Envkon. Scl. Technol. l S W , 27, 1176-1 181
Environmental Factors Influencing Methanogenesis from Refuse in Landfill Samples K. Rao Gurijalat and Joseph M. Suflita' Department of Botany and Microbiology, 770 Van Vleet Oval, The University of Oklahoma, Norman, Oklahoma 73019 Samples were excavated from the Fresh Kills Landfill and used to identify some of the environmental factors influencing refuse methanogenesis. The endogenous rate of methane production in these samples was significantly correlated with the moisture content, but not as highly correlated with other variables. Increasing moisture accelerated methanogenesis in 67% of the samples, but the rates decreased or remained unchanged in other samples. Aqueous refuse extracts were also assayed for the presence of alternate electron acceptors. Nitrate was not detected, but all samples had soluble sulfate levels ranging from 1.2 to 79.7 mmol/dry kg of garbage. Manipulation of the electron donor, acceptor, and metabolic inhibitor status in selected samples suggested that sulfate could inhibit refuse methanogenesis. An assay of individual refuse components revealed that paper and textile samples were associated with the highest concentrations of sulfate, but this anion was not detected when fresh refuse materials were similarly analyzed. Our findings suggestthat moisture, pH extremes, and high sulfate levels influence the landfill methanogenesis and that absorbent refuse components may represent reservoirs of sulfate in a landfill. Introduction Over 150 million tons of municipal solid waste (MSW) is generated every year in the United States, and more than 70% of it gets deposited in landfills (1, 2). Land disposal of solid waste has been practiced for centuries (3) with the assumption that microorganisms putrefy and degrade such wastes into more environmentally acceptable products. However, recent findings have indicated that even readily degradable materials can sometimes persist for surprisingly long periods of time in landfills (2). Such observations call into question exactly how efficient this waste disposal technology actually is. Despite the importance of landfills, there is relatively little information on the environmental factors that serve to stimulate or inhibit MSW biodegradation. Most available information focuses on the methanogenic fermentation of MSW, since methane is an economically desirable byproduct of refuse fermentation (4,5). Studies show that the absence of essential microorganisms is unlikely to limit refuse methanogenesis (2,4,6-8), while a moisture content up to 5040% (4,9,IO),a temperature of 40 OC (IO),small particle size (II), and circumneutral pH values (12)favor refuse decomposition and biogas (i.e., CH4 and COa) formation. Leachate recycle with neutralization has also been used to accelerate refuse decomposition (13,14). Methane production from fresh refuse could also be stimulated with an amendment of anaerobically degraded refuse (15, 16) but not with a sewage sludge inoculum (4). ~~
* To whom correspondence should be addressed. f
Present address: Betz Paperchem Inc.,Jacksonville, FL 32256.
1176 Envlron. Scl. Technol., Vol. 27. No. 6, 1993
These factors notwithstanding, the actual methane recovery based on the biodegradable portion of refuse is typically only 1 4 0 % of that theoretically expected (4). As yet, no systematic understanding exists of refuse decompositionprocessesthat allow for reliable predictions of methane yields from landfills. The fermentation of refuse linked to the consumption of other terminal electron acceptors has been suggested as one of the reasons for the failure to recover the expected amounts of methane (17). There may also be a potential for the anaerobic oxidation of methane in landfills (18). We investigated the endogenous rates of methane production in 34 samples obtained from the Fresh Kills Landfill. Initial results revealed a large variation in methane production rates in these samples as a function of moisture content (2). Some samples were found to contain high concentrations of sulfate (2). This study was designed to further investigate the role of moisture and sulfate in the methanogenic fermentation of municipal refuse. We also sought to identify the source of sulfate in the landfill. Experimental Section
