II ADI

Report 2b

ANALYSIS OF CJUTIN IN CONTAMINATED FUELS

September 1975 r

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1. S. ARMY MBILITY [iilPNEiT RESEARCH All iEVELIPNT CENTERFliT BELVIII, VIRGINIAISIIYEUPETRSAC I EEIUI

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PREFACE

The work was accomplished by G. Ernst, D. A. Emeric, and S. Levine under thelirection of Emil J. York, Chief, Materials Engineering Div;-on, Laboratory 4000,JSAMERDC. Technical contribution was made by Vincent J. Bagdon, Materials Engi-ieering Division.

CONTENTS

Section Title Page

PREFACE iii

I INTRODUCTION

1. Statement of the Problem 12. Background 1

II EXPERIMENTAL PROCEDURE

3. Approach to the Problem I

III RESULTS

4. Laboratory 25. Field 4

IV DISCUSSION

6. Discussion 5

V CONCLUSIONS

7. Conclusions 5

:1IBLIOGRAPHY 7

APIYFNDIX - Analytical Procedure for the Determinationof Chitin As Glucosamine 8

iv

ANALYSIS OF CHITIN IN CONTAMINATED FUELS

1. INTRODUCTION

1. Statement of the Problem. To provide a rapid and convenient chemicalanalytical technique which may be utilized for early detection of fungus growth indebris- and non-debris-contaminated fuel samples.

2. Background. The Armed Forces have utilized fuels in tropical and semi-

tropical areas where the optimum conditions for fungus growth during storage and/orfield use exist.

An analytical procedure has been developed which could be used as a rapidand convenient control technique for indicating an early stage of biodegradation ofhydrocarbon fuels. In addition, this technique may be used to evaluate fuel samplesfor their aervice potential during long storage periods and as a diagnostic aid for in-vestigating corrosion problems in the field. This procedure is a modification of a tech-

nique by Rondle and Morgan' 2 and is based upon the reaction of aminosugars withacetylacetone, in an alkaline medium, to form a chromogen that reacts with Ehrlich'sreagent (p-dimethylaminobenzaldehyde-HC dissolved in ethyl alcohol and concen-trated HC 1) to produce n pink color. The intensity of the color is proportional to theconcentration of the aminosugar.

Chitin, a polymer of glucosamine. is present in the cell wall of most fungi3

and, thus, if determined quantitatively serves to indicate the degree of fungus infiltra-tion.

II. EXPERIMENTAL PROCEDURE

3. Approach to the Problem. Experimentally contaminate clean JP-5 (to whichBushnell-Haas medium' has been added) with Cladosporium resinae and incubate until

I L E. Elson and W. T. J. Morgan, 1933, "A Colorimetric Method for the Determination of Glucosuine andChomndroeunne," Blochem. J. 27: pp. 15241828.

2 C. j. M. Rondle and W. T. J. Morgan, 1955, "The Determination of Glucosamine and Galactosamine," Biochem.

J. 61: pp. S86-H.3 Jackm W. Foster. 1949. Chemial Actidti of Fsrq, Academic Press. Inc., New York, N.Y., p. 90.4 The Buhd-m 01edk= has the following compoitin:

/ distled water

Mapiesima Sulfate 0.2Cle'W= Chloride 0.02Moopotudm Phosphate 1.0Ammoimnm Nitrate 1.0Ferrte Chloride 0.05

growth becomes visible. Recover growth from the interface, and determine chitin con-tent. After a procedure has been established, locate and sample a variety of fuel-storagesites. Analyze these samples for chitin, and correlate with standard microbiologicaltechniques.

