rn-AOBB8 055 NAVAL RESEARCH LAB WASHINGTON DC F/f 1/F MICROSIAL DETERIORATION OF HYDROCARBON FUELS FROM OIL SHALE. CO-(ETCtW)

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CONTENTS

INTRODUCTION

MATERIALS AND METHODS I

Fuel I

Base and Acid Extractions 2

Anaerobic Bacterial Test Units 2Fungal Test Units 3

RESULTS AND DISCUSSION 4

Anaerobic Studies 4

Fungal Growth 5

Identification of Fungal Inhibitors 5

Aqueous Media Effects 6

CONCLUS IONS 6

ACKNOWLEDGMENT 7

REFERENCES 8

FIGURES 9

TABLES 12

APPENDIX 23

ACCESSION for

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JUSTI ICATION

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MICROBIAL DETERIORATION OF HYDROCARBON FUELS FROM OIL SHALE, COAL, ANDPETROLEUM. II. GROWTH AND INHIBITION OF BACTERIA AND FUNGI.

INTRODUCE ION

As a part of the Energy Conversion Synthetic Fuels Program todecrease United States dependency on crude petroleum from foreignsources, the Naval Air Systems Command has the task of evaluating jetaircraft fuels originating from alternate domestic sources such ascoal and oil shale (8). In addition to physical and chemical studiesto assess the suitability of these synthetic fuels as replacements forconventional fuels, it has also appeared necessary to assess their sus-ceptibility to contamination by microorganisms because of recurringproblems with conventional fuels.

In an earlier report (7) it was shown that many typical microbialcontaminants, including fungi, yeast and bacteria, were inhibited inthe presence of JP-5 type fuels derived from crudes produced fromwestern Kentucky coal by the Char Oil Energy Development process(COED-5) and from oil shale by the Paraho process (Shale I) (I). Anexception was a fungus, Fusarium sp., which grew as well in the syn-fuels as in petroleum JP-5. Also microbial growth with mixtures of25% synfuel/75% petroleum fuel was generally as great as with 100%

petroleum fuel.

In 1979 fuels from a second shale oil production run at the Toledorefinery of Sohio became available (Shale II) (10). Hydrocracking andacid extraction resulted in a JP-5 fraction with a greatly lowerednitrogen content compared to the fuel from Shale I (9,12). Because theconcentration of nitrogen-containing compounds may influence microbialgrowth (see Reference 7), a major objective of the present investigationwas to compare the susceptibility to microbial contamination of ShaleII JP-5 with petroleum JP-5 and the synthetic fuels already examined.The microbial species employed has been extended to sulfate-reducingbacteria, a common fuel storage tank contaminant not previously includedin this study (7). An effort has also been made to identify the natureof the constituents in coal and Shale I JP-5 which are responsible forfungal inhibition and to assess the reasons for the unfavorable fungalgrowth in seawater media noted in the preceding report (7).

MATERIALS AND METHODS

Fuels

The petroleum-base fuel, designated Jet-A, was the same as that

Manucript submitted June 6, 1980

1 9

used in previous work (7). It contained no additives and was receivedin January,1976 from Naval Air Propulsion Test Center, Trenton, NJ.

Coal-derived fuel, also used in the earlier study (7) was pro-duced from Western Kentucky Coal by Sun Oil Co. using high pressurecatalytic hydrogenation (4). The finished fuel met most of the re-quirements for JP-5.

The oil shale products were from crude produced by the Parahoprocess and subsequently refined to military fuels (2). The JP-5fraction from the first 10,000 barrel operation (Shale I) had a veryhigh freezing point and a high nitrogen content, 976 ppm, of which 860ppm was acid extractable (12). JP-5 from the second 73,000 barreloperation (Shale II) was hydrogenated and acid extracted and had onlyone ppm nitrogen in the finished fuel. Studies of the composition ofthese fuels appear elsewhere (9,12,13).

Selected nitrogen-containing compounds typical of those in Shale Iwere added to petroleum JP-5 in some of the test systems used here todetermine whether the microbial inhibition of Shale I could be attri-buted to any of them. These were: pyridine (99%), 5-ethyl-2-methylpyridine (99%), 2,6 lutidine (96%), 2 -picoline (98%), 2-ethyl pyridine(98%), 4-tertiary-butyl pyridine (99%), 4-benzyl pyridine (99%) andquinaldine (97%) from Aldrich Chemical Co. Quinoline was reagentgrade from Fisher Scientific Co.