Sample Collection. Samples of MSW were collected from various sites and depths at the Fresh Kills Landfill, Staten Island, NY, in October 1989 (2). Some of the characteristics associated with these samples are shown in Table I. A bucket auger was used to drill down to the desired depth in order to obtain the samples (2). Fourteen boreholes were drilled in the landfill, and samples were collected at approximately 3-m depth intervals. The refuse was passed through a coarse sieve (5 X 5 cm) before being collected in plastic buckets containing an O-ring sealing lid. The headspace of the refuse-filled buckets was exchanged with 02-free nitrogen prior to their transport by overland courier to the laboratory, where they were stored at room temperature. Incubation of MSW Samples. Anaerobic refuse incubation vessels were constructed by joining a standard plumbing end cap (PVC plastic, 7.6 cm) to a reducing union (7.6 to 5 cm) (Figure 1). Refuse material (200-300 g) was placed in the vessels while they were inside a portable anaerobic glovebag (AtmosBag, Aldrich Chemical Co., Milwaukee, WI), which was constantly purged with nitrogen. The headspace of each vessel was initially oxygen-free N2. The vessels were incubated at room temperature. The headspace methane content of the vessels was monitored by gas chromatography. In all experiments, unless otherwisenoted, duplicate vessels were used in each treatment. The standard deviation associated with methane production was never more than 10% of the mean. The effect of moisture on methanogenesis was evaluated by amending the samples with various amounts of sterile water. Methane in the headspace of the incubation vessel was then monitored periodically. 0013-936X/93/0927-1176$04.00/0
0 1993 Arnerlcan Chemlcal Soclety
Table I. Selected Characteristics of Municipal Refuse Samples Collected from the Fresh Kills Landfill
("C)
rate of methanogenesis [pmol (dry kg)-I day']
moisture (% wt/wt)
sulfate (mmol/kg)
PH
year of burial
29.4 42.2 NDb 37.2 37.7 ND ND 22.2 25 14.4 14.4 ND 16.1 22.2 22.2 18.3 21.1 28.9 27.8 21.1 ND 43.3 40.5 ND 21.7 21.7 21.7 21.7 31.7 10.5 57.8 45.5 62.8 45.5
0 262.5 0 335.2 272.3 0 1.6 520.3 435.1 124.9 297.5 116.1 219.3 371.8 302 274.9 0 0 302 0 79.9 56.8 149.8 26.3 702.5 17.2 52.2 148.1 377.8 316.3 109.4 24.1 0 298.3
19.0 27.8 16.0 37.6 31.2 21.4 17.9 73.4 42 35.6 33.6 32.1 43.5 41.1 21.5 43.4 37.0 32.6 52.1 35.8 37.9 40.1 32.2 25.0 74.7 29.8 54.2 23.9 32.3 52.4 22.5 32.8 10.1 50.6
10.06 6.45 23.54 15.9 10.06 30.7 6.06 8.60 13.43 12.89 7.61 51.4 10.39 11.38 6.22 4.96 25.69 14.39 4.41 31.94 12.23 16.08 11.92 15.55 28.1 5.76 79.69 8.98 7.40 45.97 15.43 17.86 2.27 21.38
6.2 6.7 6.3 7.4 7.1 5.8 6.4 6.9 7.5 7.4 7.0 6.7 6.8 6.9 6.8 7.2 6.3 1.2 7.4 6.2 7.4 7.3 6.9 7.1 7.1 6.8 6.8 6.9 6.9 7.9 7.2 7.9 7.2 8.1
1984 1984 1984 1984 1981 1985 1985 1980 1980 1973 1973 ND 1981 1965 1965 ND 1986 1986 1965 1986 1988 1988 (1980?) ND 1971 1972 1971 1971 1969-1970 ND 1976 1974 ND ND
sample ID, no.
sampling depth (m)
temp
1-2 1-3 1-4 1-5 1-6 2-1 2-2 3-2 3-3 3-4A 3-4B 3-5 4-1 5-2 5-2A 5-3 6-2 6-3 6-4 7-1 8-1 8-2 8-3 8-4 9-2 10-2 11-2 12-2 13-4 13-5 14-2 14-4 14-S 14-M
3.08 6.15 9.23 13.54 15.69 4.62 10.15 4.31 6.77 9.23 9.23 ND 3.38 3.38 3.38 6.76 3.08 6.15 9.85 3.08 3.08 6.76 9.23 15.38 4.62 3.08 4.92 5.23 8.62 9.80 13.85 20.31 12.31 21.53
0
remarks"
black black; wet NH3 odor; wet black refuse leachate, 12 m leachate, 4 m
C&D debrisc C&D debris wet textiles noted oil odor; wet
wet soil lens? wet, muddy
Field observations. ND = not determined. Construction and demolition debris. Wire
m
Crimp Seal Rubber StoDDer Balch Tube bber Stopper
,m a-pl
Incubation Vessel
Flgure 1. Schematic representation of the vessel constructed for refuse incubations.
