-P137 177 MICROBIL DEERIORTIN OF HYDROCRRBON FUELS FROM

OIL i/iSHALE COAL AiND PETR..(U) NAVAL RESEARCH LAB WASHINGTONDC R A NEIHOF ET AL. 16 JAN 84 NRL-MR-5253

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MICROCOPY RESOLUTION TEST CH-ARTP4AMINAL BUREAU Of STANDARDlS 1i9- A

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* NIL Memorandum Report 5253

Dt~rtertou of Hydrocarbon Fuels FromOR S9hae, coal and Petroleum

NB. hlbthuof Fungi by Fuels From CoalItA. Niulop and M.E. MAY*

Colllllen and Fuela BranchChemisty Division

AWJWaiin InfermglN SjYuew Branch

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16, 1964

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REPORT DOCUMENTATION PAGE BEFORE COMPLETING FORM

-Ys I RIP"ET N~mu R -2 GOVT ACCESSION NO3 REC~IPIENT'S CATALOG OER

NRL Memorandum Report 5253 AD tI7- '7 "I TITf..E (&,,I S.6tley S TYPE OF REPORT A PERIOD COVERED

MICROBIAL DETERIORATION OF HYDROCARBON FUELSFROM OIL SHALE, COAL AND PETROLEUM Final report

III. Inhibition of Fungi by Fuels From Coal , PERFORMING OG. REPORT NuldBER

?. AUTmtOR(sj I. CONTRACT OR GRANT NLM*UERltsJ

R.A. Neihof and M.E. May

S PERFORMING ORGANIZA'ION NAME AND ADORESS 10. PROGRAM ELEMENT. PROJECT. TASKAREA WORIK UNIT NUMUEr1S

Naval Research Laboratory-'".''.'--': 43-1120-0-0. Washington, DC 20375

11. CONTROLLING OFFICE NAME ANO ADORESS 13. REPORT DATE

Naval Air Propulsion Test Center January 16, 1984Trenton, NJ 03628 13. NUMOER OF PAGES

26S14. MONITORING AGENCY NAME A AOORESSII different from Controlling Office) IS. SECURITY CLASS. (of tie repot)

UNCLASSIFIED-S.. DECLASSIFICATION/DOWNGRADING

SCHEOULE

1S. OISTRI@UTIOm STATEMENT (of ltfo.opott)

Approved for public release; distribution unlin. ited.

17. OISTR.JUTION STATEMENT (of rhe absraoct onroe,"i St ock 20. It dlifferen from Report)

I. SUPPLEMLNTARY NOTES

This work was supported by the Naval Air Propulsion Center.

It. KEY WOROS (Continue an feavore ads It necessary and Identify by block number)

Coal Fuel Candida sp.Petroleum Fungi Microbial deteriorationJP.5 Yeast Microbial contaminationSynthetic fuel Cladosporium resinae Inhibition

0. ABSTRACT (Contine an reverse aid* It necessary and ide.tify by black n umbee)

e anticipated future need for hydrocarbo, fuels from sources other than petroleum hasimpelled a thorough evaluation of the properties of such fuels, including their susceptibility tomicrobial contamination. The present work confirmed an earlier finding that a JP-5 fuel derivedfrom coal by the Char Oil Energy Development (COED) process was inhibitory to typical fungal

contaminants and showed that the inhibition extended to fuels produced by solvent refining aswell as to a variety of COED fuels refined in different ways. The inhibition was not due to

"lContinues)

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ack of a suitable aliphatic carbon source. Extraction of COED JP-5 fuel with aqueoussolutions showed that the inhibitor(s) had a very low water solubility and was notmarkedly concentrated at the water/fuel interface. An experiment with silica gel asadsorbent indicated that solid adsorbents may furnish a means of removing and concentratingsufficient amounts of the inhibitor for identification. Additional work to identify thesource of the fungal inhibition in coal fuel is worthwhile not only because of thepronounced and selective effects produced but because a novel inhibitor may be foundwhich would be useful as a fuel-compatible biocide for controlling microbial contaminationin any stored hydrocarbon fue

SEC URI TY CL.ASSIICATION OF~ THIS PAMI lhen Do#& Enlto d)

inii

CONTENTS

INTRODUCTION ............................................. 1

MATERIALS AND METHODS ................................... 1

Fuels .................................................... 1Microorganisms ............................................. 2Test U nits ................................................. 2Extractions ................................................. 3Silica Gel Fractionation ....................................... 3Estimation of Fungal Growth ................................... 4