Base and Acid Extractions

Base extractions were made by shaking 850 ml portions of fuel and10 ml of aqueous 0.1 N NaOH in a one-liter separatory funnel for threeone-minute intervals separated by one-minute rests. The aqueous layerwas drained off and the extraction process repeated an additional twotimes. The fuel was then washed three times with 100 ml portions ofdistilled water using the same shaking schedule. Titration of thecombined aqueous layers with standard acid showed titratable aciditiesof 0.44 meq/l for Shale I, 0.18 meq/l for Shale II and 0.068 meq/l forCOED #5.

Acid extractions of Shale II and COED JP-5, were made by shaking850 ml portions of fuel with 10 ml portions of 0.1 N HCl followed bythree washes with 100 ml portions of distilled water using the sameprocedure as for the base extraction. In order to remove all the acidextractables from the Shale I fuel it was necessary to increase theconcentration of HCl to 10.0 N; subsequent washing was with seven 100ml portions of water. Titratable basicities of 56.2 meq/l for Shale I,0.19 meq/l for Shale II and 0.174 meq/l for coal were obtained. Basiccompounds from Shale I were isolated by acid extraction followed byneutralization of the HCI adducts (9).

Anaerobic Bacterial Test Units

The anaerobic inocula, consisting of mixed microbial populations

2

of sulfate-reducing bacteria and associated bacteria, came originallyfrom three sources:

(1) Laboratory continuous culture - infected Avgas storage tanks ofthe aircraft carrier, USS YORKTOWN, was the source of the bacteriasubsequently maintained as a semi-continuous culture in the labora-tory for 10 years (5).

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

(3) Potomac River sediment - near the Blue Plains Sewage Plant.

Small amounts of material from the above three sources were cul-tured in Sisler and Zobell triple strength medium (Sisler's 3X) (6)and allowed to develop dense populations. A 1:10 dilution was madeunder n-heptane in filtered seawater (0.45 ,m Millipore) that had beenpreviously deaerated with N2 . To remove any H2S present, an addi-tional deaeration of 10 minutes with N2 was made. The dilution wasallowed to rest for 3 to 4 hours before removing aliquots for inocu-lation of the test units.

The test units consisted of sterile 50 ml screw-top test tubesto which 40 ml of the appropriate fuel was added. Ten ml of anaqueous medium consisting of a supplemented trypticase soy broth (BBL)(5) was pipetted under the fuel. One ml of the bacterial inoculumwas thin added. The tubes were tightly capped and incubated in thedark at 250 C. All tests were done in duplicate.

The growth of sulfate-reducing bacteria in the anaerobic testunits was estimated from the degree of blackening developed from re-action of microbially generated H2S with ferrous iron in the growthmedium to form FeS. The rating system developed by Klemme and Leonard(5) was used where 0 = no blackening, I = slightly grey, to 4 whichwas intense opaque black.

Fungal Test Units

Sources of fungi were as follows:(I) Cladosporium resinae DK was isolated from JP-5 storage tank at

Naval Air Station, Lemoore, CA.

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

being adapted to growth in seawater.

(3) Cladosporium resinae P-1 was isolated from sludge from a centri-fugal purifier on the USS PETERSON.

(4) Cladosporium resinae APPC-IIS was isolated from newly creosotedrailroad ties near the Alexandria Potomac Power Co., Alexandria, VA.and found to use Shale I JP-5 as a carbon source.

(5) Candida sp. was isolated from water with a skim of oil on thesurface which had collected in an exposed boiler room of a naval shipin the process of being scrapped at Curtis Bay, MD.

3

The fungi and yeast were grown on potato-dextrose agar (Difco)slants with the addition of 0.5% yeast extract (Difco). For inocu-lation, stock suspensions of the organisms were prepared by dispersingsurface growth on a slant in 10 ml of a solution of 0.05% Tween 80 indistillgd water. Tge viable count in these suspensions ranged from11 x 10 to 56 x 10 colony forming units per ml.

Fungal test units with fresh-water mineral salts media were of

two types: One followed the formulation of Bushnell and Haas (3) witha pH of approximately 6.5 after sterilization (referred to as FW B-H)while the other was that of Klausmeier as modified by Park (11) with apH of approximately 5.0 after sterilization (referred to as FW K-P).

