Journal ofApplied Bacteriology 1917, 42, 285-288

SHORT COMMUNICATION Chemical and Microbiological Studies on Leachates from a Waste Tip R. D. RILEY, R. G. BENEDICT, R. W. SEABLOOM D. A. CARLSON


Department of Civil Engineering, University of Washington, Seattle, Washington 98195, U.S.A. Received 2 September 1 9 7 6 and accepted 19 November 1 9 7 6 1. Some chemical and microbial parameters were examined in a 12 month study of leachate

from a waste tip. 2. Anaerobic decomposition within the tip yielded large amounts of aliphatic acids in raw leachate, which exerted a bacteriostatic action on coliforms within the tip. 3. Heterotrophic aerobes subcultured from the four sampling areas included representatives from 11 bacterial genera and one mould. 4. Many o f the isolated bacteria grew slowly and produced colonies much smaller than normal. The pollutants in raw leachate probably suppressed the growth of these organisms.

SOMEFACTORS affecting the chemistry and microbiology of waste tips are rainfall, surface water runoff, ground cover, types of tip and types of soil within the tip. Previous studies by Qasim & Burchinal (1970), on both simulated and natural tips, showed that anaerobic activity released CO,, H,, N, and CH,, and from acetone extracted tip solids, they obtained several aliphatic acids, ranging from acetic to isocaproic. As part of that study, Cook et al. (1967) isolated from leachates, Escherichia, Bacillus, Lactobacillus, Streptomyces, Micromonospora, Thermomonospora, Nocardia and Clostridium spp. Isolated fungi consisted mainly of Aspergillus and Penicillium spp. Engelbrecht et al. ( 1 974) noted that faecal and total coliform counts of 103/ml, at 5 5 days in a simulated tip study, had decreased to 2-3 celldm1 at 110 days. Faecal streptococci persisted for 47 days after leaching began, but none could be found at 86 days. When they added faecal coliforms, faecal streptococci, Salmonella typhimurium and polio virus, the latter disappeared rapidly, but S.typhimurium outlived the other bacterial cells. The current study involved sample collections at four sites surrounding the 920 acre Cedarhills tip, located about 40 km southeast of the University of Washington campus. Established in 1964, at an altitude of 180 m, it has an annual rainfall of 100-125 cm and receives ca. 830 x lo3kg/day solid wastes, consisting mainly of paper, animal and vegetable wastes, grass clippings, metal cans and glass.

Materials and Methods Sampling sites Raw leachate, L-I, which collected in a buried 333 m long perforated aluminium pipe, discharged into a small holding pond ca. 6 m from the tip. Leachate I1 (L-11) was 12851



collected as it emerged from top soil in a steep ravine about 450 m from the north end of the tip. Except in the dry summer months, the stream, located about 50 m from L-TI, contained some L-I1 diluted with surface runoff water. The control spring emerged 450 m west of the tip, and had no contact with seepage waters. Samples remained in a chill chest no more than 4 h before analysis for dissolved oxygen, BOD, and NH,-N (Anon. 1971).

Chromatographicstudies Ether extraction of the malodorous compounds in 100ml of L-I readjusted from pH 5 . 0 to 3.0, gave about 2.5 ml of a mixed acidic residue, after solvent evaporation. The ammonium salts of these acids separated well in the organic phases of 1 . 5 NNH,OH :n-butanol (1 : 1 v/v) or in 3 N-NH,OH :ethanol :n-butanol (5 : 1 :4) (Sherma & Zweig 1971). To reveal the acid spots, formed and dried sheets of Whatman Nos. 1 or 3 paper were first sprayed with a formalin :ethanol (1 :5) bromo-cresol purple mixture, adjusted to pH 5 .O, and then exposed to NH, vapours. For bioautographic studies, 130 pl L-I was spotted as the ammonium salts in a narrow band across the base of Whatman No. 3 strips, 3 cm wide and 30.5 cm long. A mixed standard of equal concentrations of ammonium propionate and butyrate (5Opl) was similarly treated on a second sheet. Solvent equilbration (24 h), preceded a 5 h forming in 1.5 NH,OH :IZbutanol (1 : 1). The dried strips were placed on a large assay tray, containing 1% nutrient agar, pH5.0, seeded with 1.0ml Escherichia coli broth/lOOml agar. A contact time of 15 min preceded the removal of the strips and incubation of the plate for 20 h at 30 "C. A gas chromatographic analysis of the leachate concentrate was made on a HewlettPackard 5700A gas chromatograph, with a 0.31 x 183 cm copper column packed with Porapak Q, 80-100 mesh. manufactured by Waters Associates, Inc., Framingham, Massachusetts. A column temperature of 200°C and a flame ionization detector at 250 "C were used. Nitrogen gas served as the carrier, with the flow rate set at 32 ml/min.

