BIOTECHNOLOGY TECHNIQUES Volume 7 No.3 (March 1993)pp.213-216 Received lOth January

QUANTITATIVE MEASUREMENTS OF COAL SOLUBlUZATION BY FUNGI

Bailey Ward Department of Biology

The University of Mississippi University, Mississippi 38677 USA

SUMMARY

A quantitative method for measurement of coal solubilization by fungi is descril;)ed. The method was applied to comparative assays of lignite solubilization by four fungal strains Isolated from weathered coal outcrops.

INTRODUCTION

Several reports (Cohen and Gabdele, 1982; Scott et a/., 1986; Ward and Sanders, 1989;

Qulgley et al., 1989a, 1989b) have established that diverse fungal taxa are capable of

converting some weathered (oxidized) coals directly to water-soluble products. After one to

several days' contact with well-deve~oped fungal mats, coal particles form aqueous pools of

solubillzed-coal products that accumulate on the surface. This surface-solubillzation

phenomenon has been useful for initial screening for coal-solubilizlng organisms, and for

collection of the solubillzed product for subsequent analysis. However, the factors of coal

heterogeneity, variable effects of culture environment on microbial activity, and water content

of solubllized coal can markedly affect evaluations of biosolubilization based on visual

estimates. The surface-solublllzation method does not provide a reproducible, quantitative

measurement of rate and degree of coal biosolubilization activity.

I describe here applications of a diffusion-zone assay for reproducible, quantitative

measurements of lignite biosolubilization by four different fungal strains. The assay was

developed in preliminary studies to generate data for comparative measurements of effects on

coal biosolubilization of coal rank, weathering, pretreatments, and microbial diversity.

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METHODS

When coal pieces are placed on a nutrient agar medium prior to inoculation with fungal spores or hyphal fragments, developing mycelia cover the coal, after which solubilized products diffuse Into the agar around and beneath each coal unit. The diffusing products form darkened, circular zones that clearly are visible through the underside of a transparent culture dish. Measurements of diameter (or area) of the zones of diffusion as a function of time provide time-course data on coal blosolubllization. Factors of temperature, relative humidity, medium density and depthl particle size, and coal type directly affect diffusion of solubilized coal products. Thus, I established standardized assay conditions that would minimize effects of physical variables, yet would allow for vigorous growth of a variety of fungi. For the data reported here, I used the methods described following.

Coals ~lnd Preoaration. The lignites were semiweathered (5-10 cm below the exposed and weathered surface) samples from Claibome or Wilcox Group outcrops located respectively In Panola or Lauderdale Counties, Mississippi. To minimize effects of coal heterogeneity, 0.5 to 1o0 Kg of each prewashed and air-dried coal sample was pulverized and thoroughly mixed. The samples then were sized by being passed through a 30-mesh sieve (A.S.T.M. E-11 Spec. No. 32) and collected on a 60-mesh sieve (A.S.T.M. E-11 Spec. No. 60) to yield a particle size range of between 0;25 and 0.50 mm diameter. Sized coals were soaked for eight hours in a dilute basal mineral mix (Ward, 1985) to provide a uniform mineral environment for growth of fungi on coals of possibly diverse mineral content. Decanted coals were collected on preparative filter paper, then autoclaved wet in wide-mouth Jars at 1.1 Kg/cm 2, 121~ for 15 min.

Oraanisms. The test organisms were two unidentified species of Cunninghamella (strains YML-1 and YML-21), a Candida sp. (ML-13), and an Acremonlum sp., all Isolated from weathered lignites as described eadler (Ward, 1985; 1990). Stock and experimental cultures were grown on Sabouraud Dextrose 1.5% agar (SDA, Difco Laboratories, USA), pH 5.6.

