A97 MICROBIA DEEIORATIO OF" MDIESEL FUEL FROM OIL SHALE. (ti

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MICROBIAL DETERIORATION OF D.,IESEL FUEL FI] et1'' / FROM9 LSH 4. PE IFOR 0r ORG. REPORT NUM99ER

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IS. SUPPLEMENTARY NOTES

IS. KCEY WORDS (Co.Innu an rvere side It n*eesar mnd Identify by block rumlber)

Microbial deterioration DFM Cladosporium resinaeOil shale Synthetic fuel *QNjd&Sp.ACoal Fungi Seawater

Petroleum Yeast~Diesel fuel marine Sulfate-reducing bacteria

252 STRACT (Coilinte - -ewes side It neceooy mtd Ideni~gfy by biock lmm&.)

Recurring problems with conventional ship fuels caused by microorganisms have prompted anevaluation of the susceptibility of a recently produced synthetic diesel fuel from oil shale to microbialcontamination. The growth of typical microbial contaminants of hydrocarbon fuels has been determinedover a four month period in two-phase systems consisting of fresh and se water media overaid with fuel.Anaerobic, sulfate-reducing bacteria and a yeast (Canidasp.) grew as well in the synthetic fuel as in fuelderived from petroleum. Growth of certain strains of the fungus, Cladosporium resinae, was initially - - _

(Continues)

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Z0. AeSTRACT (Contued)

ielayed in the synthetic diesel fuel but after 8-13 weeks the growth was generally comparable to thatia petroleum-derived fuel. This finding indicated that G. j~na may require time for adaptation toconstituents in the ol shale fuel. Ultimately, however, it appears that the synthetic diesel fuel isdikely to be as susceptible to microbial contamination as conventional diesel fuel has been. Experienceacquired with available synthetic fuels shows that their ability to support growth of microbialcontaminants varies widely depending on both the source of the crude oil and the refining processesused.

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CONTENTS

INTRODUCTION 1

MATERIALS AND METHODS 2

Fuels 2

Anaerobic Bacterial Test Units 2

Fungal Test Units 3

RESULTS AND DISCUSSION 4

Anaerobic Studies 4

Studies with Fungi 4

CONCLUSION5

REFERENCES6

MICROBIAL DETERIORATION OF MARINE DIESEL FUEL FROM4 OIL SHALE

INTRODUCTION

The decreasing availability of secure sources of crude petroleum andthe almost total dependence of military ships, aircraft and vehicles onliquid hydrocarbon fuels has forced an evaluation of fuels derived fromalternate sources under domestic control.

Studies of the physical and chemical properties of fuels from oilshale, coal and tar sands are being made to determine their suitabilityas replacements or extenders for conventional fuels (1,4,15,16,17). Ithas also appeared advisable to assess the susceptibility of these newsynthetic fuels to microbial contamination, inasmuch as serious problemsfrom this cause have arisen in the past with petroleum-base fuels (13).The possibility that the different chemical composition of the syntheticfuels may favor the growth of microbial contaminants should be exploredbefore these fuels come into wider use.

Problems from microbial fuel contamination generally arise from twodistinctly different groups of microorganisms, sulfate-reducing bacteriaand fungi (3,8,13). In an oxygen depleted two-phase fuel/water system,the bacteria thrive by the reduction of sulfate to sulfides, which accele-rates corrosion in storage tanks and fuel handling systems and generatesparticulate matter. The fungi, on the other hand, require oxygen, and themost troublesome species tend to form coherent mats at water/fuel inter-faces which can clog filters and orifices. The isolation of differentspecies and strains of both groups of microorganisms from conventionalfuel systems and from natural sources has been previously described (9,10).

Growth responses of various microorganisms to jet fuels from oil shaleand coal have been studied and shown to be markedly different (9,10). AFusariuM fungus grew as well in all the synthetic fuels as in petroleum3P-5. Sulfate-reducing bacteria were relatively inhibited only in a shalefuel containing considerable concentrations of basic nitrogen compounds.This fuel was also inhibitory to the fungus,Cladosporium resina., and ayeast (Candida sp.) but no inhibition was noted with another shale oilfuel from which the nitrogen constituents ware almost completely removedby hydrogenation and acid extraction. The coal-derived fuels examinedwere highly inhibitory to fungi. Apparently microbial growth varies withthe species of organism, the source of the fuel and the refining processesused.

Manuacript submitted February 12, 1981.