Fungal test units were also set ug with seawater obtained from

the Mediterranean Sea (salinity = 37.1 /oo) during a cruise and agedfor over a year in the dark at 40 C before use. In all cases the sea-water had 0.05% peptone (Difco) and 0.05% yeast (Difco) added. The pHof the seawater peptone-yeast after autoclaving was 8.00 ± 0.02 FSW+PY(8)7. In some cases the seawater medium was adjusted with IN HCIto 6 1SW+PY(6)1.

All test units consisted of 250-ml Erlenmeyer flasks with cottonplugs. Fifty ml of the water phase were dispensed into each flask andautoclaved for 20 minutes at 120 0 C. Fifty ml of fuel were then addedand the unit was allowed to rest overnight. The pH of each flask wasreadjusted if needed before the addition of 0.5 ml of the inoculum ofC. resinae or yeast. The flask plugs were loosely covered with alumi-num foil and the test units were incubated in the dark at room tempera-ture (220 -250 C). All experiments were done in duplicate.

The test units inoculated with fungi and yeast were visually in-spected for growth at appropriate time intervals. The rating systemranged from 0 for no growth, i for spore germination to 6 for a matthicker than 0.5 cm over the entire interface (Figure 1). A study oftle dry weight of microbial material corresponding to these visualratings showed that an increase of one unit in the visual rating cor-responded approximately to a doubling of the amount of growth (seeAppendix A). At the conclusion of each experiment, viability studieswere made on those units showing growth ratings of 0 or I by spreadingapproximately 0.5 ml of the water/fuel interface on potato dextroseagar + 0.57. yeast extract. The agar surface of these plates had previ-

ously been allowed to dry so that this large amount of inoculum couldbe spread without having too wet a surface during incubation. Also atthe end of these experiments, pH measurements were made on the waterphase in all test units using a glass electrode.

RESULTS AND DISCUSSION

Anaerobic studies on the sulfate-reducing bacteria in syntheticfuels. The results of the tests on the sulfate-reducing bacteriaafter 68 hours incubation are given in Table I. The sulfate reducersfrom the USS MERRILL showed the most vigorous growth probably because

4

S - .. - - --- ... .---.---.------ .- -- . . . .- -- - -

aI

they came from a fuel/water system similar to that in the test units andhad adapted to it. Inocula from the Potomac River sediment showed theslowest growth, but eventually those units also became intensely black.The sulfate reducers grew very well under all of the fuels.

Fungal growth in synthetic fuels. Tables 2 through 5 show thegrowth, survival and final aqueous pH for three different strains ofC. resinae (DK, DK/adapted, and P-l) and for the yeast, Candida sp.As was found in the preliminary experiments (7), JP-5 made from coalinhibits C. resinae and there is a gradual loss of spore viability,Shale I is also inhibitory but to a lesser extent, and viability wasretained almost as well as with petroleum JP-5. Under Shale II therewas a slight delay in growth in some C. resinae strains, but generallythe growth paralleled that under petroleum JP-5 except for strain P-1which grew less in the fresh water media under Shale II JP-5.

Candida sp. (Table 5) behaved about the same as in the preliminaryexperiments (7). It did not grow with Shale I or coal JP-5 except inone case where the seawater pH was lowered to 6 (coal JP-5/SW+PY(6));and here the growth was atypical consisting only of balls of hyphae atthe bottom of the flasks. Candida grew with Shale II JP-5 almost aswell as with petroleum JP-5.

Identification of fungal inhibitors in JP-5 from coal and oilshale. It is apparent from the data that growth of the fungal organ-isms employed here depended not only on the fuel but also on the com-position and pH of the aqueous phase. In general, it was noted thatthe different strains of C. resinae grew better in the fresh waterthan in the seawater systems while the Candida grew more luxuriouslyin the seawater media than in either of the two fresh water media.Additional consideration of aqueous effects is postponed until afterthe main effects produced by the different fuels have been discussed.

Neither acid nor base extractions of Shale II made substantialdifferences in fungal growth except for the P-1 strain of C. resinaewhich grew better after both extractions when fresh water media were

used (Table 4).

Acid and base extractions of coal JP-5 made no difference in theinhibition which this fuel shows for all C. resinae strains andCandida; also the loss in viability of the inoculum remained high(Tables 2-5).

