Vous êtes sur la page 1sur 11

927

Methanogens and sulfate-reducing bacteria in oil sands fine tailings waste Fervone M. Holowenko, Michael D.
Methanogens and sulfate-reducing bacteria in oil sands fine tailings waste Fervone M. Holowenko, Michael D.
Methanogens and sulfate-reducing bacteria in oil sands fine tailings waste
Methanogens and sulfate-reducing bacteria in oil sands fine tailings waste

Methanogens and sulfate-reducing bacteria in oil sands fine tailings waste

Fervone M. Holowenko, Michael D. MacKinnon, and Phillip M. Fedorak

Fervone M. Holowenko, Michael D. MacKinnon, and Phillip M. Fedorak

bacteria in oil sands fine tailings waste Fervone M. Holowenko, Michael D. MacKinnon, and Phillip M.
bacteria in oil sands fine tailings waste Fervone M. Holowenko, Michael D. MacKinnon, and Phillip M.

Abstract: In the past decade, the large tailings pond (Mildred Lake Settling Basin) on the Syncrude Canada Ltd. lease near Fort McMurray, Alta., has gone methanogenic. Currently, about 60%–80% of the flux of gas across the surface of the tailings pond is methane. As well as adding to greenhouse gas emissions, the production of methane in the fine tailings zone of this and other settling basins may affect the performance of these settling basins and impact reclama- tion options. Enumeration studies found methanogens (10 5 –10 6 MPN/g) within the fine tailings zone of various oil sands waste settling basins. SRB were also present (10 4 –10 5 MPN/g) with elevated numbers when sulfate was avail- able. The methanogenic population was robust, and sample storage up to 9 months at 4°C did not cause the MPN val- ues to change. Nor was the ability of the consortium to produce methane delayed or less efficient after storage. Under laboratory conditions, fine tailings samples released 0.10–0.25 mL CH 4 (at STP)/mL fine tailings. The addition of sul- fate inhibited methanogenesis by stimulating bacterial competition.

Key words: fine tailings, methanogens, sulfate-reducing bacteria, methane, oil sands.

Résumé : Au cours de la dernière décennie, le parc de rejets miniers grossiers (Mildred Lake Settling Basin) de la Syncrude Canada Ltd Lease, près de Fort McMurray en Alberta, est devenu générateur de méthane. Normalement 60 à 80 % du flux des gaz à la surface des parcs à rejets sont du méthane. En plus d’amplifier le problème des gaz à effet de serre, la production de méthane dans la zone de rejets fins dans ce bassin de décantation et probablement dans d’autres peut diminuer la performance de ces bassins et nuire aux possibilités de restauration. Des dénombrements ont déjà confirmé la présence de microorganismes méthanogènes (10 5 à 10 6 NPP/g) dans les rejets fins de divers parcs à rejets de sables bitumineux. On a aussi observé des quantités élevées de bactéries sulfatoréductrices SRB (10 4 à 10 5 NPP/g) en présence de sulfate. La population de microorganismes méthanogènes s’est révélée résistante puisque des échantillons entreposés à 4°C pendant 9 mois sont demeurés stables au niveau du dénombrement NPP. La capacité du consortium bactérien à produire du méthane n’était pas diminuée ou retardée suite à la période d’entreposage. Dans des conditions de laboratoire, les échantillons de rejets fins produisaient 0.10 à 0.25 mL de CH 4 (à STP)/mL de rejets fins. L’addition de sulfate inhibait la méthanogénèse en stimulant la compétition bactérienne.

Mots clés : rejets fins, méthanogènes, bactéries sulfatoréductrices, sables bitumineux.

[Traduit par la Rédaction]

Holowenko et al.

937

Introduction

The presence of oil sands in northeastern Alberta has been documented since the 1780s. It is estimated that there are over 1.7 trillion barrels of bitumen contained in the Athabasca Basin making it one of the largest reserves of hy- drocarbons in the world (MacLean 1998), and likely the largest accumulation of biodegraded oil in the world (Hunt 1979). Presently, there are two companies strip mining the oil sands and extracting bitumen from them. Both companies

Received March 22, 2000. Revision received June 21, 2000. Accepted July 11, 2000. Published on NRC Research Press web site on September 15, 2000.

Fervone M. Holowenko and Phillip M. Fedorak. 1 Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada. Michael D. MacKinnon. Syncrude Canada Ltd., Edmonton Research Centre, 9421-17 Avenue, Edmonton, AB T6N 1H4, Canada.

1 Author to whom all correspondence should be addressed (e-mail: phil.fedorak@ualberta.ca).

Can. J. Microbiol. 46: 927–937 (2000)

are undertaking massive expansions to increase production, and a third company is starting to construct a mine and an extraction plant. The oil sands industry produces over 120 million barrels of synthetic crude oil yearly, but within the next decade this is expected to increase to 400 million bar- rels per year. In the Clark caustic hot water extraction process, the oil sand is digested with a mixture of hot water, NaOH, and steam (Clark and Pasternak 1932). The bitumen is separated from the sand as a froth and refined. Every cubic metre of mined oil sand requires 3 m 3 water, and produces on average 4 m 3 of waste. The tailings slurry waste consists mainly of solids (sand and clays), water, organics, and residual bitu- men (MacKinnon 1989; Mikula et al. 1996). The oil sands companies do not release any extraction wastes from their leases, consequently, all process-affected waters and fine tailings are contained on site, primarily in large settling ponds. Currently, Syncrude Canada Ltd., which is the largest single producer of synthetic oil from the oil sands, has in ex- cess of 300 million m 3 of fine tailings contained in these ponds, and this volume will continue to increase as mining operations proceed. One of the major reclamation plans for