RESULTS AND DISCUSSION ................................... 4

Inhibition by Different Coal Fuels ............................... 4

Stability of Coal Fuel Inhibition ................................. 5Experiments with an n-Alkane Source of Carbon Added to Coal Fuel ... 5Mixtures of COED-5 and Petroleum JP-5 (Jet-A) .................... 5Extractions ................................................. 5Silica Gel Adsorption ......................................... 6

CONCLUSIONS ............................................... 7

ACKNOWLEDGMENTS ......................................... 7

REFERENCES ............................................... 23

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MICROBIAL DETERIORATION OF HYDROCARBON FUELS FROMOIL SHALE, COAL AND PETROLEUM

III. Inhibition of Fungi by Fuels From Coal

INTRODUCTION

Under the Energy Conversion Synthetic Fuels Program theNaval Air Systems Command is evaluating jet aircraft fuelsderived from alternate domestic sources such as coal and oilshale (6). In a-dition to studies of the physical and chemicalproperties of these fuels, it has also appeared necessary toassess their susceptibility to contamination by microorganisms

Sbecause of recurring problems from this source with conventionalfuels.

In earlier reports it was shown that an inhibition to growthof typical fungal fuel contaminants by a shale oil-derived fuelwas due to nitrogenous constituents which imparted an excessivelyhigh pH to the aqueous phase of the test system (4, 5). When thenitrogenous constituents were removed from this fuel by acidextraction, fungal inhibition disappeared. Also a more highlyrefined shale oil JP-5 containing only 1 ppm nitrogen did notshow any microbial in'iIbition.

Marked inhibition of two principal fuel contaminants,Cladosporium resinae and a yeast (Candida sp.), also occurredwith coal-derived fuel produced by the Char Oil Energy Develop-ment (COED) process, but certain other microbial genera sometimesfound associated with fuels (eg., sulfate-reducing bacteria and aFusarium fungus) were not significantly affected. In this case,extractive treatments had no effect on the inhibition and itappeared that specific substances in the fuel were responsible.

2In view of the possibility that there are constituents in low

concentration in coal-derived fuels which are compatible withaircraft use and also inhibitory to important fungal contami-inants, it was deemed worthwhile to attempt to characterize thesource of the inhibition by coal fuel. Experiments carried outfor this purpose are described in this report.

MATERIALS AVD METHODS

FuelsA petroleum- ise fuel, designated Jet-A, was essentially

JP-5 without additives. It was the same control fuel as used and

described in previous work (4, 5). This fuel was sterilized inall cases by autoclaving 45 min at 121 0 C.

Crude coal fuels prepared by the COED proces- were refinedby hydrotreatment and distilled to give JP-5 grade fuels. COED-1, 3 and 5 were from Western Kentucky coals and COED-2 and 3 werefrom Utah coal. Additional descriptions have been given else-where (2, 4, 9).

Solvent refined coal fuels (1) were obtained with the helpof Mr. Forrest Schaekel (U.S. Army MERADCOM, Fort Belvoir, VA).Descriptive data on these fuels were kindly furnished through thecourtesy of Mr. S. J. Lestz and staff of U.S Army Fuels andLubricants Research laboratory, San Antonio, TX. These samples

Manuscript approved October 25, 1983.

a. . 1

were designated as follows:

AL-11222-F, EDS Distillate, solvent refined coal, mid-dis-tillate.

AL-11189-SP-F, H-Coal, naphtha91236, SRC II, naphtha- 1237, SRC II, middle distillate#1238, SRC II, heavy distillate

.0 Descriptive data on these fuels are given in Table I. The datain the last three columns may be for different samples ofnaphtha, middle and heavy distillates than those used in thepresent work, since the identifying numbers are different. Theyare included as indications of the properties to be expected.

Coal-derived fuels were usually not sterilized becausc ofthe possibility of losing volatile constituents or producingother changes. Filtration through 0.45 pm Millipore filtersmade no difference in the microbial inhibition seen in testsytems.

Dodecane (99%) was obtained from Phillips Petroleum Co.All fuels were stored at 40 C after receipt by the Naval

Research Laboratory.

MicroorganismsThe fungus, Cladosporium resinae DK, was isolated from a

JP-5 storage tank at a naval air station.Cladosporium resinae DK/adapted is the above organism after

adaptation to growth in seawater.Candida sp. was a yeast isolated from water with a skim of

oil on the surface which had collected in an exposed boiler roomof a partially scrapped naval ship.