To evaluate the influence of sulfate on methane production, the electron donor, acceptor, and inhibitor status of selected refuse samples were adjusted with acetate (20 mmol/kg), methanol (20 mmol/kg), sulfate (10 mmol/kg), molybdate (5 mmol/kg), and bromoethanesulfonic acid (BESA, 5 mmol/kg). Sterile anoxic distilled water was added to these systems (1:lwt/wt) to ensure that methane production was not adversely limited by the moisture content. The samples were thoroughly mixed by hand with the amendments before they were placed in the incubation vessels. This method of mixing was chosen over others
since preliminary dye studies (not shown) indicated that the amendments were more evenly distributed throughout the refuse samples. Analytical. Methane in the headspace of the incubation vessels was measured by flame ionization gas chromatography as previously described (19). Electron acceptors were assayed by extracting the refuse samples with an equivalent weight of water for 1 h. The refuse slurries were then centrifuged at 20000g for 25 min, and the supernatant was analyzed by anion-exchange highpressure liquid chromatography as previously described (19).SamplepH wasmeasuredin 1:l(wt/wt) refuse/water slurries using a pH electrode (Orion Model SA 720). The percent moisture was determined as weight loss on incubating triplicate refuse samples at 110 "C for 48 h. Refuse temperatures and depth of sample collection below the landfill surface were recorded in the field. Sulfatereducing and methanogenic bacteria were analyzed as previously described (2).
Results Characterization of Refuse Samples. The endogenous rate of methane production in all the samples obtained from the Fresh Kills Landfill is shown in Table I. This rate ranged from undetectable to over 700 pmol (kg of dry wt)-' day'. Samples were classified into four groups based on their methane production rate [pmol (kg of dry wt1-l day1]: either no methane production, low (300) rates. An approximately equal number of samples fell into each Environ. Sci. Technol., Vol. 27, No. 6, lQQ3 1177
0
25
0
1 1
75
100
0.50
150
25
1 67
50
75
100
125
150
+ 19
/ C
0.75
125
mi
404
1.XI
50
* 4635
---t 56
METHANE: NONE A G E 1984
* --f.
54
-60
0.25 0.00
0
25
50
100
75
125
150
DAYS Flgure 2. Effect of water amendments on the endogenous methane productionrate in samples: (A) 10-2, (6) 5-3, and (C) 1-2. The curves are labeled with the final concentration of moisture ( % wt/wt).
category. Methanogens could be cultured from all samples, including those not producing methane (data not shown). We questioned why such variations in methane production rates were encountered in samples collected from the same landfill. As noted in an earlier paper (2), moisture was identified as a significant factor influencing the methanogenic fermentation of refuse. There was a significant correlation between the methane production rate and the moisture content of the sample (r = 0.69; p = 0.0001) (2). Other factors such as pH, depth, age of sample, temperature, cellulose content, volatile solids, degradable organic matter, etc. (2) were not as highly correlated with the rate of methane production. However, this analysis cannot be interpreted as an indication of the relative importance of these variables on landfill methanogenesis. Effect of Moisture Amendments on Methanogenesis. When the moisture status of landfill samples was experimentally manipulated, a range of responses was noted. Figure 2A illustrates the effect of moisture on methanogenesis in a sample that was buried in the early 1970s, which initially contained about 33 % moisture and fell into the low methane production category. An increase in the amount of methane produced in this sample as a function of the moisture status was evident up to the maximum value assayed of 67 % Figure 2B shows the effect of moisture on a sample similar in age and moisture content to that above except
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Environ. Sci. Technol., Vol. 27, No. 6, 1993
that the endogenous rate of methanogenesis was medium rather than low. Increasing the moisture content over the same range did not significantly influence the methane production rate. Similar results were obtained with other samples that were already reasonably high in moisture content (>40%) and had a high endogenous methane production rate (data not shown). The effect of moisture on methanogenesis in a refuse sample that initially did not produce methane is shown in Figure 2C. A slight increase in methane production was noted only at the very highest moisture levelexamined. However, it is important to note that this is still a very slow rate of refuse methanogenesis. Based on these findings and on literature reports, a moisture content of at least 50% is generally considered desirable for refuse methanogenesis. However, the addition of water (l:l, wt:wt) to 33 refuse samples