© 2000 NRC Canada

928

fine tailings waste is a wet landscape approach (Gulley and MacKinnon 1993; List and Lord 1997). With this approach, the fine tailings would be transferred into an abandoned mined-out pit, over which a layer of water would be placed, establishing a water-cap over the fine tailings base (Boerger et al. 1992). Foght et al. (1985) examined the microbial content and activities in samples from the Mildred Lake Settling Basin (MLSB), which is the primary tailings pond for Syncrude Canada Ltd. Methane production was observed in samples from the 15-m depth that were supplemented with acetate or glucose and incubated at 37°C. However, no methane was detected in the supplemented cultures incubated for 47 d at 15°C, which was the temperature of the tailings in the MLSB. Foght et al. (1985) concluded that it was unlikely that methane evolution would be adequate to disrupt the consolidation of the tailings. In the early 1990s, nitrate-, iron-, and sulfate-reducing bacteria (SRB) were detected in samples from the MLSB, but methanogens were below de- tectable limits using the five-tube most probable number (MPN) assay (Sobolewski 1992). Over the next 5 years, visi- ble bubbling activity was observed on the surface of the MLSB. Enumeration of the MLSB samples collected in 1996 detected methanogens (Sobolewski 1997). Methane evolution from the MLSB was originally noted on the south side of the tailings pond in the early 1990s, and has spread northward. Recent analyses indicate that 2%–5% of the volume of the MLSB is comprised of dissolved and entrapped gases, containing 20%–60% methane. The ob- served gas flux to the atmosphere across the surface of the MLSB is 60%–80% methane. As of 1999, about 40%–60% of the 12-km 2 water surface area of the MLSB was consid- ered an active bubble zone, with an estimated daily flux of 12 g CH 4 /m 2 . Considering that one cow produces about 200 L of CH 4 /d (Miller 1991), the MLSB produces as much methane as 0.5 million cows each day. Methane is an important greenhouse gas and has an im- portant role in atmospheric chemistry influencing the ability of the atmosphere to oxidize other chemical pollutants (Al- exander 1999). Methanogenesis in the MLSB may pose po- tential problems for the remediation of the fine tailings, particularly with the wet landscape approach. Of primary concern is that methane percolating into the overlying water cap from the fine tailings will provide a faster transport of toxic compounds (primarily naphthenic acids) from the pore water of the fine tailings into the capping water layers (Gulley and MacKinnon 1993). As well, methane in the wa- ter could be consumed by aerobic methanotrophs, possibly leading to anoxic conditions particularly during periods of ice cover (Sinke et al. 1992; Wang et al. 1996). Low oxygen levels in the water cap could prevent the establishment of a lake ecosystem with higher pelagic forms of life, which may hinder remediation by the wet landscape approach. This paper presents a survey of the methanogenic popula- tion in the fine tailings. Both methanogens and SRB were enumerated in a variety of samples. The effects of sulfate additions and prolonged sample storage on methanogenesis were also evaluated with serum bottle microcosms. Methane yield values were determined from total methane produced in batch microcosms monitored for over a year.

Can. J. Microbiol. Vol. 46, 2000

Materials and methods

Samples

Samples were collected in August 1997 and July 1998 from the Syncrude Canada Ltd. lease (Fort McMurray, Alta.). Sterile 4-L glass bottles were plugged with a size 5 or 6 rubber stopper equipped with an eye hook screw attached to a string. A bottle was lowered to the desired depth at which time the stopper was dis- lodged by pulling the string, and the bottle filled with sample from that depth. Once air bubbles from filling the bottle ceased to break at the water surface, the bottle was considered full and brought to the surface. Because of the small size of the bottle mouth, it was very unlikely that the bulk sample was contaminated by fine tail- ings from other depths during the ascent of the sample bottle to the sampling boat. Three sources of fine tailings were sampled; the MLSB, the Base Mine Lake, and the Demonstration Pond. MLSB and Base Mine Lake are both actively methanogenic settling basins while the Demonstration Pond, which has no visible gas production and was created to evaluate the water capping option of the wet landscape reclamation approach. Created in 1993, the base of the 4 ha Dem- onstration Pond consisted of mature fine tailings from the north end of the MLSB, and before methanogenesis was significant in the fine tailings. This base was then capped with approximately 2 m of water. At the time of sampling, the surface water of the Demonstration Pond was clear, and plants and sedges were grow- ing in and around the pond. Birds were nesting successfully in the area. No methanogenic activity in the pond was visible as there was no bubble activity at the water surface. The fine tailings sam- ples collected from a depth of 5 m, below the water cap zone were fairly fluid and subsamples collected contained little trapped gas. In 1997 and 1998, the water:tailings interface was 2.5–3 m below the water surface. The Base Mine Lake is in the preliminary stages of the full- scale wet landscape remediation project and is contained within the old mined-out pit on the Syncrude Canada Ltd. lease. Mature fine tailings from the MLSB are being pumped to the Base Mine Lake via pipeline at a rate of up to 2.0 × 10 5 L/min. After the pit has been filled, plans are to continue with remediation by capping the fine tailings with water. The Base Mine Lake consists of two sepa- rate areas, denoted InPit 1 and InPit 2. Samples were collected from InPit 1 in August 1997 and from InPit 2 in July 1998. Bubble activity in the Base Mine Lake was visible. In 1997, the water:tail- ings interface was at 2.5 m below the water surface. Collection of the 10-m sample was complicated, as penetration through the fine tailings was difficult. When the empty sample bottle was first low- ered into the fine tailings, it would not penetrate past 9 m, however, as it began to fill up, the bottle penetrated down to 10 and 11 m and at one point was down 12 m. Therefore, the 10 m assignment is an approximate designation. In 1998, the water:tailings interface was 5 m below the water surface, so a 5-m sample was not col- lected because this depth was within the water:tailings interface. In 1997, two sites on the MLSB were sampled, one in the north region and the other in the central region. Samples were taken from depths of 1, 5, 10, 15, and 20 m below the water surface. In 1998, one site in the central region of the MLSB was chosen to sample. However, after obtaining samples from 1, 5, and 8 m, it was impos- sible to penetrate the sampling device deeper into the fine tailings. Thus, the boat was moved approximately 300 m to a new location and samples were taken from 5, 10, 15, and 20 m. The first 1998 location is designated MLSB(1), and the second location is desig- nated MLSB(2). The water:tailings interface was at about 4 m in 1997, and the 5-m sample was mainly fine tailings. In 1998 the wa- ter:tailings interface was 2–2.5 m below the water surface. In some cases, subsamples of the MLSB samples were taken for dissolved gas analyses. These subsamples were drawn through a

© 2000 NRC Canada

Holowenko et al.

929

Fig. 1. Depth profiles of dissolved and entrapped methane in fine tailings from the MLSB. Samples were taken at two different loca- tions in 1997 and 1998.

were taken at two different loca- tions in 1997 and 1998. short piece of Tygon tubing
were taken at two different loca- tions in 1997 and 1998. short piece of Tygon tubing
were taken at two different loca- tions in 1997 and 1998. short piece of Tygon tubing

short piece of Tygon tubing into a 60-mL plastic syringe. The sy- ringe was then sealed and the subsamples were analyzed by Maxxam Analytics Inc. (Edmonton, Alta.) using vacuum stripping and gas chromatography as outlined by ASTM (1990).