Zon~ Diameter Assav. The SDA medium was poured to a uniform depth of 5.5 mm In 100 mm diameter x 75 mm deep Coming No. 3250 culture vessels. The dishes provide ample room for fungi that develop erect sporanglophores (2-3 cm for strains YML-1 and YML-21), and help to maintain constant relative humidity In the headspace over the cultureS. The agar surface should be free of condensation, which hampers placement and maintenance of uniform coal units. The SDA was poured after it had cooled to near the point of solidification; excess moisture was vented from partly open dishes under HEPA-flltered air In a laminar-flow hood. Six 3-4 mm in diameter mounds (units) of sized coal per dish then were placed evenly spaced (one central unit circled by five, at 25 mm spacing) on the SDA surface. A template circle drawn on paper to the size of the culture vessel and with locations and sizes of coal units marked will aid in uniform placement of coal units on all replicate culture vessels and by all personnel. Fungal inocula consisted of spores suspended in dilute mineral medium, evenly spread over the agar surface. Three replicate culture dishes per treatment provided 18 coal units for statistical analysis of the data. Preparations were Incubated In the dark at 25~ 70% relative humidity. Controls consisted of coal-only or organism-only preparations. As growth and coal solubUization progressed with time, each numbered coal unit was measured with a vemler caliper across the same diameter at each time point. The data were corrected for diameter of coal units. Culture dishes taken from warm, humid Incubators into cooler environments for measurements tend to form condensation, which can be prevented by keeping the vessels under incandescent lamps during data recording.

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RESULTS AND DISCUSSION

Within 1-3 days after inoculation, fungal hyphae began to cover much of the agar surface,

and to colonize the coal mounds. After 3-5 days, mycelial mats were developing and

solubilized coal products were beginning to diffuse into the agar beneath each coal mound.

Figure 1A presents results of the assays to compare biosolubUization activity by three

different fungi tested on the Claiborne lignite. Figure 1B compares solubilization activity on

the two different lignites by the Acremonium sp. As the diffusion zones increased in diameter,

the outer margins became less distinct, thus limiting the precision of measurements after

several days' biosolubilization activity. No diffusing products were evident in the coal-only or

organism-only preparations. The rate and degree of solubilization of any one coal often will

be different for different fungi, and any one fungus usually will exhibit different activities on

different coals. Different coals can produce product zones of different color and intensity;

some allow for longer time of assay and more precise measurements than do others.

12

10

E E 8

IN

< 6

I,t.I z O N 4

0

A �9 = YML-1

n = YML-21 ##

I I I I I

5 10 15 20 25 30

B S T R A I N I D M L - 1 2

WILCOX - 0

CLAIBORN F ==I"I

r

,I,, 1 ;!-T , . . / l l T

o, jg ! I I I I

5 10 15 20 25 30

TIME (days) TIME (days)

Fig. 1. (A) Solubilization of Claiborne lignite by three different strains of fungi. (B) Solubilization of two different lignites by Acremonium sp. Assay conditions: 25~ 70% relative humidity, 1.5% agar. Error bars = 95% confidence levels.

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The diffusion-zone assay allows for direct and convenient quantitative measurements of coal

blosolubiiization by fungi without sacrifice of cultures or disruption of activity. The

biosolubilized coal products can be extracted from the agar medium for subsequent

Instrumental analysis. Such variables as temperature, agar density and depth, and relative

humidity can affect accuracy, uniformity, and repeatability of measurements; exact conditions

of assay should be reported for each application.

ACKNOWLEDGMENT

This work was supported by the U.S. Department of Energy, Pittsburgh Energy Technology Center, grant No. DE-FG22-85PC80913.

REFERENCES

Cohen, M.S., and Gabriele, P.D. (1982) Appl. Environ. Mlcroblol. 44, 23-27.

Quigley, D. R., Ward, B., Cmwford, D. L, Hatcher, H. J., and Dugan, P. R. (1989) AppL Biochem. Biotech. 20/21,753-763.

Quigley, D. R., Breckenridge, C. R., Dugan, P. R., and Ward, B. (1989)J. Energyand Fuels 3, 571-574.

Scott, C.D., Stmndberg, G.W., and Lewis, S.N. (1986) Biotechnol. Progress 2, 131-139.

Ward, B. (1985) System. Appl. Microbiol. 6, 236-238.

Ward, B. (1990) Isolation and application of coalosolubilizing microorganisms. In: Bioprocessing and B/otreatment of Coal, D. L. Wise, ed., pp. 303-321, New York: Marcel Dekker.

Ward, B., and Sanders, A. (1989) Solubillzation of coals by fungi. In: Symposium on Biological Processing of Coal and Coal-Derived Substances. Electric Power Research Institute Pub. No. ER-6572, pp. 3/57-3/67, Palo Alto, California.

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