Microbiological studies Heterotrophic aerobes were counted on 'Cytophaga' agar (Anaker & Ordal 1959). Dried plates, in triplicate, received 0.1 ml of lo-', or lo-' dilutions of leachate or control. Surface spread plates, in sets of nine, were incubated for two weeks at 25 "C. Strict anaerobes were processed in deep BBL Trypticase Soy agar (TS), and in the deep cellulose agar of Rautela & Cowling (1966), then incubated in museum jars with a depleted oxygen atmosphere. The subculture of methane-utilizing organisms occurred on plates of the carbon-free basal medium of Leadbetter & Foster (1958), in an atmosphere of 50% CH, and 50% air. Twelve to sixteen colonies on countable 'Cytophaga' agar plates, picked in February 1973 as representative isolates of the flora in each of the four sampling areas, and whose colony sizes, morphology and chromogenesis had been recorded before isolation, were incubated in 'Cytophaga' broth for two weeks at 25 "C. Following motility and Gram stains (Skerman 1967), identification was facilitated by use of many differential taxonomic media and tests. To determine closely related isolates. taxonomic results for each strain were transferred to Indecks Research



Deck Cards. The number of unit characters compared ranged from 87 to 96/operational taxonomic unit; Coliform counts in lactose broth were done by the standard MPN method (Anon. 1971). Czapek and BBL Sabouraud's Maltose agars served as nutrients for isolation of fungi.

Results and Discussion No significant differences occurred in temperatures recorded for samples collected from the four sites, with winter lows at 6.5"C and summer highs at 12.5OC. Comparative levels of dissolved O,, BOD, and NH,-N are shown in Table 1. Passage of L-IT through soil had stripped the odorous acids, resulting in a substantial rise in pH. Viable cell counts of heterotrophic aerobes and anaerobes are recorded in the Table. Except for those from the spring, 50 to 57% of the 59 colonies of aerobes selected for study measured less than 1 mm in diameter. Their small sizes could not be attributed to overcrowding on the plates. Despite efforts to find temperature and nutrient combinations more suitable to them, 11 isolates grew so slowly or proved so hypersensitive to standard manipulations that nothing determinative could be done with them. This suggests a probable 'division of labour' occurring among the diverse microbial flora involved in catabolic processes within the tip. Genera identified among 38 isolates were Pseudomonas (1 6), Flavobacterium (7), Bacillus (9, Alcaligenes (4), Sarcina (2), Xanthomonas (2), Streptomyces (1) and Penicillium (1). Ten isolates could not be classified. The motile, CH, utilizing Gram negative rods, growing on Leadbetter-Foster medium appeared to be Methylomonas spp. As taxonomic aids, we used Bergey's Manual (Breed et al. 1957), and Skerman's Guide (Skerman 1967). Fewer numbers of L-I tubes in Set 1 (1 ml in 10 ml lactose broth) showed gas than those in Set 2 (0.1 rnl sample in 9.9 ml broth). The reverse was always true for L-I1 tubes, suggesting that raw leachate contained inhibitory substances. Assay of L-I using 12.7 mm diameter Schleicher and Schuell assay discs on nutrient agar plates seeded with E. coli, gave small, hazy zones of growth inhibition, whereas ether-extracted acid concentrates from 100 ml leachate produced sharp zones measuring 33 mm in

TABLE1 Mean values ofparameters in leachates and control L-l Dissolved 0, (mg I BOD, (mg 1.') NH,N (rng 1-I)



Viable Heterotrophic Aerobes (ml-') Viable Heterotrophic Anaerobes (ml-') MPN Presumptive Coliforms/lO* ml

{iCs t :

* Trypticase Soy Agar. -?

0 11 760 67.1 53 299 x lo6 340 x 103 8 5 x lo3 307 x lo2

Rautela & Cowling (1966) deep cellulose agar.