Enumerations of methanogens and SRB

The standard five-tube MPN method (Standard Methods 1985) was employed to enumerate methanogens and SRB. Ten-fold serial dilutions of the samples were prepared in sterile pre-reduced dilu- tion blanks containing anaerobic bicarbonate-buffered medium pre- pared by adding 4.0 g NaOH to 1 L boiled H 2 O under a constant stream of O 2 -free 30% CO 2 balance N 2 . Once equilibrated (pH 7.2–7.3), 2.0 g yeast extract (Difco Laboratories, Detroit, Mich.), 2.0 g trypticase peptones (Difco), 0.5 g mercaptoethane sulfonic acid (Jain et al. 1991), 14 mL mineral solution I (Fedorak and Hrudey 1984), 1.4 mL mineral solution II (Fedorak and Hrudey 1984), and 14 mL 0.1 g/L resazurin were added. Each dilu- tion bottle received 18 mL medium, and after being sterilized by autoclave for 20 min at 121°C, each received 0.2 mL of freshly prepared 2.5% (w/v) Na 2 S·9H 2 O. Methanogen MPNs (Jain et al. 1991), were done in 15-mL Hungate tubes (Belco, Vineland, N.J.) which were flushed with 30% CO 2 balance N 2 , and received 9 mL anaerobic bicarbonate- buffered medium. The tubes were sealed and autoclaved for 20 min at 121°C. After cooling, 0.1 mL of filter-sterilized vitamin B solu- tion (Fedorak and Hrudey 1984) and 0.1 mL of 2.5% (w/v) Na 2 S·9H 2 O were added to each tube. After inoculation, the tubes were incubated stationary, in the dark, at 22°C for 3 months. Meth- ane in the headspace was considered a positive result. SRB MPNs were determined as outlined by Fedorak et al. (1987). Tubes contained modified Butlin’s medium (Butlin et al. 1949) containing 10 mM lactate plus two iron finishing nails, and after inoculation were incubated stationary in the dark at 22°C for 1 month. The formation of a black iron sulfide precipitate was con- sidered a positive result.

Freshly collected samples were used for the surveys of micro- bial numbers in the tailings ponds. The MPN/mL results were con- verted to MPN/g dry weight and compared using the statistical method of Cochran (1950) (P < 0.05). The MPN method provided reproducible data for fine tailings when replicate samples were tested.

Microcosm preparation

Methanogenic microcosms were prepared using the modified Hungate method for serum bottles (Miller and Wolin 1974). Each 125-mL serum bottle was flushed with 30% CO 2 balance N 2 and received a 1:1 (v/v) ratio of anaerobic bicarbonate-buffer medium to fine tailings with final slurry volumes between 30 and 40 mL. Anaerobic medium was prepared and dispensed into bottles con- taining amendment (treatments) or water (controls). The bottles were capped with butyl rubber stoppers and crimped closed and autoclaved. Vitamin B solution (0.1 mL) and freshly prepared 2.5% (w/v) Na 2 S·9H 2 O (0.1 mL) were added prior to inoculation. Bottles were re-opened and were inoculated under a constant stream of 30% CO 2 balance N 2 . Microcosms were prepared in trip- licate and incubated in the dark at 22°C. Treatments included the addition of 1000 mg/L acetate as sodium acetate and up to 8000 mg/L sulfate as sodium sulfate. The headspace gas of the mi- crocosms was monitored for methane. Methane concentrations in the treatments were compared to those in the controls using the statistical method of Dunnett (1955) (P < 0.05).

Chemical and physical analyses

Pore water analyses were performed by Syncrude Canada Ltd. using standard industry methods (Syncrude 1995). Chloride, sul- fate, and ammonium were separated by ion chromatography and detected with a conductivity detector (Dionex-DX 300 series, Dionex Canada Ltd., Oakville, Ont.). Cations were analyzed by an Inductively Coupled Argon Plasma Atomic Emission Spectrometer (model 3580, Applied Research Laboratories). Bicarbonate con-

© 2000 NRC Canada

930

Can. J. Microbiol. Vol. 46, 2000

Table 1. Properties of fine tailings samples collected from different locations in the central region of the MLSB in 1997 and 1998.

Parameter

August 1997 samples

 

July 1998 samples

 

Depth (m) pH Temp (°C) Solids (g/100g) Na + (mg/L) K + (mg/L) Mg 2+ (mg/L) Ca 2+ (mg/L) Cl (mg/L) SO 4 2 (mg/L) HCO 3 (mg/L) NH 4 + (mg/L) Naphthenic acids (mg/L) Bitumen (g/100g)

1

5

10

15

20

1

5

8

10

15

20

8.1

a

8.3

8.3

8.3

8.5

8.5

20.2

17.7

17.8

21.2

20.6

22.0

14.0

13.5

12.5

11.0

11.5

b

19.9

33.2

39.0

39.8

b

15.5

22.4

25.0

38.8

47.7

782

533

624

627

656

895

590

667

541

438

444

9.1

10.3

12.6

12.5

12.0

12.9

15

18.5

14.6

11.9

12.2

4.3

2.6

5.0

4.4

4.1

9.7

7.3

6.6

8.0

6.9

5.7

5.9

3.4

5.8

5.3

5.1

7.3

6.1

5.2

6.0

4.6

4.4

536

195

265

284

298

634

386

307

301

150

127

146

4.9

2

3.3

3.9

173

87

68

68

17

7

960

1070

1240

1250

1020

1020

1010

1030

970

1000

1080

6.7

8.8

7.6

7.5

7.5

9.0

10

10

10

9.3

8.7

84

63

65

88

86

61

79

66

63

0.9

1.3

1.3

1.5

0.62

0.93

1.8

3.1

2.6

a Parameter was not measured on this sample.

b Overlaying water with very low suspended solids.

centrations were measured by titration or by calculating the value from dissolved inorganic carbon values. Naphthenic acids concen- trations were determined with a FT-IR spectroscopy method (Jivraj et al. 1995). Naphtha content was determined by solvent extraction and GC analysis with a Hewlett Packard 5890 GC-FID with two columns of complementary polarity. Bitumen and solids content were determined concurrently. Solvent extraction isolated the bitu- men which was dispensed onto filter paper and quantified gravi- metrically and the solids were collected and dried to a constant weight. Dissolved organic carbon was analyzed by Maxxam Ana- lytics Inc. through UV oxidation and IR detection of CO 2 . Sulfate measurements were performed on filter-sterilized (0.22 µ m filter) supernatants of subsamples from microcosms. Sulfate was separated from the other ions in the sample by ion chromatography and then detected and quantified with a conductivity detector. A Dionex model 2000i ion chromatograph with a IonPac AS4A-SC column (Dionex) was used. The mobile phase was 1.7 mM sodium bicarbonate and 1.8 mM sodium carbonate.

Methane analysis

Methane was analyzed using a Hewlett Packard 5700A GC with a flame ionization detector fitted with a 2 m × 0.3 cm column packed with Tenax GC (60/80 mesh). N 2 was the carrier gas at

50

mL/min, and H 2 and air were set at 35 mL/min and

300

mL/min, respectively. The injector, oven and detector tempera-

tures were 40°C, 40°C, and 250°C, respectively. Microcosms were

shaken, and 0.1 mL of headspace gas was removed for analysis. Chromatograms and peak areas were obtained using a HP 3380A integrator.