In order to understand the relationship between synthetic fuel compo-sition and the growth response of microbial contaminants in fuel/watersystems it is necessary to evaluate each new fuel as it becomes available.In this report we present the results of an investigation of the ability ofa diesel fuel refined from an oil shale crude to support growth of typicalbacterial and fungal contaminants in fresh and sea water systems.

MATERIALS AND METHODS

Fuels

The petroleum-base fuel used was Diesel Fuel Marine (DFM, MIL-F-16884G)received from the Naval Supply Center, Norfolk, VA in 1973.

The oil shale-derived diesel fuel came from a 73,000 barrel lot ofcrude produced by the Paraho process (2) and refined into military fuelsat the Toledo refinery of Sohio in iJ79. It was received in June, 1979and stored at 40C. This fuel is designated Shale II DlM in this report.It has been characterized elsewhere (1,15).

Paraffin oil (Fisher),representing an innocuous hydrocarbon,was usedin anaerobic test units as an additional control.

Anaerobic Bacterial Test Units

The anaerobic inocula, consisting of mixed microbial populations ofsulfate-reducing bacteria and other associated bacteria, came originallyfrom three sources:

(1) Laboratory continuous culture - an infected Avgas storage tank in theaircraft carrier, USS YORKTOWN, was the source of the bacteria sub-sequently maintained as a semi-continuous culture in the laboratoryfor 10 years under Avgas (6).

(2) USS MERRILL (DD-976) - diesel fuel tank (12).

(3) Potomac River sediment - near the Blue Plains Sewage Treatment Plant,Washington, D.C.

Small amounts of material from the above three sources were culturedin Sisler and Zobell triple strength medium (Sisler's 3X) (7) and allowedto develop dense populations. A 1:10 dilution was made under n-heptane infiltered seawater (0.45 um Millipore) that had been previously deaeratedwith nitrogen. An additional deaeration with nitrogen for 10 minutes wasmade to remove excess hydrogen sulfide in the diluted inoculum. Thisinoculum was allowed to rest for 3 to 4 hours before removing aliquotsfor inoculation of the test units.

The test units consisted of sterile 50 ml screw-top test tubes towhich 40 ml of the appropriate fuel were added. Ten ml of an aqueousmedium of supplemented trypticase soy broth (BBL) (6) were pipetted underthe fuel. One ml of the bacterial inoculum was then added. The tubes were

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tightly capped and incubated in the dark at 250 C. All tests were done induplicate.

The growth of sulfate-reducing bacteria in the anaerobic test units wasestimated from the degree of blackening developed from reaction of micro-bially generated hydrogen sulfide with ferrous iron in the growth medium toform ferrous sulfide. The rating system consisted of 0 - no blackening,1 - slightly grey, to 5 which was intense, opaque black.

Fungal Test Units

Sources of fungi were as follows:

(1) Cladosporlum resinae DK was isolated from a JP-5 storage tank at NavalAir Station, Lemoore, CA.

(2) Cladosporium resinae DK/adapted is the above C. resinae after adaptation

to growth in seawater.

(3) Cladosporiom resinae P-1 was isolated from sludge from a centrifugal

purifier on the USS PETERSON (DD-969).

(4) Candida sp. was isolated from water with a film of oil on the surfacewhich had collected in an exposed boiler room of a naval ship in theprocess of being scrapped at Curtis Bay, MD.

The fungi and yeast ware grown on potato-dextrose agar (Difco) slantswith the addition of 0.5% yeast extract (Difco). For inoculation, stocksuspensions of the organisms ware prepared by dispersing surface growth ona slant in 10 ml of a solution of 0.057 polysorbate 80 (Tween 80) in dis-tilled water. The viable count in these suspensions ranged from 11 x 106

to 56 x 100 colony forming units per ml.

Fungal test units with fresh-water mineral salts media were of twotypes: One followed the formula of Bushnell and Haas (5) and had a pH ofapproximately 6.5 after sterilization (referred to as FWBH) while the otherwas that of Klausmeier as modified by Park (14) with a pH of approximately5.0 after sterilization (referred to as FwKP)

Fungal test units were also set up with seawater obtained from theMediterranean Sea (salinity - 37.10/oo) during a cruise and aged for overa year in the dark at 40C before use. In all cases 0.057. peptone (Difco)and 0.057. yeast (Difco) were added. The pH of the peptone-yeast solutionin seawater after autoclaving was 8.00 * 0.02 (SWPY8). In some cases theseawater medium was adjusted to pH 6 with IN hydrochloric acid (SWPY6).