stimulated methanogenesis in only 67% of the samples. In other samples, the rate of methanogenesis either decreased (21%) or remained unaltered (12 %). Potential Role of Alternate Electron Acceptors. Moisture alone did not account for all the variation in methane production rates. Therefore, other factors that might influence methanogenesis were examined. Ionexchange chromatography of aqueous extracts (1:lwt.wt) of the refuse samples revealed that nitrate was not detectable (not shown) but that sulfate was present in all samples in concentrations ranging from 1.2 to 80 mmol/kg of refuse. All samples also possessed sulfate-reducing bacteria (not shown). Such levels of sulfate suggested that methanogenesis might be adversely affected in some samples since it is well-established that methanogens and sulfate-reducing bacteria compete for fermentation intermediates like acetate and hydrogen. By selectively stimulating or inhibiting these groups of organisms in landfill samples, we attempted to gain insight into the potential importance of sulfate as an alternate terminal electron acceptor influencing refuse methanogenesis. The influence of various amendments on the rate of methane production in a refuse sample is shown in Figure 3. The addition of molybdate, to inhibit sulfate reduction, stimulated the production of methane (Figure 3A). This suggested that the background rate of methanogenesis in this refuse sample might be influenced to some degree by the endogenous levels of sulfate. However, the addition of sulfate further inhibited methanogenesis, as did BESA. We examined whether the theoretically expected amount of methane could be recovered from the same sample when electron donors like acetate (Figure 3B) or methanol (Figure 3C) were used as amendments. With acetate as the amendment, the molybdate-treated sample produced close to the theoretically expectedamount of methanemore rapidly relative to the control which received only acetate. The addition of a soluble sulfate source inhibited the conversion of acetate to methane, as did BESA. In contrast, high recoveries of methane were obtained when methanol was provided as an electron donor regardless of the sulfate content in the sample (Figure 3C). Molybdate tended to slow methanol methanogenesis, but close to the theoretically expected amount of methane was eventually recovered. As expected, the amendment of BESA inhibited methane production. This examination was performed on a total of four landfill samples, one from each of the methane production categories. All samples responded to the various amend-
?n
.
I
A PLASTIC
45
C OTHERS
METHANE.LOW
0
30
15
(WOOD, GLASS,CERAMIC, ALLUMINUM, RUBBER,FOAM, LEATHER, DIAPERS, AND UNIDENTIFIED)
"
0.10
10.20
20.30
0.10
40.50
30.40
20-30
10-20
E
0
30
15
30.40
40-50
TEXTILE
45
0-10
10-20
30.40
20-30
40.50
50.60
60-70
SULFAT E ( mmol/kg) Figure 4. Distribution of sulfate in various landfill constituents. The insertnumbers on the bar graphs reflect the number of samples assayed when this value exceeded the maximum abscissa value. 12
15
0
45
30
DAYS Flgure 3. Effect of sulfate, molybdate, and bromoethanesulfonicacid (BESA)additions on the productionof methane in a refuse sample (8-2) receiving no exogenous electron donor (A), acetate (B), or methanol (C). Theoretically expected amounts of methane were calculated assuming the followingstoichiometry: CH3COOH-+CH4 CO,; 4CH3OH --* 3 CH4 -k 2H20 Con.
+
+
ments in analogous fashion, except for the sample that did not produce methane. These results suggest that sulfate could be an important electron acceptor in some landfill samples and could adversely influence the methanogenic fermentation of refuse organic matter. Origin of Sulfate in Landfills. In an effort to identify the source@)of sulfate in the landfill, about 120individual refuse artifacts obtained from the landfill were assayed. The artifacts were collected by a team of collaborating archaeologists led by Dr. W. Rathje, University of Arizona, and divided into about 30 categories (2). The artifacts were extracted and analyzedfor sulfate in the samemanner as whole samples. Most of the 20 plastic samples were found to have little or no sulfate associated with their extracts (Figure 4A). Extracts that had higher levels of sulfate came from samples that also had attached debris. Similarly, as expected, extracts of 13 cans recovered from the landfill also contained uniformly low levels of sulfate (Figure 4B). Figure 4C is a composite of a number of other artifact categories; of the 26 samples analyzed, 22 extracts were also very low in sulfate content. However, most paper items recovered from the landfill and extracted had higher levels of sulfate (Figure 4D). In contrast, no sulfate was detected when fresh paper refuse items were similarly analyzed (data not shown). This analysis included an assay of newsprint, packaging, and
NEWSPRINT
.