Determining methane yields

Serum bottle (158-mL) microcosms were monitored for over a year to determine the total methane yield from 100 mL fine tail- ings. Four replicate microcosms containing 1997 samples were in- cubated at both 22°C and 14°C. At the time of preparation, the samples had been stored at 4°C for 9 months. Microcosms were also prepared with samples collected in 1998. Ten replicates were prepared within 12 d of sample collection. Each microcosm re- ceived filter-sterilized resazurin to a final concentration of 1.0 mg/L, but none received any organic or inorganic supplemen- tation. Five replicates were incubated at 22°C and five at 14°C. Gas volumes in microcosms were measured using the method of Fedorak and Hrudey (1983) and all methane volumes were ex- pressed at standard temperature and pressure (STP). A methane

yield value for each sample was determined by averaging the individual methane volumes measured on the last three to five sampling times of the 468-d or 516-d experiments. Standard devia- tions were calculated as outlined by Skoog and West (1976). Meth- ane yields were compared by Student’s t-test (Steel and Torrie 1980) (P < 0.05).

Results and discussion

Methane content of fine tailings Samples were collected from the MLSB to determine the amount of methane dissolved and entrapped in the fine tail- ings. Figure 1 shows the amounts of methane in samples from two locations in the MLSB in 1997 and 1998. Al- though the concentrations were quite variable, methane was detected in each sample. The concentrations ranged from 1– 77 mL CH 4 (at STP)/L of fine tailings (1 mL CH 4 at STP = 44.6 µmol CH 4 ), but there was no consistent trend with depth at the different locations. Many samples contained about 20 mL CH 4 /L of fine tailings (Fig. 1). While sampling on the MLSB, copious amounts of gas were released when the boat anchor was dragged through the fine tailings zone. Natural release of the methane produces a daily flux of about 17 L CH 4 (at STP)/m 2 ,or 12 g CH 4 /m 2 .

Chemical, physical, and toxicity properties of fine tailings samples At the times of sampling, the MLSB the water tempera- ture of the 1-m samples was about 20°C. The fine tailings samples, found at depths greater than 5 m, had temperatures dropping as low as 11°C (Table 1). Generally, the tempera- ture of the fine tailings does not fluctuate significantly aver- aging between 11 and 15°C year round. The pH of the samples was relatively constant ranging between 8.1 and 8.5, which is consistent with reported values (Foght et al. 1985; MacKinnon and Boerger 1986; MacKinnon 1989). The sol- ids content of the fine tailings increased with depth, result- ing from the settling and consolidating process within the MLSB. The bitumen content of the samples ranged between 0.62 and 3.1 g/100 g which are normal levels of unrecovered oil in the fine tailings. Dissolved organic carbon concentra-

© 2000 NRC Canada

Holowenko et al.

tions ranged between 43 and 57 mg/L (data not shown). Al- though two different locations on the MLSB were sampled in 1997, samples from both locations had the same chemical and physical properties, so only the central site has been documented.

Pore waters from the samples were analyzed for major cations and anions (Table 1). The concentrations of Na + , K + , Mg 2+ , Ca 2+ , and NH 4 + changed little with depth of sample or between 1997 and 1998. The concentrations of Cl were ap- proximately twice as high in the water samples (taken from a depth of 1 m) as in the fine tailings samples (taken from depths of 5 m). Bicarbonate was over 960 mg/L in all of the samples (Table 1) and was quite constant throughout the depth profile of samples, attesting to the high buffering ca- pacity of the tailings pond. The concentrations of K + , Mg 2+ , and Ca 2+ in Table 1, were essentially the same as those re- ported by MacKinnon and Boerger (1986), but the Na + , HCO 3 , and NH 4 + concentrations have increased since that report. Concentrations of sulfate were highest in the surface wa- ters (1 m) and then dropped markedly as the depth in- creased; below 5 m there was little sulfate present in the

1997 sample (Table 1). MacKinnon (1989) reported this de-

crease in sulfate concentration with depth, and also observed an increase in bicarbonate that accompanied the change in sulfate concentration. However, the increased bicarbonate concentration with depth was not observed in this study (Ta- ble 1). Naphthenic acid concentrations in the pore waters ranged from 61 to 88 mg/L (Table 1). These acids originate from the oil sands and have been shown to be a major contributor to the toxicity of the waters from the oil sands tailings ponds (MacKinnon and Boeger 1986; Schramm et al. 2000). Herman et al. (1994) demonstrated that aerobic microbial activity will decrease the toxicity of naphthenic acids, but the role of anaerobic microbial activity on these acids has not been reported.

Enumeration of methanogens and SRB All of the samples collected in 1997 and 1998 were enu- merated for methanogens. All of the 1998 data (samples only from the central region of MLSB) are presented. North and central regions of MLSB were sampled in 1997 but only data from the central region are presented because both sam- ple sites had statistically similar MPN values. In both years, the lowest methanogen MPN values were in the 1-m-depth water samples (10 3 –10 4 MPN/g) (Fig. 2). There were no significant differences for the methanogen MPN values of samples collected from 5–20 m in 1997 or

1998 (Fig. 2). Generally, for a given depth, there were no

statistical differences between the methanogen MPN values in 1997 (10 5 –10 6 /g) and 1998 (10 6 –10 7 /g) (Fig. 2). All of the 1997 samples had SRB MPN values close to 10 4 /g, except for the samples obtained from a depth of 5 m, which had MPN values of 10 7 /g (Fig. 2A). Similarly in 1998, the highest SRB MPN values were in the MLSB sam- ples from 5 and 8 m, while all the other samples had statisti- cally similar SRB MPN values (10 4 /g) (Fig. 2B). Sulfate serves as a terminal electron acceptor for SRB, therefore it is reasonable that their numbers were high at the depths where

931

Fig. 2. Depth profile of the MPN values for methanogens and SRB, and sulfate concentrations in fine tailings samples collected from the central region of MLSB in 1997 (A) and 1998 (B). Samples from 5 m have high solids content and the counts are expressed as MPN/g dry wt of solids. The samples from 1 m contained few suspended solids, so the counts are expressed as MPN/g wet wt of sample (equivalent MPN/mL). * Indicates that the number of methanogens were significantly different (P < 0.05) from the number of SRB in that sample.