111. RESULTS

4. Laboratory. Several fuel samples (200 ml of JP-5 enriched with 50 ml ofBushnell-Haas medium) were experimentally contaminated with C. resinae. Thesamples were incubated at 28°C until growth was visible. The samples were filteredthrough a 1.2-micrometer filter manufactured by the Millipore Corporation, and eachresidue was analyzed for its chitin content by the Rondle-Morgan method. No gluco-samine values (above the sample blank) were obtained. A fuel debris sample obtainedfrom an aircraft fuel (fuel-water interface) was analyzed, and a high glucosamine valuewas obtained. Microbiological analysis of the debris resulted in the isolation of afungus belonging to the genus Paecilomyces. In spite of the fact that C. resinae is fre-quently the predominant fungus' isolated from fuel, we decided, because of the posi-tive (high) glucosamine values obtained from Paecilomyces, to investigate the chitincontent of other fungi found growing in the fuel-water interface. For this purpose, weselected the following fungus species:' Aspergilius luchuensis, Cladosporium resinae,Paecilomyces varioti, Penicillium janthineuin, and a mixed culture containing all thefungus species mentioned above. A. luchuensis was isolated from a tent in NewGuinea. All of the other organisms were originally isolated from fuel.

Fuel-water samples were prepared from JP-5 and a salt-mineral medium.This medium 7 replaced the water and was composed as follows:

g/l distilled water

KH 2 PO4 2.6K2HPO 4 2.2NaNO 3 3.0MgSO4 • 7H 20 0.25Yeast Extract 0.2

It. T. Darby, . G. Simmons, and B. J. Wiley, 1968, "A Survey of Fungi in a Military Aircraft Fuel SupplySystem," International Biodeterioration Bulletin 4 (1), pp. 39-41.

6 The fungus cultures were supplied by the Pioneerh Research Laboratory, U.S. Army Natick Laboratories.Natick, Mamachusetts, 01700. The present address for obtaining cultures is: G. E. Simmons, Natick Labors.tores Culture Collection of Fungi (QM). Department of Botany, University of Masachusett, Amherst, Mama-chuses 0o1002.MA. R. Rogers and A. M. Kaplan, 1964. "A Survey of the Microbiological Contamination in a Military FuelDistribution System" Dev. Indust. Microb. 6, pp. 80.94.

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The initial pH was between 6.5 and 7.0.

The samples were incubated until growth could be noticed and then wereanalyzed for their chitin content. All the fungi with the exception of C. resinae gaveglucosamine values above the sample blank. It was decided to increase the sampleinterface volume (by increasing the number of sample bottles which were analyzed)in order to obtain higher values and increased sensitivity. It is interesting to note thatA. luchuensis and P. janthinelium gave the highest glucosamine values, and C. resinaegave the lowest glucosamine value (see Table 1).

Table 1. Glucosamine Analysis of Experimentally Contaminated Fuel

Glucosamine in Sample (mg)*

Fungi 5 Bottles/Set I Bottle/Set

Mixed Culture 6.2 1.3P. varioti QM 8465 2.4 0.3P. varioti QM 8009 3.9 0.3A. luchuensis QM 873 6.2 1.1P. janthineUum QM 8464 7.3 0.7C. resinae QM 8013 0.5 negative

* Each act incubated for 7 to 8 days

In order to further increase sensitivity, the number of bottles per set was varied (seeTable 2).

Table 2. Glucosamine Analysis of P. janthineilum QM 8464

Glucosamine in Sample (mg)

No. of Bottles 3 Days' Incubation 6-7 Days' Incubation

10 0.7* -5 0.2 7.03 0.0 2.42 0.0 1.0

*2 days' incubation

Further experiments with a mixed culture consisting of P. varioti QM 8465 and QM8009, A. luchuensis QM 873, P. janthineUum QM 8464, and C. resinae QM 8013 andvarious time frames indicated that 10 bottles/set was a practical number. The resultswere as follows:

3

pJ

Time Frame (h) Glucosamine ini Sample (mg)0 0.0

24 0.024' 0.327 0.245 0.248 0.472 2.5

20 botdmet; d otha smplae were 10 bottlee.

5. Field. Samples from the fuel-water interface were collected with a "GoldenThief" sampling ckvice (made by W & W Manufacturing Company, Chicago, Illinois) inpresterilized, widemouth glass bottles. Fuel samples collected from different sourceswere an-lyzed for their chitin content. It was found that iron interfered with the pro-cedure because of the coloration from iron (III) chloride (FeCl 3). Tin (11) chloride(SnC 2) was used to reduce the iron, but this also interfered with normal color develop-ment. In order to eliminate the iron interference, the residue was washed with 6N HCIand then rinsed several times with distilled water. Diesel fuel (DF-2) samples with nofungal contamination yielded upon analysis a light-yellow or a light-brown solution,which absorbed strongly at 530 nanometers. These yellow-colored solutions led tohigh readings and had to be discarded. The presence of even very low fungal levels inDF-2 produced upon analysis the customary pink-colored solution.

