhelger, P. J. Kinney, nd D. K. Button Hydrocrbon biodegrdtion in Alsk ters, p In D. G. Ahern nd S. P. Meyers (ed.), The microbil degrdtion of oil pollutnts. Publ. no. LSU-SG-73-1, Louisin Stte University, Bton Rouge. 17. Roslycky, E. B Relible procedure for silic gel preprtion. Appl. Microbiol. 24: Seki, H Silic gel medium for enumertion of petroleumlytic microorgnisms in the mrine environment. Appl. Microbiol. 26: Sommer, L. E., nd R. F. Hrris Routine preprtion of silic gel medi using silicte solutions of vrying ph. J. Bcteriol. 95: Tylor, C. B An improved method for the preprtion of silic gel medi for microbiologicl purposes. J. Gen. Microbiol. 4: Temple, K. L A ne method for the preprtion of silic gel pltes. J. Bcteriol. 57: Thtcher, R. C., nd T. L. Wever Simplified method for the preprtion of silic gel medi. Appl. Microbiol. 28: Wlker, J. D., nd R. R. Colell Microbil ecology of petroleum utilition in Chespeke By, p In API/EPA/USCG conference on prevention nd control of oil spills. Americn Petroleum Institute, Wshington, D.C. 24. Wlker, J. D., nd R. R. Colell Degrdtion of hydrocrbons nd mixed hydrocrbon substrte by microorgnisms from Chespeke By. Adv. Wter Pollut. Res. 7: Wlker, J. D., nd R. R. Colell Fctors ffecting enumertion nd isoltion of ctinomycetes from Chespeke By nd Southestern Atlntic Ocen sediments. Mr. Biol. 3: Wlker, J. D., nd R. R. Colell Micro_il degrdtion of model petroleum t lo tempertures. Microb. Ecol. 1: Wlker, J. D., nd R. R. Colell Microbil petroleum degrdtion: The use of mixed hydrocrbon substrtes. Appl. Microbiol. 27: Wlker, J. D., nd R. R. Colell Mesuring the potentil ctivity of hydrocrbon-degrding bcteri. Appl. Environ. Microbiol. 31: Wlker, J. D., R. R. Colell, M. C. Hmming, nd H. T. Ford Extrction of petroleum hydrocrbons from oil-contminted sediments. Bull. Environ. Contm. Toxicol. 13: Wlker, J. D., B. F. Conrd, P. A. Seesmn, nd R. R. Colell Comprison of membrne filter counts on heterotrophic nd oil gr used to estimte popultions of yest, fungi nd bcteri. In Symposium on recovery of indictor orgnisms employing membrne filters. Americn Society for Testing nd Mterils, Fort Luderdle, Fl. In press. 31. Winogrdsky, S Microbiologie du sol. Msson et Cie, Pris. 32. ZoBell, C. E Microbil modifiction of crude oil in the se, p In API/FWPCA conference on prevention nd control of oil spills. Americn Petroleum Institute, Wshington, D.C. 33. ZoBell, C. E., nd J. F. Prokop Microbil oxidtion of minerl oils in Brtri By bottom deposits. Z. Allg. Mikrobiol. 6: Donloded from on September 23, 218 by guest