( P < 0.05) from the number of SRB in that sample. sulfate consumption was occurring,

sulfate consumption was occurring, and lower where sulfate was depleted (Fig. 2). Winfrey and Zeikus (1977) studied the sulfate profile in a freshwater lake and observed a sharp decrease in concentra- tion near the water–sediment interface. The profile was very similar to that shown in Fig. 2A. Although microbial counts were not done in the study by Winfrey and Zeikus (1977), there was an increase in methane concentration (indicating the activity of methanogens) after the sulfate was depleted. Furthermore, Raskin et al. (1996), using biofilm reactors, showed that population dynamics between methanogen and SRB are affected by the concentration of sulfate. When sul- fate was not available, the biofilm contained 25% methanogens and 15% SRB. Adding sulfate to the medium resulted in an immediate decrease in methane concentrations

© 2000 NRC Canada

932

followed by a decrease in the size of the methanogen popu- lation to 8% and an increase in the size of the SRB popula- tion to 30–40% of the biofilm. Similarly, a reactor that received continuous sulfate had 30–40% SRB and <10% methanogens in the biofilm, and when sulfate was removed from the medium, methane concentrations increased and the methanogen population increased and accounted for 18% of the biofilm. In the 8-m and 15-m samples, SRB and methanogens both had MPN values of 10 5 /g (Fig. 2B). The co-existence of large SRB and methanogen populations at depths where the SRB have strong competitive advantages (sulfate is avail- able) may suggest that there is an abundance of acetate and H 2 present to support both populations or that the two popu- lations are using different substrates and are not in direct competition. It is interesting to note that although a sharp drop in sul- fate concentration from the surface water to the settled fine tailings in the MLSB was observed by MacKinnon (1989), it took several years before bubbles of methane were observed at the surface of the MLSB. This implies that a considerable amount of time was required before the methanogenic pro- cess became established in the fine tailings and that the com- petition between SRB and methanogens was not the only factor curtailing methanogenesis.

SRB and methanogens in other samples Only one fine tailings sample (5 m) was collected from the Demonstration Pond each year and these samples had the lowest methanogen MPN values of all the collected samples. Yet these values did increase from 5.4 × 10 4 /g (1997) to >4.4 × 10 5 /g (1998). SRB MPN values in these 5-m samples varied little between 1997 (7.4 × 10 3 /g) and 1998 (1.4 × 10 4 /g) but were two orders of magnitude lower than the MPN values for the 5-m samples collected from the MLSB (Fig. 2). Sulfate concentrations in these samples were <10 mg/L. The Demonstration Pond is a pilot project for the wet landscape remediation plan, and the low methanogen numbers and activity are promising. If methanogen numbers continue to increase, then the feasibility of water capping may be compromised, because methane in the water may fa- cilitate the establishment of an active methanotrophic popu- lation. Methanotrophs will utilize the methane and deplete the water of oxygen, producing conditions that may impact the viability of the lake ecosystem. The two fine tailings samples collected from the Base Mine Lake were taken from the 5-m depth of InPit 1 (1997) and from the 10-m depth of InPit 2 (1998). Both samples had similar methanogen MPN values (10 7 /g) which were significantly higher than those obtained from the MLSB (10 5 –10 6 /g). SRB MPN values dropped from 8.9 × 10 6 /g in 1997 to 2.3 × 10 4 /g in 1998. Sulfate concentrations in both the 1997 and 1998 samples were <3 mg/L. The Base Mine Lake is the start of the full-scale wet landscape remediation plan, containing mature fine tailings pumped from the MLSB. That methanogen numbers in the Base Mine Lake are higher than in the fine tailings from the MLSB was un- expected and their activities may delay the creation of a self- sufficient lake ecosystem.

Can. J. Microbiol. Vol. 46, 2000

Effects of prolonged sample storage on methanogens and SRB During this project (Holowenko 2000), new experiments were established throughout the year, and at times fresh samples were not available. During the winter, bodies of wa- ter in northern Alberta are ice covered for approximately 6 months, thus samples from the MLSB can only be conve- niently and safely collected during the summer. Therefore, after collection, samples were stored at 4°C until required for various experiments (Holowenko 2000). There was con- cern that long-term storage at 4°C might affect the consor- tium which could alter methanogenesis and interfere in the interpretation of the results obtained. To determine whether storage affected the viability and performance of the inoculum, MPNs and acetate-stimulated methanogenesis were monitored in samples stored at 4°C for up to 9 months. MPN tubes were inoculated with selected samples stored

at 4°C for 0, 1, 3, 6, and 9 months. These samples were col-

lected in 1998 from the 5-m and 15-m depths of the MLSB and the 10-m depth of the Base Mine Lake InPit 2. Methanogens and SRB were enumerated. The MPN values did not drop with prolonged storage, and remained in the range of 10 4 –10 6 MPN/g, indicating that population sizes of methanogens and SRB were not affected by the long-term storage.

Microcosm studies tested the effect of prolonged sample storage on acetate-stimulated methanogenesis. Microcosms established with fresh samples and 1000 mg/L acetate pro- duced 8.0–8.9 mL methane. After storage of the fine tailings samples, acetate-supplementation resulted in at least 8.0 mL methane being produced, and ultimate methane production did not change significantly (P < 0.05) with storage time (data not shown), as evaluated by the method of Dunnett (1955). All microcosms that received acetate had lag times between 16–23 d before significant methane production, but there was no consistent change in lag time with storage time. Overall, prolonged storage had no adverse effects on acetate- stimulated methanogenesis in the fine tailings. Methane production in microcosms without added acetate (controls) was not affected by prolonged sample storage. A fresh fine tailings sample had a 19-d lag before methane pro- duction reached 0.60 mL CH 4 , a maximum rate of methane production of 0.24 mL/d and a final yield of 3.7 mL CH 4 af- ter 60 d incubation. A sample stored for 9 months had a 23-

d lag before methanogenesis, with a maximum rate of CH 4

production of 0.21 mL CH 4 /d and a total methane yield of 3.1 mL after 60 d incubation. Thus the ability of the consor- tium to produce methane was not delayed nor was the rate of methane production hampered by 9 months of storage at 4°C. The same fine tailings samples were also stored at 14°C and 22°C. MPN count and microcosm studies yielded the same results as obtained from the samples stored at 4°C.

Effects of mixing active and non-active samples

The gradual spread of methanogenesis in the MLSB from the south part of the lake prompted us to test whether mixing of a sample from a site that was actively methanogenic with

a sample from a site that was not visibly producing gas,

would lead to the formation of methane. An active sample from the 15-m depth of the MLSB (containing 4.3 × 10 6