All organisms were grown on slants of potato-dextrose agar(Difco) with the addition of 0.5% yeast extract (Difco). Forinoculation, stock suspensions of the organisms were prepared bydispersing surface growth on a slant in 10 ml of a solution of0.05% Tween 80 in distilled water. The viable count in thesesuspensions approximated 40 /ml and inoculation volumes wereadjusted to give about 10 colony forming units per test unit.

Test UnitsTest units with freshwater mineral salts media were of two

types: a formulation of Bushnell and Haas (3) with a pH ofapproximately 6.0 after sterilization (referred to as WBH) andanother following the formulation of Klausmeier as mo, fled byPark (8) with a pH of approximately 5.0 after sterilization(referred to as FWKP).

Test units with seawater as the aqueous phase were also oftwo types, differing only in the pH. Aged seawater (salinity,37.1% ) with additions of 0.05% peptone (Difco) and 0.05 % yeastextract (Difco) was used for both. The pH was unadjusted in onecase (pH - 8.0)(SWPY8) and in the ther I M HCI was added to givea pH of 6.0 (SWPY6).

Test units in most cases were 250 ml Erlenmever flasks withcotton plugs. Fifty ml of the water phase were dispensed in eachflask and autoclaved for 20 min at 1200 C. Fifty ml of fuel were

2

V'

7

then added and the unit was allowed to rest overnight. The pH ofeach flask was readjusted if needed before the addition of n.2 to0.5 ml of the inoculum of fungi or yeast. The flask plugs wereloosely covered with aluminum foil and the test units wereincubated in the dark at room temperature (22 0 -25 0 C).

In order to conserve fuel, later experiments were carriedout similarly but in 50 ml flasks with 10 ml each of aaueous andfuel phases. Results agreed with those carried out with thelarger test units.

ExtractionsEquilibration: 150 ml of COED-5 JP-5 were shaken continu-

ously in a 500 ml separatory funnel for 6 hrs with 150 ml FWKP orSWPY8 media. Inhibition of fungal growth was determined in testunits with the extracted fuel and fresh FWKP and SWPY6 media.The inhibition of the aqueous extractants was similarly deter-mined with Jet-A as the fuel phase.

Repetitive extraction: 150 ml of COED-5 JP-5 was extractedwith ten successive 500 ml portions of FWKP or SWPY8 mediadiluted to 10% of their normal concentrations. The inhibition ofthe extracted fuel was then determined in test units with freshFWKP or SWPY6 media.

Repetitive, prolonged extraction: 800 ml of COED-5 JP-5 fuelwas extracted with twelve 100 ml portions of FWKP or SWPY8 mediadiluted to 10% of their normal concentrations and shakingintermittently for at least one hour for each extraction. Theextracted fuel was tested for fungal inhibition in test unitswith fresh FWKP and SWPY6 media. The aqueous extractants werepooled and concentrated ten-fold in a rotary evaporator. Thisconcentrate and portions of it diluted 1:3 with fresh FWKP andSWPY6 media were examined for fungal inhibition in test unitswith Jet A fuel. The interfacial material from each extraction

was collected, pooled and added to test units with Jet A as thefuel phase.

Acidic and basic extractions: 200 ml quantities of COED-5JP-5 were extracted with three 15 ml portions of 0.1 M HCI

aallowing 5 min shaking for each extraction. This was followed by.U three successive 15 ml extractions with 0.1 M NaOH and three

washes with water. The procedure was repeated with the basicextraction preceding the acid extraction. A similar treatmentwas also carried out using nine successive extractions withdistilled water. The interfacial materials from the acidic andbasic extractions were combined and tested separately in systems. ith Jet-A as the fuel.

* Silica sel fractionationOne-hundred ml of COED-5 were added to a column (5 cm

,. diameter) of 800 g activated silica gel (Davison Chemical, Grade923, 100-200 mesh) suspended in pentane. A bed volume (500 ml)

*. of pentane was passed through the column during which timefraction #1 (475 ml) was collected. Then 100 ml of hexanefollowed by 500 ml hexane/benzene (3:1) and 800 ml methanol werepassed through the column. During this time successive fractionsof 400 ml (fraction #2) and 450 ml (#3) were collected.