CORRUGATED PAPER
8-
c n -
i3 -1
N0N.PACKAGING PAPER
1
PACKAGING PAPER
1
J
SULFATE (mmollkg) Figure 5. Distribution of sulfate in various paper artifacts collected from the landfill.
nonpackaging paper samples. Similarly, Figure 4E shows that a variety of textiles recovered from the landfill were also associated with sometimes high levels of sulfate. Figure 5 shows a more detailed examination of the sulfate levels associated with various types of paper artifacts obtained from the landfill. While small sample size limited firm conclusions, newspaper extracts seemed to be frequently associated with relatively larger amounts of sulfate.
Discussion The microbiological analysis of an environment as complex and heterogeneousas a municipal landfill presents many methodological difficulties. Most obvious in this respect is the collection of representative samples. We used a systematic sampling effort that allowed us to collect refuse materials from every major section of the landfill, and the dates of recovered newspapers spanned the full age of the landfill (2). To reduce heterogeneity for Environ. Sci. Technol., Vol. 27, No. 6, 1993
1170
microbiological analysis, it was necessary to sieve each refuse sample, which effectively removed large refuse components (tires, lumber, bricks, etc.), allowing us to concentrate our efforts on refuse fractions more likely to be microbiologically important. However, it should be noted that all findings and inferences reported here are associated with sieved refuse samples. This study, as well as earlier ones (2,4, 7-9), identified the importance of moisture as a environmental variable influencing refuse methanogenesis. Three lines of evidence are present that reinforce this conclusion. First, a significant positive correlation between the moisture content of the refuse samples and the endogenous rate of methane production was noted with these samples (2). Second, several samples produced more methane in response to incremental moisture amendments (Figure 2). Third, the majority of the refuse samples (67%) produced more methane after amendment with an equivalent weight of water. The latter observation is consistent with other studies indicating that refuse samples possessing 55 % (wt/ wt) moisture produced increased amounts of methane, while those that contained less than 33% moisture did not produce methane (4). Several of the samples (21%) produced less methane when amended with water. It may be that the moisture amendment solubilized substance(s) that inhibit methanogenesis, including alternate electron acceptors (see below). Alternately, the added moisture may have stimulated initial fermentation processes, resulting in the net production of low molecular weight fatty acids. The accumulation of such fermentation products could have depressed the sample pH enough to inhibit methanogenesis (14). Neutralization of leachates containing high fatty acid concentrations and the subsequent recycling of the leachates over refuse has been shown to enhance methane production (4). While moisture content is a significant factor influencing methane production in landfills, the variation in this parameter does not account for the differences in refuse methanogenesis rates. Another factor could be the refuse pH. Kasali et al. (12) reported that a pH of 6.8-7.2 is optimal for methane production in municipal refuse samples. Examination of Table I shows that many samples which fell outside a circumneutralpH range produced little or no methane. Yet another factor that may impact refuse methanogenesis is the availability of alternate electron acceptors. Our assay of refuse revealed that all samples contained some soluble sulfate (Table I). Inhibition of methanogenesis by sulfate has been observed in a variety of environments (19-23) and with defined bacterial cultures (24). The generalexplanation for the inhibition of methane production in sulfate-rich environments is that sulfatereducing bacteria tend to outcompete methanogenic bacteria for electron donors like Hz or acetate (25-30). However, while these groups of organisms exhibit overlapping substrate specificities, they do not compete to an ecologically significant extent for other electron donors like methanol (31). The contention that sulfate could inhibit refuse methanogenesis was supported. When sulfate reduction was presumably inhibited by the addition of molybdate, a faster endogenous rate of methanogenesis was observed in some samples (e.g., Figure 3a). The same observation was made when an exogenous electron donor, acetate, was added to 1180 Environ. Sci. Technol., Vol. 27, No. 6, 1993