© 2000 NRC Canada

Holowenko et al. 933 Fig. 3. The effect of mixing a fine tailings sample from
Holowenko et al.
933
Fig. 3. The effect of mixing a fine tailings sample from an ac-
tive methane-producing site with fine tailings from a non-active
site on methanogenesis in microcosms. Microcosms were
unsupplemented (A) or received 1000 mg/L acetate (B). Each
point represents the mean of triplicate microcosms and bars for
controls represent one standard deviation.
Fig. 4. Total volume of methane produced in microcosms con-
taining 100 mL fine tailings samples collected from the 5-m
depth of the MLSB in 1997 and incubated at 22°C or 14°C.
Each point represents a mean of triplicate microcosms.
true when H 2 was added to another series of microcosms
(data not shown). These data suggest that the methanogens
present in both samples are able to use both acetate and H 2
and that the difference between an active and a non-active
sample is likely influenced by the in situ abundance of
methanogenic substrates.
Estimating methane yields from fine tailings samples
Microcosm studies were done to determine how much
methane would be released from endogenous substrates in
methanogens/g, 4.5 × 10 4 SRB/g, and 3.3 mg/L sulfate) was
mixed in equal volume with an non-active sample from the
5-m depth of the Demonstration Pond (containing 5.4 × 10 4
methanogens/g, 7.4 × 10 3 SRB/g, and 9 mg/L sulfate).
Methane production in microcosms containing the mixture
was compared to that in microcosms that contained the
individual tailings samples (Fig. 3). The non-active sample did
not suppress methanogenesis in the mixed samples (Fig. 3A),
and methane was observed after only 2 d of incubation at
22°C. The microcosms than contained only the fine tailings
from the non-active Demonstration Pond, began to produce
methane after 45 d of incubation (Fig. 3A), and the rate of
methanogenesis paralleled methanogenesis in the micro-
cosms containing the active sample.
When acetate (1000 mg/L) was added to another series of
microcosms, all of them produced methane at essentially the
same rate after a 30-d lag period, regardless of whether they
contained the active fine tailings (Fig. 3B). The same was
the fine tailings from the MLSB, the Base Mine Lake and
the Demonstration Pond, and to estimate how long methane
production might occur. Figure 4 shows some typical results
of methane released in 100-mL microcosms containing only
fine tailings inoculum (no medium and no substrates added)
and monitored for 516 d. Some microcosms were incubated
at 22°C and others were incubated at 14°C. Most methane
production occurred in the first 200 d when the microcosms
were incubated at 22°C, while methane production was
slower in the microcosms incubated at 14°C. Because of
time constraints, these experiments were terminated after
516 d, at which time the amounts of methane in the micro-
cosms incubated at 14°C were essentially the same as the
amounts of methane in those incubated at 22°C (Fig. 4). Sta-
tistical analyses of the data showed that there was no differ-
ence (P < 0.05) in the total amount of methane produced.
The total methane produced in the microcosms was used
to determine methane yield values (Table 2). The data from
1997 show that the calculated yields, expressed as mL
CH 4 /g of tailings, were essentially the same regardless of the
temperature of incubation. For example after 516 d of incu-
bation, the central MLSB sample yielded 0.64 ± 0.34 mL

© 2000 NRC Canada

934

Can. J. Microbiol. Vol. 46, 2000

Table 2. Methane produced in microcosms containing 100 mL of fine tailings from various sources. Means of triplicate microcosms ± one standard deviation.

 

Sample

Incubation

Dry CH 4 produced at STP (mL)

Mass of fine tailings used (g, dry wt)

CH 4 yield (mL/g)

Year

Sample

depth (m)

temperature (°C)

1997

a

Central MLSB Central MLSB North MLSB North MLSB Base Mine Lake–InPit 1 Base Mine Lake–InPit 1 Demonstration Pond Demonstration Pond

5

22

9.6±5.1

15

0.64±0.34

 

5

14

7.9±2.9

15

0.53±0.19

5

22

10±1.6

26

0.38±0.06

5

14

9.9±6.2

26

0.38±0.24

5

22

18±9.9

32

0.56±0.31

5

14

11±7.3

32

0.34±0.23

5

22

2.0±3.0

40

0.05±0.07

5

14

0.74±0.59

40

0.02±0.01

1998

b

MLSB(1)

5

22

25±1.4

26

0.96±0.05

 

MLSB(1)

8

22

18±0.56

24

0.75±0.02

MLSB(2)

5

22

17±1.0

26

0.65±0.04

MLSB(2)

15

22

20±3.1

35

0.57±0.09

Base Mine Lake–InPit 2

10

22

23±2.3

23

1.00±.010

a Microcosms incubated for 516 d.

b Microcosms incubated for 468 d.

CH 4 /g and 0.53 ± 0.19 mL CH 4 /g at 22°C and 14°C, respec- tively. In situ, methane is trapped in the layers of fine tailings in the MLSB (as shown in Fig. 1), thus if methane was trapped within the samples in the microcosms, it would lead to inac- curate methane yields. After final methane measurements (day 516), a few microcosms were exposed to vigorous me- chanical shaking to release any trapped gas. Less than 2% of the total methane produced by these microcosms was recov- ered after shaking and therefore does not constitute a signifi- cant reserve of unaccounted-for methane. Except for the Demonstration Pond samples and the North MLSB 5-m sample (Table 2), all the methane yields fell within a statistically indistinguishable range of 0.53– 1.00 mL CH 4 /g. The microcosms containing the 1998 sam- ples were established within 12 d of their collection, whereas the microcosms containing the 1997 samples were stored at 4°C for 9 months prior to establishing the micro- cosms. The methane yields from the MLSB samples in the two experiments did not differ markedly, indicating that the pool of usable substrates in the 1997 samples was not signif- icantly depleted during 9 months of storage. The rate of methanogenesis was faster in the room- temperature-incubated microcosms than those incubated at 14°C which is similar to the in situ temperature of the fine tailings in the settling basins. This is not surprising, as methanogens tend to have increased activities at higher than in situ temperatures (Conrad and Wetter 1990; Zeikus and Winfrey 1976). Metabolism by methanogens is likely slower at 14°C as enzyme activity, which is affected by tempera- ture, is likely lower (Atlas and Bartha 1993). Furthermore, the rate of metabolism by the consortium, which provides methanogenic substrate, maybe slower at lower temperatures (Zeikus and Winfrey 1976). While it is understood that the rate of methanogenesis will be slower at low temperatures, few studies report on the ex- tent of methanogenesis at low temperatures. Studies of peatlands by Updegraff et al. (1998) demonstrated that methanogenesis slowed when temperatures were lowered but

that the total amount of methane produced was not affected by temperature. This is consistent with the data presented in Fig. 4. Total methane produced in the 14°C-incubated mi- crocosms was the same as those incubated at 22°C (Fig. 4) and thus the total amount of substrate shunted to methane production was not influenced by incubation at the lower temperature. Samples from the Demonstration Pond produced signifi- cantly lower amounts of methane than the other samples studied. The fine tailings from the Demonstration Pond gen- erated 0.02 and 0.05 mL CH 4 /g, and the temperature of incu- bation had little effect on methane yield (Table 2). These samples had the lowest methanogen numbers (10 4 /g) and were the least active samples collected. Therefore, it is not surprising that the amount of methane produced in these mi- crocosms was low compared to the samples taken from the active tailings ponds.

The total amount of methane produced in the microcosms was divided by the mass of fine tailings added to obtain the methane yield values of mL CH 4 /g fine tailings reported (Ta- ble 2). When considering the volume (100 mL) of fine tail- ings rather than the mass of fine tailings from the MLSB used in the microcosms, the methane yield range of 0.53– 1.00 mL CH 4 /g fine tailings converts to 0.10–0.25 mL CH 4 /mL fine tailings. To produce 0.10–0.25 mL CH 4 /mL fine tailings, the amount of carbon converted to CH 4 is 54–

134 µg/mL fine tailings. The average dissolved organic car-

bon values in the 1997 MLSB samples was 50 µg C/mL.