Debris-contaminated fuel samples (JP4 and DF-2) were obtained at differentareas at the U.S. Army Mobility Equipment Research and Development Center(USAMERDC) and were analyzed for their chitin content. The results were negative(no chitin). However, microbiological analyses of the fuel samples indicated thepresence of fungi. Contaminated fuels (JP-4 and MoGas) from the Crash Rescue Areaat Davison Army Airfield in Fort Belvoir, Virginia, were analyzed for their chitin con-tent. The results were also negative. Microbiological analyses of these fuel sampl-salso indicated the presence of fungi.

The negative chitin values obtained by the chemical method as against posi-tive findings (fungi were isolated by microbiological technique) may be explained bythe fact that the chemical method is apparently unable to determine chitin in spores.However, chitin can be determined when mycelium is present. In order to hasten theappearance of the mycelium in the fuel if spores are present, sterile Bushnell-Haas solu-tion was added to the field-obtained fuel samples, and the mixture was placed on ashaker for 24 hours prior to filtration and chitin analysis. Diesel fuel samples wereanalyzed chemically and bacteriologically for the presence of fungi. The results arecorrelated in Table 3.

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Table 3. Correlation of Chemical and MicrobiologicalMethods for Fungal Contamination

Sample Results, Growth, Incubation PeriodNo. Chemical Method Microbiological Method (days)

1 Neg No 52 Neg No 53 Neg No 54 Pos Yes 35 Pos Yes 36" Neg No 5

20 bottles/met; aD other smpla were 10 bottles/set.

IV. DISCUSSION

6. Dicussion. Fuel-water samples that were heavily laden with debris werefound to quickly clog the 1.2-micrometer Millipore filter. A Millipore prefilter (AP 20)was helpful but did not solve the "clogging" problems. Filtration of the heavily con-taminated fuel-water samples was accomplished by the judicious use of untreatedcott -i osnaburg cut to fit the Millipore, filter holder (see figure on page 6). The resi-due oi- the cotton filter was removed for analysis by means of an ultrasonic dismem-brator (see analytical procedure in appendix).$

V. CONCLUSIONS

7. Conclusions. Based on the work contained in this report, it is concludedthat:

a. It is possible to detect low levels of fungus contamination in fuel-waterinterfaces.

b. A rapid and convenient technique has been established to detect thepresence of low levels of fungus growth in the fuel-water interface even when heavilycontaminated with inorganic and organic debris.

G. Ernst, D. A. Eneric, and S. Levine. Quutiative Chemid Esamy of Chitn as Mme,. of Fuw* Infdlveriatin Cehdok MteriA To be Published.

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Cotton usnaburg, 40 by 30 yara in3 , having a weiht of 0.8 g/ft2.

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BIBLIOGRAPHY

Blumental, H-. J., and Roseman, S., 1957. "Quantitative Estimation of Chitin inFungi." J. Bact. 74: 222-224.

Boas, N. F., 1953. "Method for the Determination of Hexosamines in Tissues." J. of

Biol. Chem. 204: 553-563.

Foster, A. B., and Weber, J. M., 1960. "Chitin ." Adv. Garb. Chem. 15: 371-393.

Hubbell, D. H., A3sistant Professor, University of Florida, Gainesville. Written Com-munication, June 3, 197 1; August 14, 1972.

Mea, D. J., Hubbell, 1D. H., and Richards, B. N., 1971. Quantitative Chemical Essayfor Fungal Infection. Paper presented at the Annual Meeting of The AmericanSociety of Microbiology, Minneapolis, Minn.

Roseman, S., and Daffner, J., 1956. "Colorimetric Method for Determination of Glu-cosamine and Galactosamine." Anal. Chem. 28: 1743-1746.