refuse samples. The conversion of acetate to methane was faster in molybdate-treated samplesrelative to controls amended with acetate alone (e.g., Figure 3b). Moreover, methanogenesis from either endogenous or exogenous electron donors was inhibited by the addition of sulfate relative to the sulfate-unamended control (Figure 3a,b). In contrast, methanol was converted to close to the theoretically expected amount of methane, regardless of the sulfate status of the sample (Figure 3c). As expected, BESA severely limited refuse methanogenesis whenever it was used as an experimental treatment. The results obtained from examination of the effect of sulfate, acetate, methanol, molybdate, or BESA on other refuse samples were analogous, suggesting that these effects may not necessarily be limited to select landfill samples. Future research will explore this point with refuse obtained from other landfills. Our findings argue that refuse methanogenesis may be limited to an unknown degree by the availability of sulfate. However, this interpretation must be tempered with the recognition that the electron donors undergoing biodegradation in a landfill and the electron acceptors exist in both soluble and insoluble forms. The relative importance of the various forms and the quantitative influence of alternate electron acceptors on refuse methanogenesis remains to be fully explored. While acid-labile sulfides could be detected in the refuse samples, conclusions on when or where such products were formed or the rate of sulfide formation could not be made with certainty without employing radiochemical techniques. Methods to quantitatively assess the rates of sulfate reduction and sulfide formation in landfill samples are currently being developed. However, it is well-established that sulfates emanating from MSW landfills can have a major impact on the biogeochemistry of leachates and adjacent groundwater andsurface water (32,331. It, therefore, seems reasonable to presume that sulfate reduction occurs to some degree in the anaerobic and organic matter-rich environment of a landfill. We assayed a variety of artifacts recovered from the landfill to determine the source of sulfate in landfills. This preliminary analysis revealed that aqueous extracts of papers or textiles were high in sulfate relative to other landfill components. However, sulfate was not detected when fresh paper and textile components were analyzed similarly. Our current hypothesis is that absorbent materials such as paper and textiles may act as reservoirs of sulfate, but the sulfate may originate from other refuse fractions. In this respect, it should be noted that the Fresh Kills Landfill contained up to 14 % construction and demolition debris (2). This refuse category undoubtedly included gypsum building materials. It may be that as water percolates over insoluble sources of sulfate like gypsum, a fraction of the electron acceptor is mobilized which can then be absorbed by materials like paper and textiles. This mobilization may effectively put the electron acceptor in the immediate vicinity of labile carbon sources like the cellulose and hemicelluloseresidues of paper. The sulfate levels could stimulate sulfate reduction to the detriment of methanogenesis. In this respect, it is interesting that building materials were easily observed in the field when samples 6-2 and 6-3 were recovered (Table I); methanogenesis was not detected from either sample. Since paper can represent over 40% of the volume of landfills (2),the