This concentration is less than the carbon needed to generate the amount of methane produced in the microcosms (54–

134 µg C/mL fine tailings). Thus, some amount of undis-

solved organic carbon, must be solubilized to provide methanogenic substrates. A majority of the carbon in the fine tailings is residual bi- tumen. Assuming that bitumen is 83% carbon (Boyd and Montgomery 1962; Camp 1973), and is the primary, but not only source of carbon in the tailings ponds, we can approxi- mate how much of the total carbon in the tailings pond is be- ing converted to methane. If the fine tailings contain on

© 2000 NRC Canada

Holowenko et al.

935

Table 3. The effects of the addition of 8000 mg sulfate/L in microcosms containing fine tailing samples taken in 1997. Microcosms were incubated in the dark for 220 d at 14°C.

 

Initial SRB

Lag time b before inhibition of methanogenesis (d)

Final CH 4 concen- tration in sulfate- amended microcosms (%vol)

Final CH 4 concentration in unamended controls (%vol)

Sulfate

numbers

consumed

Sample

Depth (m)

(MPN/g) a

(mg/L)

Central MLSB North MLSB Base Mine Lake–InPit 1 Central MLSB Central MLSB

5

1.57×10

7x

7

0.7

9.1

3620±1400

5

8.86×10

6x

14

0.6

9.6

3000±30

5

8.90×10

6x

18

1.1

10.8

3360±340

15

2.25×10

5y

45

3.6

9.6

980±780

15

4.52×10

4y

45

5.7

8.6

980±730

a MPN values with the same superscript letter were not statistically different from each other but were different from those with a different letter (P < 0.05).

b Inhibition of methanogenesis was determined by comparing methane concentrations in the sulfate-amended microcosms with those in the unsupplemented control microcosms using Dunnett’s method (P < 0.05).

average 2% (w/v) bitumen, then there are 20 g bitu- men/1000 mL fine tailings. Multiplying this by 83% and converting to mg carbon, there are approximately 17 mg C/mL fine tailings. If 54–134 µg C/mL fine tailings is needed to produce the measured volume of methane, re- leased methane accounts for <1% of the total carbon in the fine tailings. To date, the indentities of the compounds in the fine tailings that are converted to methanogenic substrates are unknown. It is not known how long the fine tailings were at the vari- ous depths of the MLSB prior to our collecting samples. The greater the sample depth, the longer the material has been in the tailings pond, which has received inputs since 1978. The ages of the fine tailing would affect how much of the fer- mentable material had been converted to methane prior to our sampling. The methane yields generated in the labora- tory may only represent a fraction of the methane actually produced by a given volume of freshly extracted fine tailings which are still being added to the MLSB. However, our data demonstrate that methanogenesis in the fine tailings appears

to be is a finite process, slowing when usable substrate is de-

pleted.

Sulfate addition to fine tailings samples It is well established that methanogenesis is inhibited by the presence of other terminal electron acceptors such as O 2 , nitrate, Fe(III), and sulfate (D’Anglo and Reddy 1999; Klueber and Conrad 1998; Londry and Suflita 1999). Holowenko (2000) demonstrated that methane production from fine tailings samples was severely inhibited over a 300-

d incubation in microcosms that contained 1800 mg/L ni-

trate. However, nitrate addition was deemed impractical for the control of methanogenesis in the oil sands fine tailings.

As shown in Fig. 2, the numbers of methanogens were lower where the sulfate concentrations in the MLSB were high. Thus, it was presumed that the presence of elevated sulfate concentrations would control the activities of the methanogens. Microcosm studies were used to test the abil- ity of sulfate to inhibit methanogenesis. The data from ex- periments with 8000 mg/L sulfate added to five sets of microcosms with different fine tailings samples are summa- rized in Table 3. Methanogenesis was not stopped immedi- ately by the addition of sulfate. The time before methane production in the sulfate-amended microcosms was signifi-

cantly less (P < 0.05) than the methane production in the un- amended microcosms ranged from 7–45 d (Table 3). This type of delay has been reported by D’Anglo and Reddy (1999) who observed a 13-d lag before inhibition of methane production in a North Carolina Belhaven soil slurry amended with sulfate. The inhibition of methanogenesis by sulfate arises from the competition between SRB and methanogens for sub- strates such as H 2 and acetate. SRB are better competitors because they obtain more energy from the substrates than the methanogens (Holland et al. 1987; Zinder 1993). Thus, one would predict that the larger the initial population of SRB, the sooner inhibition would occur. This trend was ob- served. Inhibition occurred quickly (7–18 d) in samples with large SRB populations (MPN values >10 6 /g) and took longer (45 d) for samples with smaller SRB populations (MPN val- ues 10 4 –10 5 /g) (Table 3). Consumption of sulfate in all the microcosms (Table 3) indicated that SRB were active, and the reduced methane concentrations in the sulfate-amended microcosms indicated that the SRB out-competed the methanogens for substrates. Sulfate was added as sodium sulfate, resulting in a sodium concentration of 2900 mg/L, which is below the 4800 mg/L sodium tolerance limit documented for methanogens (Kugelman and Chin 1971). Cation interference was not likely a factor contributing to inhibition of methanogenesis. Although sulfate was shown to efficiently inhibit methano- genesis in the fine tailings, its use to prevent methanogenesis in the MLSB is not feasible. For inhibition to be successful, there would have to be sufficient mixing of sulfate through- out the MLSB and considering the huge volume of the tail- ings in the MLSB this is not plausible. Furthermore, the amount of sulfate that would need to be added to the pond would be enormous and would have significant financial costs associated with it. Sulfate is being used in a new technology developed to minimize the large volume of tailings waste generated. Com- posite tailings (CT) is a process in which gypsum (mainly Ca 2 SO 4 ·H 2 O) is added to fine and coarse tailings. Cation ex- change between Ca 2+ and Na + in the clays occurs, which causes aggregation and increases slurry viscosity (List and Lord 1997). Using the CT process, the tailings dewater rela- tively quickly, which further consolidates the tailings (List and Lord 1997). Initially, the released water and the pore

© 2000 NRC Canada

936

water contains high concentrations of sulfate (1300– 8000 mg/L), which is derived from the gypsum. Although the CT process was designed as a method of quickly reduc- ing the volume of tailings, the added sulfate should help control methane production. Studies of the microbial pro- cesses in the CT material are underway in our laboratories.