Each test unit consisted of a 250-ml Erlenmeyer flask with a cottonplug. Fifty ml of the water phase were dispensed into the flask and auto-claved for 20 minutes at 1200 C. Fifty ml of the fuel were then added andthe unit was allowed to stand overnight at room temperature. The pH ofeach flask was readjusted if needed and 0.5 ml of the inoculum of C. resinaeor yeast was added. The flask plugs were loosely covered with aluinum foiland the test units were incubated in the dark at room temperature (220-25°C).All experiments were done in duplicate.

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The test units inoculated with fungi and yeast were inspected for growthat appropriate time intervals. The rating system was as follows:

0 no germination1 germination and minute amount of growth, no mat formation2 slight interfacial growth, no mat formation3 a mat formation over one-third of the interface4 - mat formation over two-thirds of the interface5 - mat formation over entire interface6 - mat formation over entire interface with a thickness of 0.5 cm or more.

At the conclusion of each experiment, pH measurements were made on thewater phase of each test unit with a glass electrode. A test for viabilitywas made on those test units showing growth ratings of 0 or 1 by spreadingapproximately 0.5 ml of the liquid taken from the water/fuel interface onpotato dextrose agar + 0.57. yeast extract. The agar surface of these plateshad previously been allowed to dry so that this large amount of inoculumcould be spread and absorbed by the agar without having too wet a surfacefor discrete colony growth during subsequent incubation.

RESULTS AND DISCUSSION

Anaerobic Studies

The growth of the sulfate-reducing bacteria after 68 hours incubationin test units containing paraffin oil and the two DFM fuels is given inTable I. The sulfate reducers from all three sources were able to grow withthe Shale II DFM as well as with petroleum DFM or paraffin oil. Growth wasslower in those test units inoculated with sulfate reducers from the PotomacRiver sediment, probably because they were not adapted to the fuel/watersystem used,whereas the organisms from the other two sources were isolatedfrom fuel systems. That an adaption to fuel by the organisms from the riversediment may be occurring is indicated by the observation that after 18 days,growth ratings increased to 3, 5 and 3/5 for petroleum DFM, Shale II DFMand paraffin oil, respectively.

Studies with Fungi

Tables 2-5 show the growth, survival and final aqueous pH for thethree different strains of C. resinae (DK, DK/adapted and P-1) and for theyeast (Candida sp.). Where growth in duplicate test units was unequal,ratings for both are shown.

With a few exceptions these organisms had nearly the same growth rating,pH and viability after four months under Shale II DFM as under petroleum DFM.The rates at which the C. resinae strains grew in the early weeks of theexperiments tended to be slower in the Shale I DFM. This may signify thata period of adaption of the organisms to the synthetic fuel system exists.No such delay was noted for Candida.

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The generally poor and erratic growth of C. resinae in SWIY8 media has

been shown elsewhere to be due to the high pH of the seawater (11) (illustra-ted in Fig. 1). The C. resinae DK strain even lost viability in this medium.The failure of the DK and P-1 strains to grow under Shale II DFM in SWPY8may be due to the combination of unfavorable pH and the factor in the fuelwhich caused the retarded growth rate. Decreasing the initial pH of theseawater to six allowed much improved growth of C. resinae in all instances.

It will be noted that with one exception (Candida under Shale II DFMwith FWBH media, Table 5) heavy growth of fungal organisms also resulted ina greatly lowered final pH after four months. This supports an hypothesisadvanced elsewhere (11) that Candida may promote the growth of C. resinaewhen they occur together as co-contaminants in seawater/fuel systems bylowering the pH sufficiently, at least in microenvironments, to allow C.resinae to initiate growth and generate its own acidic products. Thesuperior ability of Candida sp. to grow in high pH situations may thus bea major factor in promoting the growth of the more troublesome C. resinae.This situation appears to apply to Shale II DFM as well as to petroleumfuels.

CONCLUS ION

In no case was the growth of a microbial fuel contaminant promotedsignificantly by the presence of a diesel fuel from oil shale as comparedwith conventional petroleum-derived diesel fuel. Certain of the importantfungal contaminants were initially inhibited somewhat by the syntheticdiesel fuel which indicated that a period of adaptation was necessary be-fore the growth rate became comparable to that observed in the presence ofpetroleum fuel. Thus the use of a synthetic diesel fuel like the oneevaluated here either alone or in mixtures with conventional fuel is notlikely to lead to microbial contamination problems significantly differentfrom those encountered in the past.