effect of sulfate on landfill methanogenesis could be substantial. Further research will be directed toward an assessment of how generalizing these observations are and an evaluation of the quantitative importance of the various factors influencing methanogenesis in landfills. Acknowledgments
We thank W. J. Rathje, F. Concannon, K. Ramanand, and V. K. Bhupathiraju for help in obtaining the refuse artifacts, M. Mormile for the assay of methanogens and sulfate-reducing bacteria, N. Wofford for graphics assistance, and R. Tanner for helpful discussions. This work was supported by a consortium of private foundations and industries including the following: Councilfor Solid Waste Solutions, Exxon Chemical Co., Procter and Gamble Co., and Scott Paper Co. Literature Cited (1) US.Environmental Protection Agency. The Solid Waste Dilema: an agendaforaction;FinalReport of theMunicipd Solid Waste Task Force; EPA/530-SW-89-019; US. Government Printing Office: Washington, DC, 1989; 70 pp. (2) Suflita, J. M.; Gerba, C. P.; Ham, R. K.; Palmisano, A. C.; Rathje, W. L.; Robinson, J. A. Enuiron. Sci. Technol. 1992, 26,1486-1495. (3) Senior, E. In Microbiology of Landfill Sites; Senior, E., Ed.; CRC Press, Inc: Boca Raton, FL, 1990; pp 1-15. (4) Barlaz,M. A.; Ham,R. K.; Schaefer,D.M. Crit.Rev. Environ. Control 1990, 19, 557-584. (5) Senior, E.; Watson-Craik, I. A.; Kasali, G. B. Crit. Rev. Biotechnol. 1990, 10, 93-118. (6) Barlaz, M. A,; Schaefer, D. M.; Ham, R. K. Appl. Environ. Microbiol. 1989, 55, 55-65. (7) Jones, K. L.; Grainger, J. M. Eur. J. Appl. Microbiol. Biotechnol. 1983, 18, 181-185. (8) Jones, K. L.; Rees, J. F.; Grainger, J. M. Eur. J. Appl. Microbiol. Biotechnol. 1983, 18, 242-245. (9) Leckie, J. 0.;Pacey, J. G.; Halvadakis, C. J. Environ. Eng. Diu. (Am. SOC.Civ. Eng.) 1979, 105, 337-355. (10) Kasali, G. B.;Senior,E. J.Chem. Technol.Biotechnol. 1989, 44,31-41. (11) DeWalle,F. B.; Chian,E. S. K.;Hammerberg, E. J.Environ. Eng. Diu. (Am. SOC.Civ. Eng.) 1978, 104, 415-432.
(12) Kasali, G. B.; Senior, E.; Watson-Craik, I. A. J. Appl. Bacteriol. 1988, 65, 231-239. (13) Barlaz, M. A.; Ham, R. K.; Schaefer, D. M. Waste Manage. Res. 1992, 10, 257-267. (14) Pohland, F. G. J. Environ. Eng. Diu. (Am. SOC.Civ. Eng.) 1980,107, 1057-1071. (15) Barlaz, M. A.; Milke, M. W.; Ham,R. K. Waste Manage. Res. 1987, 5, 27-39. (16) Stegmann, R. Waste Manage. Res. 1983, 1, 201. (17) Barlaz, M. A.; Ham, R. K.; Schaefer, D. M. J.Environ. Eng. (N.Y.)1989,115, 1088-1102. (18) Dalton, H.; Hocknall, M. In Proceedings of Landfill Microbiology R&D WorkshopZI;Heathrow,England; 1991;
Evans, S. A., Lawson, P. S., Eds.; Harwell Laboratories: Oxfordshire, England, 1991; pp 53-66. (19) Beeman, R. E.; Suflita,J. M. Microb. Ecol. 1987,14,39-54. (20) Winfrey, M. R.; Zeikus, J. G. Appl. Environ. Microbiol. 1977,33, 275-281. (21) Yadav, V. K.; Archer, D. B. Appl. Microbiol. Biotechnol. 1989, 31, 103-106. (22) Cappenberg,T. E. Antonie van Leeuwenhoek 1974,40,285295. (23) Oremland,R. S.;Polcin, S. Appl. Environ. Microbiol. 1982, 44, 1270-1276. (24) Phelps, T. J.; Conrad, R.; Zeikus, J. G. Appl. Enuiron. Microbiol. 1986,50, 589-594. (25) Kristjansson, J. K.; Schonheit, P.; Thauer, R. K. Arch. Microbiol. 1982, 131, 278-282. (26) Robinson, J. A.; Tiedje, J. M. Arch. Microbiol. 1984, 137, 26-32. (27) Schonheit, P.; Kristjansson, J. K.; Thauer, R. K. Arch. Microbiol. 1982, 132, 285-288. (28) Winfrey, M. R.; Zeikus, J. G. Appl. Environ. Microbiol. 1977,33,275-281.
(29) Abram, J. W.; Nedwell, D. B. Arch. Microbiol. 1978, 117, 89-92. (30) Lovley, D. R.; Klug, M. J. Appl. Environ. Microbiol. 1983, 45, 187-192. (31) King, G. M. Geomicrobiol. J. 1984,3, 275-306. (32) Baedecker, M. J.; Back, W. J. Hydrol. 1979,43, 393-414. (33) Strebel, T; Schiifer, W.; Peiffer, S. Aquat. Sci. 1991, 53, 346-366.
Received for review S e p t e m b e r 23, 1992. A c c e p t e d February 12,1993.
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