3

Subsequently an oily, yellow fraction was collected (44) followedby the methanol washout (#5). The volatile solvents were removedfrom the fractions by evaporation with gentle heat (40-450 C for-41,2 and 3 and up to 600 C for #4 and #5). The non-volatile

li', *d remaining in each fraction was: #1, 6.5 ml; #2, 68 ml; #3,7.5 ml; 44, 15ml; #5, 15ml. Each fraction was divided in halfand used to set up duplicate test units with enough FWKP or S% PYAmedia and sterile Jet A to make each phase 50 ml. Fractions 01,#2, #3 and #4 were soluble in the fuel phase while Y5 was solublein the water phase.

Estimation of fungal growthThe test units inoculated with fungi or yeast were visually

inspected for growth at appropriate time intervals. The ratingsystem ranged from 0 for no growth and 1 for spore germination to6 for mats thicker than 0.5 cm over the entire interface. Astudy of the dry weights of microbial material corresponding tothese visual ratings showed that an increase of one unit in thevisual rating corresponded approximately to a doubling of theamount of growth (5). At the conclusion of each experiment,viability studies were made on those units showing growth ratingsof 0 or 1 by spreading approximately 0.5 ml from the water/fuelinterface on potato dextrose agar containing 0.5 % yeast extract.The agar surface of these plates had previously been allowed todry so that this large amount of inoculum could be spread without

having too wet a surface during incubation. Also at the end ofthese experiments, the pH of the water phase of all test unitswas determined with a glass electrode.

RESULTS AND DISCUSSION

Inhibition by different coal fuelsThe inhibition to fungal growth by a number of different

coal-derived fuels has been studied to determine the generalityof the phenomenon. Tables 2-A and 2-B show the results withCOED-i to 5 and two C. resinae strains. Jet-A petroleum-derivedfuel was used as a non-inhibitory control in this and subsequentexperiments.

COED-5 was completely inhibitory in most instances and afterfour months the organisms lost viabIlity in two out of fourcases. COED-2 was slightly les inhibitory overall but signifi-cantly delayed growth did occur after 15 weeks with C. resinae DKadapted and FWKP media. COED-3 and 4 appear to be least inhibi-tory especially with fresh water media. This may be associatedwith the more severe hydrotreatment given to these fuels. It

should be pointed out, however, that these COED samples were overfive years old and were kept at ambient tmnperatures for aportion of that time.

Table 3 shows that all of the solvent refined coal liquidswere extremely inhibitory and lethal to C. resinae DK. Thisrefining process is so different from the COED process that noclaim can be made that the inhibitory mechanism is the same withthe two kinds of fuels.

Thus all coal-derived fuels examined so far inhibit fungal

4

. growth but there are differences which may depend on the refiningprocess and coal source.

Stability of coal fuel inhibitionTo determine whether the factor in coal fuel causing inhibi-

tion of fungal growth was stable, experiments were carried outwith COED-5 fuel aged 15 months at room temperature and afterautoclaving 45 min at 121 0 C. Table 4 indicates that neither ofthese treatments has any effect 4n alleviating the inhibition ofgrowth of the test organisms.

Experiments with an n-alkane source of carbon added to coal fuelInasmuch as the n-alkane content of COED fuels is low, the

• "possibility existed that there was an insufficient concentrationof carbon substrate in the fuel to support the growth of fungi.To evaluate this, test units were set up with dodecane additionsto COED-5 (Table 5). While growth was rapid and prolific with100% dodecane, the addition of 20% COED-5 caused a cessation ofgrowth for all organisms and a loss of viability in many cases.A real inhibition and not a lack of suitable carbon sourceappeared to exist.

This conclusion was further supported by the observationthat a zone of inhibition surrounded a small well or padcontaining COED-5 in a agar plate whose surface was seeded withfungal spores.

Table 6 shows that although very small amounts of Jet-A ordodecane were sufficient to support considerable growth of fungiand yeast, the same small amounts of COED-5 were completelyinhibitory. Small amounts of toxic material in COED-5 wereapparently sufficient to produce inhibition.

Mixtures of COED-5 and petroleum JP-5 (Jet-A)Tables 7-A, B and C summarize results carried out with

mixtures of COED-5 in Jet-A. Inhibition was generally nearlycomplete only at 80% COED-5 except for C. resinae in FWKP wheresignificant growth occurred. The inhibition is obviously moremarked with the least favorable aqueous media (SWPY8). It issurprising that Jet-A dilutes out the inhibition of the coal fuelso readily. The result is not in agreement with the experimentswith dodecate/COED-5 mixtures where 20% COED-5 was completelyinhibitory (Table 5).