REFERENCES

1. Affens, W.A., J.M. Hall, E. Beal, R.N. Hazlett, C.J. Nowack, andC. Speck. Aug. 1980. Relationship between fuel composition andproperties. III. Physical properties of U.S. Navy Shale II fuels.Preprints Amer. Chem. Soc.,Div. Fuel Chem.

2. Anon. 1977. Energy fact book. Tetra-Tech Report No. TT-A-642-77-306.

3. Bailey, C.A., and M.E. May. 1979. Evaluation of microbiological testkits for hydrocarbon fuel systems. Appl. Environ. Microbiol.37.,871-877.

4. Brinkman, D.W., M.L. Wisman, and J.N. Bowden. 1979. Stabilitycharacteristics of hydrocarbon fuels from alternative sources.BETC/RI-78/23. National Technical Information Service, U.S. Dept.of Commerce, Springfield, VA 22161.

5. Bushnell, L.D., and H.F. Haas. 1941. The utilization of certainhydrocarbons by microorganisms. J. Bacteriol. 41:653-673.

6. Kleme, D.E., and J.M. Leonard. 1971. Inhibitors for marine sulfate-reducing bacteria in shipboard fuel storage tanks. Naval ResearchLaboratory Memo Report 2324.

7. Klemm, D.E., and R.A. Neihof. 1969. Control of marine sulfate-reducing bacteria in water-displaced shipboard fuel storage tanks.Naval Research Laboratory Memo Report 2069.

8. Klemme, D.E., and R.A. Neihof. 1976. An evaluation in large-scaletest systeme of biocides for control of sulfate-reducing bacteria inshipboard fuel tanks. Naval Research Laboratory Memo Report 3212.

9. May, M.E., and R.A. Neihof. 1979. Microbial deterioration of hydro-carbon fuels from oil shale, coal, and petroleum. 1. Exploratoryexperiments. Naval Research Laboratory Memo Report 4060.

10. May, M.E., and R.A. Neihof. 1980. Microbial deterioration of hydro-carbon fuels from oil shale, coal, and petroleum. II. Growth andinhibition of bacteria and fungi. Naval Research Laboratory MemoReport 4294.

11. May, M.E., and R.A. Neihof. 1981. Growth of Cladosporium resinaein seawater/fuel systems. Dev. Ind. Microbiol., Vol. 22 (in press).

12. Neihof, R.A., and M.E. May. 1979. Microbiological analysis ofsamples from fuel tanks of USS MERRILL (DD-976). Naval ResearchLaboratory Letter Report 8353-238. 25 June.

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13. Parbery, D.G. 1971. Biological problems in Jet aviation fuel and thebiology of Amorphotheca resinae. Mater. Organismen 6:161-208.

14. Park, P.B. 1975. Biodeterioration in aircraft fuel systems. Soc.Appl. Bacteriol., Tech. Series 9:105-126.

15. Solash, J., R.N. Hazlett, J.C. Burnett, E. Beal, and J.M. Hall.Aug. 1980. Relation between fuel properties and chemical composition.II. Chemical characterization of U.S. Navy Shale II fuels. PreprintsAmer. Chem. Soc.,Div. Fuel Chem.

16. Solash, J., R.N. Hazlett, J.M. Hall, and C.J. Nowack. 1978.Relation between fuel properties and chemical composition. I. Jetfuels from coal, oil shale and tar sands. Fuel 57:521-528.

17. Solash, J., C.J. Nowack, and R.J. Delfosse. 1976. Evaluation of aJP-5 type fuel derived from oil shale. NAPTC-PE-82. Naval AirPropulsion Test Center, Trenton, N.J. 08628.

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Table 1. Growth of sulfate-reducing bacteria in aqueous assay mediumafter 68 hours incubation under conventional and synthetic

Diesel Fuel Marine (DFM) and paraffin oila

Source of sulfate-reducing bacteria

Fuel Laboratory Potomac River Diesel Fuel TankContinuous Culture Sediment USS MERRILL

Petroleum DFM 4 2 5

Shale II DFM 5 2 5

Paraffin Oil 4 2 5

aRating system based on blackening in assay medium where I = slightly

grey, to 5 which was intense opaque black.

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Fig. I - Growth of Cladosporium resinae DK in two-phase water/Shale II DFMfuel systems. There are four different water phases, startingfrom the left: Bushnell-Haas mineral salts (FWBH), Klausmeier-Park mineral salts (FWKP), seawater + peptone yeast pH 8 (SWPY8)and seawater + peptone yeast pH 6 (SWPY6).

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