Base extraction of Shale I did not change its inhibitory qualities.After acid extraction, however, growth was as good with all organismsas under petroleum JP-5 (Tables 2-5 and Figure 2). Removal of a largeamount of organic bases would appear to be responsible for this dif-ference. It was surprising, therefore, to find that microbial growthin the presence of petroleum JP-5 with added acid extracted materialfrom Shale I was as good as in neat petroleum JP-5 (Table 6). Possiblereasons for this are discussed below.

5

In additional attempts to identify a particular class of inhibitor

in Shale I, non-basic nitrogen residues from a silica gel column (Table6) and a number of pyridine and quinoline derivatives (Table 7) wereadded back to petroleum JP-5. No inhibition was observed except with500 ppm of 4-benzyl pyridine (both DK/adapted and APPC-IIS strains)and 500 ppm of 4-tertiary butyl pyridine (APPC-IIS strain). It is un-likely that these compounds were present in Shale I in these concen-trations although there may be cumulative effects from all nitrogencompounds present and other unidentified inhibitory compounds couldplay a role.

Aqueous media effects on C. resinae growth. If the initial pH of

the seawater media is lowered by the addition of acid (HCI), growth ofall strains of C. resinae can occur except under fuel from Coal andShale I. This is illustrated in Tables 2,3,4 and 6 by comparison ofgrowth in SW + PY (8) and SW + PY (6). The normal high pH of the sea-water clearly appears to be inhibitory particularly for the DK, P-1and APPC-11S strains. Supporting evidence that pH is the crucial vari-able in C. resinae inhibition in seawater comes from the finding thatgrowth in seawater at an initial pH of 6 (SW + PY (6)) under petroleum,Shale II and acid-extracted Shale I was comparable to growth in freshwater systems (FW B-H, FW K-P).

Although it will be noted that C. resinae did not grow underShale I fuel in seawater initially adjusted to pH 6, the terminal pHof the aqueous phase in these test units was at least 7.3. This isdue to the gradual diffusion of basic materials from the fuel into thewater and the poor growth can again be attributed mainly to an unfavor-ably high pH. If this simple interpretation is valid it follows thatthe basic materials removed by acid extraction from Shale I should notbe inhibitory to C. resinae when neutralized and added back to a systemwith petroleum JP-5 so long as the pH of the aqueous phase is favorable.This was generally true (cf. Table 6).

C. resinae DK/adapted was the only strain to show appreciable

growth in normal seawater media (Table 3). This growth was character-istically slow to develop, however, and duplicates did not always growat the same rate (see Shale II, SW + PW (8)). Growth could start atone place in the interface, usually along the side of the flask asshown in Fig. 3 and from this point the growth could continue over theentire interface. Occasionally the DK strain grew similarily in normalseawater (see Footnote in Table 6). The problem in initiating growthmay be in lowering the pH enough in a microenvironment at some point inthe interface to allow growth to proceed. Once begun, the considerablecapacity of the fungus to produce acid within a colony is sufficient tolower pH in a larger volume and allow growth to spread.

CONCLUSIONS

1. The anaerobic sulfate-reducing bacteria were able to grow as wellwith all of the synthetic JP-5 fuels as with the petroleum JP-5. Thus

6

future problems from contamination by sulfate-reducing bacteria ofsynthetic fuels of the type examined here would be expected to besimilar to those experienced in the past with petroleum JP-5.

2. Shale II JP-5 supported good growth of the normal fuel tank con-taminating microorganisms. This was in striking contrast to thesevere inhibition to fungi seen with Shale I. The difference appearsto be due mainly to the additional refining of Shale II fuel that re-moved basic materials which in Shale I tended to keep the aqueous pHat too high a level for fungal growth.

3. Coal JP-5 was highly inhibitory to all of the yeast and fungaltest organisms. The inhibition was not due to acidic or basic ex-tractables or to the lack of a favorable aqueous pH. In view of thepossibility that there are constituents in this fuel which are com-patible with aircraft use and also inhibitory to fungi at low concen-trations, it appears worthwhile to attempt to isolate and characterizethese materials.

4. Shale I JP-5 was inhibitory to yeast and fungal test organismsbecause of the basic extractables in this fuel. However, except fortwo substituted pyridine compounds at high concentration (500 ppm),the nitrogenous compounds present in Shale I did not cause an in-hibition. Most of the inhibition of Shale I appears to be due to thetendency of the nitrogenous constituents to keep the pH of the aqueousphase at too high a level for fungal growth.

5. Different strains of the fuel fungus, C. resinae, showed differentpreferences for fresh or salt water media but all were inhibited bythe normally high pH of seawater. Growth of this organism in seawatermay depend on initiation of reduced pH in local microenvironments.