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APPENDIX

ANALYTICAL PROCEDURE FOR THE DETERMINATION OvCHITIN AS GLUCOSAMINE

1. Equipment and Materials:

a. Apparatus:

(1) Spectrophotometer or colorimeter (wavelength: 530 nanometers)(2) Heaters(3) Refluxing apparatus (preferably with ground-glass joints)(4) 10 ml test tubes graduated in 0.2 ml divisions with ground-glass

stoppers(5) Ultrasonic dismembrator(6) Shaking apparatus

b. Reagents:

(1) 12N hydrochloric acid(2) 2N hydrochloric acid(3) 0.5N hydrochloric acid(4) ION sodium hydroxide(5) IN sodium hydroxide(6) Phenolphthalein solution (1%)(7) 2, 4-pentanedione (acetylacetone). This reagent must be re-

distilled every 2 weeks.(8) 0.5N sodium carbonate(9) Ethanol (absolute)

(10) Ehrlich's reagent (1.336 grams of p-dimethylaminobenzaldehyde isdissolved in 50 ml of ethanol followed by the addition of 50 ml of 12N hydrochloricacid). Store at -10°C.

(11) Bushnell-Haas medium(12) Glucosamine hydrochloride (Iml = 8.6 mg glucosamine). Store

frozen at -IOOC.

2. Procedure.

a. Take at least 10 bottles (bottle dimensions are 5 inches high and 2%inches wide at the base) of the suspected fuel sample collected from the interface. Addsterile Bushnell-Haas medium to the field-obtained fuel samples. Place the mixture on

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a shaker for 24 hours. A matrix control must be analyzed simultaneously with thesample.

b. Filter the fuel sample through a prefilter (Millipore AP2004700) orthrough cotton osnaburg. If cotton osnaburg is used, dislodge the residue with theultrasonic dismembrator by passing the probe back and forth across the residue area atleast three times.

c. Add an equal volume of 12N hydrochloric acid to the volume of watercontaining tlc residue, and transfer the total volume to the refluxing apparatus. Milli-pore or prefilters containing the filtered residue may be placed directly into 6N hydro-chloric acid. In either case, the final volume of the acid refluxing medium should notexceed 50 ml.

d. Reflux for 6 hours.

e. Cool the contents and filter through a Whatman 41-H filter paper orequivalent. Collect the filtrate into a 100-nl volumetric flask.

f. Rinse with distilled water, and bring flask up to volume. (The analyssmay be interrupted at this time for a maximum of 24 hours.) A reagent blank andglucosamine must be carried throughout the procedure.

g. A 1.0-wil aliquot is taken from the test solution(s) and pipetted into a10-ml test tube(s). Analysis must be carried out at least in duplicate.

h. Add a drop of phenolphthalein, and slowly titrate with ION NaOHuntil a pink color appears; then, back-titrate with 2N HCI until the pink color is dis-charged. Back-titrate with IN NaOH until a pink color appears, and then back-titratewith 0.5N HCI until the pink color is discharged.

i. Pipet 1.0 ml of acetylacetone (1.0 ml of acetylacetone dissolved in 50.0ml of 0.5N sodium carbonate). Prepare daily. The final volume at this point shouldbe 3.0 ± 0.2 ml; bring to volume with distilled water if necessary.

j. Place a glass tube condenser into a test tube. The condenser must nottouch the contents of the test tube. NOTE: An elongated glass tube with one sealedend or a conical test tube filled with water will act as a loose stopper and condenser toprevent the low of acetylacetone.

k. Place the test tube containing the condenser in a vigorously boilingwater bath for 20 minutes.

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1. Bring the contents of the test tube to room temperature, and addethanol (absolute) up to the 9.0-ml mark.

m. Add 1.0 ml of Ehrlich's reagent, and thoroughly mix the contents ofthe tube.

n. Place the test tube in a 65"C water bath for 10 minutes to acceleratethe liberation of carbon dioxide. Bring the test tube to room temperature, and bringup to the 10-ml mark with alcohol if necessary.

o. Spectrophotometer is set to 100% T with a reagent blank at a wave-length of 530 nanometers.

p. Transfer contents to a spectrophotometer cell. Color must be readwithin hour to avoid error due to fading.

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