49 0 454 435 0.89 1 10 6.63 6 43 128 x 103 50.9 x 103 321 X 103 13 5 x 103 16 x lo2 5 x 102

Spring (Control) 10.0

0.73 0 006 6.60 7.9 x 102 -


R. D. RILEY ET A L . diameter. Paper chromatograms of concentrates showed the presence of propionic, abutyric, n-valeric and n-caproic acids. Gas chromatography confirmed the above and in addition revealed acetic and traces of isovaleric acid. Later quantitative studies on the aliphatic acids in L-I by Johansen (1975) indicated that propionic and butyric acids accounted for more than half of the 16 g/l total. Unequivocal proof on bioautographic plates that an acetic-propionic-butyric acid combination alone caused the bacteriostatic effect in coliform MPN tubes could not be attained. Presumptive evidence that these acids are the inhibitors must reside in our finding that complete removal of the noxious odours occurred with ether extraction and that the ‘stripped’ leachate no longer inhibited coliform bacteria. Certain anaerobes other than Clostridium spp. are capable of producing the aliphatic acids encountered in L-I and the substantial quantities found there suggest that production is faster than assimilation by anaerobic methane bacteria. When this occurs, the pH drops, making conditions unfavourable for growth of the methane producers and further increasing the rate at which these acids accumulate. The absence of Arthrobucter spp., and actinomycetes, particularly Streptomyces and Nocurdia spp., also indicates a marked shift from a normal pattern. Fungal colony numbers were scant, and mostly represented species of Penicillium. This work was supported by a Solid Wastes Management Training Grant No. 6 TO1 EC0093-01A1. We thank Miss Betsy Williams for the gas chromatographic analysis; Professor J. T. Staley, Microbiology, for assistance and encouragement throughout the project and Professor D. E. Stuntz, Botany, for help in identifying the fungal isolates.

References ANAKER, R. L. & ORDAL,E. J. 1959 Studies on the Myxobacterium Chondrococcus columnaris. I. Serological Typing. Journal of Bacteriology 78,25-32. ANON. 1971 Standard Methods for the Examination of Water and Wastewater 13th edn, American Public Health Association, Water Pollution Control Federation. BREED,R. S., MURRAY, E. G. D. & SMITH,N. R. 1957 Bergey’s Manual of Determinative Bacteriology 7th edn, Baltimore: The Williams & Wikins Co. COOK,H. A., CROMWELL, D. L. & WILSON,H. A. 1967 Microorganisms in household refuse and seepage water from sanitary landfills. Proceedings West Virginia Academy of Science 39,107-1 14. ENCELBKECHT, R. S., WEBER,M. J., ARMIRHOR, P., FOSTER, D. H. & L A ROSA, D. 1974 Biological properties of sanitary landfill leachate. In Virus Survival in Water and Wastewater ed. Malina, J. F., Jr. & Sagik, B. P. pp. 201-217, Water Resource Symposium No. 7, University of Texas Center for Research on Water Sources, Austin. JOHANSEN, o.-J.G. 1975 Rensing av sigevann fra Grennzo soppe~ylplass.Norsk Institut for vannforskning. Boks 333 Blindern, Oslo 3. LEADBETTER, E. R. & FOSTER,J. W. 1958 Studies on some methane utilizing bacteria. Archives f u r Mikrobiologie 30,91-I 18. QASIM,S. R. & BURCHINAL, J. C. 1970 Leaching of pollutants from refuse beds. Journal ofthe Sanitary Engineering Division, American Society of Civil Engineering 96,49-58. RAUTELA.G. S. & COWLING, E. B. 1966 Simple cultural test for relative cellulolytic activity of fungi. Applied Microbiology 14,892-898. SHERMA,J. & ZWEIG, G. 1971 Paper Chromatography and Electrophoresis. Paper Chromatography vol. 11, pp. 177-178 and 192-193, New York: Academic Press. V. B. D. 1967 A Guide to Identification of the Genera of Bacteria 2nd edn., BalSKERMAN, timore: The Williams and Wilkins Co.

Chemical and microbiological studies on leachates from a waste tip.

Journal ofApplied Bacteriology 1917, 42, 285-288 SHORT COMMUNICATION Chemical and Microbiological Studies on Leachates from a Waste Tip R. D. RILEY,...
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