6. In the present stage of synthetic fuel development it is ncZ yetpossible to predict with certainty the probable susceptibility of aproduct to microbial contamination, especially by fungi, from aknowledge of the refining processes used or the conventional proper-ties of the fuel.

ACKNOWLEDGMENT

The authors thank D.G. Keenan for the typing of this manuscript.

7

REFERENCES

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

2. Bartick, H., K. Kunchal, D. Switzer, R. Bowen, and R. Edwards.1975. Final Report on Office of Naval Research Contract No.N-00014-75-C-0055. Applied Systems Corp., Vienna, VA.

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

4. Eisen, F.S. 1975. Preparation of gas turbine engine fuel fromsynthetic crude oil derived from coal. Sun Oil Co. Final Reporton Navy Contract N-00140-74-C-0568.

5. Klemme, D.E., and J.M. Leonard. 1971. Inhibitors for marine

sulfate-reducing bacteria in shipboard fuel storage tanks. NavalResearch Laboratory Memo Report 2324.

6. Klemme, 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.

7. May, M.E., and R.A. Neihof. 1979. Microbial deterioration ofhydrocarbon fuels from oil shale, coal, and petroleum. I. Explo-ratory experiments. Naval Research Laboratory Report 4060.

8. Naval Air Propulsion Center, Trenton, NJ. 1979. Fuel flexibility/

synthetic fuels. Detailed program plan.

9. Nowack, C.J., R.J. Del Fosse, G. Speck, J. Solash, and R.N. Hazlett.Aug. 1980. Relation between fuel properties and chemical compo-sition. IV. Stability of oil shale derived jet fuel. PreprintsAmer. Chem. Soc., Div. Fuel Chem.

10. Office of Naval Research. 1978. ONR Contract N-00014-79-C-0001.

11. Park, P.B. 1975. Biodeterioration in aircraft fuel systems.

Soc. Appl. Bacteriol., Tech. Series 9:105-126.

12. Solash, J., R.N. Hazlett, J.C. Burnett, E. Beal, and J.M. Hall.Aug. 1980. Relation between fuel properties and chemical com-

position. II. Chemical characterization of U.S. Navy Shale IIfuels. Preprints Amer. Chem. Soc., Div. Fuel Chem.

13. 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.

8

a 9

Fig. 1 - Illustration of the rating system for Cladosporium resinaegrowing at the interface of a two-phase fuel/water system; 0 = nogrowth to 6 = mat thicker than 0.5 cm.

9

(a)

(b)

Fig. 2 - (a) Cladosporium resinae DK in a two-phase system of seawater +peptone yeast (pH = 6) under, from the left, Shale I, base extracted and

acid extracted Shale I. (b) C. resinee DK/adapted in a two-phase systemof fresh water (Klausmeier-Park's mineral salts) under, from the left,

Shale I, base and acid extracted Shale I.

10

Fig. 3 - Duplicate flasks containing seawater -r peptone yeast (pH8)

under JP-5 showing Cladosporium resinae growth starting in the right

flask on the side at the interface. Growth in this flask eventually

covered the interface. The left flask exhibited no growth.

11

Table 1. Growth of sulfate-reducing bacteria in assay medium underconventional and synthetic jet fuels after 68 hours inci-bation

Fuel Source of sulfate-reducing bacteria

Laboratory Potomac River DieselContinuous culture Sediment Fuel Tank

USS MERRILL

Petroleum JP-5 4 1 5

Coal JP-5 4/5 1 5

Shale I JP-5 2 2 4/5

Shale II JP-5 4/5 2 5

Mineral Oil 4 2 5

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20

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22

APPENDIX A

Key to the rating system for growth in two-phase fuel/water systemsincluding approximate dry weights

Rating Dry Weight Description(mg)

0 0.34 ± 0.12a No germination, growth or mat formationdetected.

1 0.34 ± 0.23 Germination and minute amount ofgrowth detected, no mat formation.

2 1.5 ± 0.52 Slight growth at interface.

3 9.7 ± 3.5 Mat formation over 1/3 of interface.

4 26.0 ± 0.3 Mat formation over 2/3 of interface.

5 64.0 ±17.9 Mat formation over entire interface.

6 115.0 : 41.0 Mat formation over entire interfacewith a thickness of at least 0.5 cm.

aMean values ± standard deviation.

23

FIIE

DI