ExtractionsBecause the growth of microorganisms depends on the presence

of water, it appears clear that the inhibition to fungal growthby COED-5 fuel is due to some constituent which is able todissolve in the water media to a small extent or is at leastactive against organisms growing near the interface of the fueland water. Thus it appeared reasonable to expect that theinhibitory constituent could be removed and perhaps isolated byaqueous extractions.

COED-5 fuel which was shaken 6 hrs with an equal volume (150ml) of FWKP or SWPY8 was still inhibitory (Table 8-A). Repeti-tive extraction of 150 mi COED-5 fuel with ten 500 ml portions of

p5

S*" S o' .. '' . *. .• . ... .. . '. ' " . . ,. ' .. ,. - - ' -'- ., -

FWKP or SWPY6 (diluted to 10% of normal concentration) also did

not remove inhibition to C. resinae DK (Table 8-A). Extractionof 800 ml of COED-5 fuel twelve times with 100 ml portions ofFWKP or SWPY8 diluted to 10% did not alleviate the inhibition inspite of the prolonged shaking time (1 hr or more) alio,7ed f,)reach extra-tion (Table 8-A). Tables 8-B and C show t c'.t tl',

IN% failure to remove COED-5 inhibition to C. resinae DW b,:extraction extends also to C. resinae DK/adapted and Candidasp. It is interesting but inexplicable that viability of alltest organisms is lost in the extracted fuel but not in the COED-5 unextracted control.

In an effort to improve the extraction efficiencies ofinhibitory constituents from COED-5, acidic (0.1 M HCI) followedby basic (0.1 M NaOH) extractions (and the inverse order) weremade (Tables 9-A and B). Again no reduction in inhibition to thegrowth of the two C. resinae strains occurred nor were distilledwater extractions effective. Collected interfacial material fromthe acid and base extractions added to Jet-A containing systemsgave significant inhibition in FWKP for C. resinae but not inSWPY6. Thus some inhibitory constituent may be present in theinterfacial material or there may be enough transfer of COED-5fuel with the interfacial material to cause the observedinhibition with FWKP.

Although it was not possible to remove enough inhibitoryconstituent(s) from COED-5 fuel by aqueous extractions to allow

-. growth of fungi, it was still reasonable to examine the aqueousextractant for the presence of inhibitory material in systems

containing JET-A as the fuel phase. Table 10 summarizes theseexperiments. FWKP from the twelve-fold extraction of 800 ml ofCOED-5 fuel was not inhibitory even when concentrated ten-fold byevaporation. A similar experiment with SWPY6 did show completeinhibition of C. resinae DK for 4 months with the ten-foldconcentrated extract. The inhibition was readily removed,however, by a three-fold dilution of the concentrate. The

,K[. combined interfacial material from these extractions was also notinhibtory with either extractant.

Thus it appears that only with diluted SWPY solution was a

significant amount of inhibitor removed from COED-5 byextraction, i.e., sufficient to produce some inhibition when theaqueous phase was tested with Jet-A, but not enough was removed

to cause a significant reduction in the inhibition of the

extracted COED-5 (Tables 8-A, B, C and 9-A, B).

Silica Gel AdsorptionNone of the fractions from the silica gel column experiment

(Table 11) appeared to be markedly inhibitory, which would

suggest that some adsorption of inhibitor on the gel is occur-ring. Fraction 4 may show a slight inhibition that might havebeen much stronger if it had not been diluted so much by Jet-A inorder to carry out the experiments. The experiment should herepeated and refined so that fractions are better defined and thetest unit size adjusted so that no dilution of fractions is

necessary.

S.-o

CONCLUSIONS

1. Coal-derived fuels prod cec' I- the COED process and bysolvent refining were generally inhibitory to growth of thefungus, Cladosporium resinae, and the yeast, Candida sp.,although differences appear in COED-5 fuels which may be relatedto the different refining treatments.

2. Inhibition of fungal growth by COED-5 is due to constit-uents in the fuel and not to lack of a suitable n-alkanesubstrate.

3. Inhibitory constituents from COED-5 may adsorb on silica

gel, however, considerable additional work is needed to establishthe conditions for adsorption and elution in concentrated form.Other adsorbents may be worth investigating.

4. It is axiomatic that the inhibiting constituents in coalfuels must be at least slightly soluble in the aqueous phase to

be effective against the microorganisms growing there. However,aqueous extractions were not highly effective in rcrnc6inginhibition from the fuels. Only with a seawater extractant wasany measurable amount of inhibitor removed. Perhaps aqueousextractants containing other solutes would be worth invest-

igating.5. Additional work to identify the source of fungal

inhibition in coal fuels appears worthwhile not only because ofthe inherent interest in an inhibitor with pronounced selectivetoxicity at low concentrations for a relatively small group of

a microorganisms but also because a new fuel-compatible biocide maybe made known which could be used to control microbial

contamination in any hydrocarbon fue..

6. In systems containing seawater as the aqueous phase,prolific growth of fungi or yeast tended to lower the pH to amuch greater extent than in fresh water systems. This inter-esting difference should be further investigated in view of itssignificance for seawater-compensated fuel storage tanks on ships(7).

ACKNOWLEDGMENTS

This work was supported by the Naval Air Propulsion Center.The authors thank Carole A Komenda for typing tedious tables andForrest Schaekel and S. J. Lestz for supplying fuel samples anddescriptive data.

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SRC II, Naphtha 0

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a Density greater than water. Test units consisted of fuel onbottom and FWKP on top.

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Ifl~~~~ BI 0 .J4 q w 0 'v 44

181

i~( 4 - r- m wa %a %a M m

I.00 0 0 N - '4(4 0 dw

a in c CD 0D CD N g (N 0D en.

*! in~ mD 0 0 m C4 - ( N 0

.4 w 0 02 C 0 CN (N 40 C'4 N 0 N~

4 r

1.4 04 0 0D C Q 14 (N4 0 (N (N 0D (N

(1

41 9"I~A 0 I I' I., (

(4 a 0I N0 4- 0(

P4 .. 4 .-.4 to

0 to' 41*I

08 2t %4 41, 80282 44

194 L

00 N 0

%D 0 0 0 0 0 -

In 0 0 0 0 0

0 0 0

uq a a 0 a Nf 0 0 0 0

iijI3, 0 0 M 0

id ' 0 'd 0 0 0w 0

~ '. i~ a 0 '4 0 0 8

-B20

.h l + + 4 . + + +4 +

kn -. 1

%DIoi O LC U. n ULAOn

* n %-l LA ininu in n -W m

v v U,, nu mi C4 AC40 fn~

-j M. C14 4, i4c0ca en

'0

C'D

-4~~~~~ ("4 ('4 M'14C4 N (N .4N.4

%4 4 +g *n I I I hi46- V 1. 1 -044 I.S6

AjI '.4

N. 21

Table 11. Growth of Cladoscariu= res unae OK in two-phase systers with EWaqueous medim and fractions of COED-5 fram a silica gel column.

CDED-5 Growth ratings after incubation (weeks).. ~~~~~Fraction A. 2. /. A 5 . /

2 2 4 4 5 5 5

2 2 2 2 2 3 4 5

3 2 3 4 4 5 5 6

4 1 2 2 3 4 4 4

5 1 1 2 3 5 5 5

Jet A fuel (control) 2 3 4 4 5 5 6/5

222%-.-

'7- T, :7 7777 7. - 2 • --

REFERENCES

I. Bodie, W.1W., and K.C. Vyas. 1974. Clean fuels from coal. Oiland Gas Journal, Aug. 26: volume 72 pages 73-88.

2. Brinkman, D.W., M.L. Whisman, and J.N. Bowden. 1979. Stabilitycharacteristics of hydrocarbon fuels from alternate sources.BETC/RI-78/23. National Technical Information Service , U.S. Dept.

Commerce, Springfield, VA 22161.

3. Bushnell, L.D., and H.F. Haas. 1941. The utilization of

certain hydrocarbons by microorganisms. J. Bacteriol. 41: 653-

673.

4. May, M.E., and R.A. Neihof, 20 August 1979. Microbial deterioration ofhydrocarbon fuels from oil shale, coal, and petroleum. I.

Exploratory experiments. Naval Research Laboratory Report 4060. ADA073 761.

5. May, M.E., and R.A. Neihof, I August 1980. Microbial Deterioration ofhydrocarbon fuels from oil shale, coal and petroleum. II. Growth

and inhibtion of bacteria and fungi. Naval Research Laboratory

Report 4294. ADA088 055.

6. Naval Air Propulsion Center, Trenton, N.J. 1979. Fuelflexibility/synthetic fuels. Detailed program plan.

7. Neihof, R.A., and M.E. May, 23 July 1982. Survey of contamination infuel tanks of DD-963 class ships. Naval Research Laboratory

Report 4853. ADA117 109.

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

9. 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. Fuels 57:521-528.

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