Conclusions Methanogenesis in the fine tailings zones of the oil sands settling basins is proceeding and is coming from biological activity. At present 2%–5% of the fine tailings volume is present as methane. A portion of this is escaping to the at- mosphere with present daily fluxes in the most active areas exceeding 10 g CH 4 /m 2 . Work is continuing to assess the significance of this biogenic gas production on both the per- formance of these active settling basins, and the viability of lake ecosystems in wet landscape reclamation approaches. The work reported here presents a survey of the methanogen and SRB populations in the fine tailings. Methanogens were present in all samples collected (10 5 –10 6 MPN/g), as were SRB (10 4 –10 5 MPN/g). There was no inhibition of methane production by an active methanogenic sample when mixed with a non-active sample. The MLSB fine tailings samples released 0.10–0.25 mL CH 4 /mL fine tailings, which ac- counts for less than 1% of the total carbon in the MLSB. Sulfate inhibited methanogenesis by stimulating bacterial competition for available substrate.

Acknowledgements

Funding for this project was provided by Syncrude Can- ada Ltd., and the Natural Sciences and Engineering Re- search Council of Canada. Technical assistance by D. Coy, B. Fung, M. Chandra, R. Eckford, and B. Rolselth is ac- knowledged.

References

Alexander, M. 1999. Biodegradation and bioremediation. 2nd ed. Academic Press, San Diego, Calif. pp. 409–433. ASTM 1990. Standard test method for analysis of gases dissolved in electrical insulating oil by gas chromatography. Annual Book of ASTM Standards. Method D-3612. pp. 375–379. Atlas, R.M., and Bartha, R. 1993. Microbial ecology: Fundamen- tals and applications. 3rd ed. The Benjamin/Cummings Pub- lishing Company, Inc. Redwood City, Calif. p. 219. Boerger, H., MacKinnon, M., Van Meer, T., and Verbeek, A. 1992. Wet landscape option for reclamation of oil sands fine tails. In Proceedings: Second International Conference on Environmental Issues and Management of Waste in Energy and Minerals Produc- tion. Edited by R.K. Singhal, A.K. Mehrotra, K. Fytas, and J.-L. Collins. A.A. Balkema. Rotterdam, Netherlands. pp. 1249–1261. Boyd, M.L., and Montgomery, D.S. 1962. Structural group analysis of the asphaltenes and resins components of the Athabasca bitu- men. Fuel, 41: 335–350. Butlin, K.R., Adams, M.E., and Thomas, M. 1949. The isolation and cultivation of sulfate-reducing bacteria. J. Gen. Microbiol. 3: 46–49. Camp, F.W. 1973. The Tar Sands of Alberta, Canada. 3rd ed. Cameron Engineers Inc. Denver, Co.

Can. J. Microbiol. Vol. 46, 2000

Clark, K.A., and Pasternak, D.S. 1932. Hot water separation of bi- tumen from Alberta bituminous sand. Ind. Eng. Chem. 24:

1410–1416.

Cochran, W.G. 1950. Estimation of bacterial densities by means of the most probable number. Biometrics, 6: 105–116. Conrad, R., and Wetter, B. 1990. Influence of temperature on energetics of hydrogen metabolism in homoacetogenic, methanogenic, and other anaerobic bacteria. Arch. Microbiol. 155: 94–98. D’Anglo, E.M., and Reddy, K.R. 1999. Regulators of heterotrophic microbial potentials in wetland soils. Soil Biol. Biochem. 31:

815–830.

Dunnett, C.W. 1955. A multiple comparison procedure for compar- ing several treatments with a control. J. Am. Statist. Assoc. 50:

1096–1121.

Fedorak, P.M., and Hrudey, S.E. 1983. A simple apparatus for measuring gas production in methanogenic cultures in serum bottles. Environ. Technol. Lett. 4: 425–432. Fedorak, P.M., and Hrudey, S.E. 1984. The effects of phenol and some alkyl phenolics on batch anaerobic methanogenesis. Water Res. 18: 361–367. Fedorak, P.M., Semple, K.M., and Westlake, D.W.S. 1987. A sta- tistical comparison of two culturing methods for enumerating sulfate-reducing bacteria. J. Microbiol. Methods, 7: 19–27. Foght, J.M., Fedorak, P.M., and Westlake, D.W.S. 1985. Microbial content and metabolic activities in Syncrude tailings pond. AOSTRA J. Res. 1: 139–146. Gulley, J.R., and MacKinnon, M. 1993. Fine tails reclamation utili- zation using a wet landscape approach. AB Chamber Resour., AOSTRA, Energy, Mines & Resources Canada, Oil Sands: Our Petroleum Future Conference, Edmonton, Alta. April 4–7, 1993. Paper F23. Herman, D.C., Fedorak, P.M., MacKinnon, M., and Costerton, J.W. 1994. Biodegradation of naphthenic acids by microbial popula- tions indigenous to oil sands tailings. Can. J. Microbiol. 40:

467–477.

Holland, K.T., Knapp, J.S., and Shoesmith, J.G. 1987. Anaerobic Bacteria. Chapman & Hall, New York. pp. 130–133. Holowenko, F.M. 2000. Methanogenesis and fine tailings waste from oil sand extraction: A microcosm-based laboratory exami- nation. M.Sc. Thesis. University of Alberta, Edmonton, Alta. Hunt, J.M. 1979. Petroleum geochemistry and geology. W.H. Free- man and Company, San Francisco, Calif. p. 390. Jain, M.K., Zeikus, J.G., and Bhatnagar, L. 1991. Methanogens. In Anaerobic microbiology: A practical approach. Edited by P.N. Levett. Oxford University Press, New York. pp. 223–235. Jivraj, M.N., MacKinnon, M., and Fung, B. 1995. Naphthenic ac- ids extraction and quantitative analyses with FT-IR spectros- copy. Syncrude Canada Ltd. Edmonton, Alta. Klueber, H.D, and Conrad, R. 1998. Effects of nitrate, nitrite, NO and N 2 on methanogenesis and other redox processes in anoxic rice field soil. FEMS Microbiol. Ecol. 25: 301–318. Kugelman, I.J., and Chin, K.K. 1971. Toxicity, synergism, and an- tagonism in anaerobic waste treatment processes. In Anaerobic biological treatment processes. Edited by F.G. Pohland ACS Ad- vances in Chemistry Series. Washington, D.C. 105: 55–90. List, B.R., and Lord, E.R.F. 1997. Syncrude’s tailings management practices from research to implementation. CIM Bulletin, 90:

39–44.

Londry, K.L., and Suflita, J.M. 1999. Use of nitrate to control sul- fide generation by sulfate-reducing bacteria associated with oily waste. J. Ind. Microbiol. Biotechnol. 22: 582–589.

© 2000 NRC Canada

Holowenko et al.

MacKinnon, M. 1989. Development of the tailings pond at Syncrude’s oil sands plant: 1978–1987. AOSTRA J. Res. 5: 109–133. MacKinnon, M., and Boerger, H. 1986. Description of two treat- ment methods for detoxifying oil sands tailings pond water. Wa- ter Pollut. Res. J. Can. 21: 496–512. MacLean, D. 1998. Syncrude facts. Syncrude Canada Ltd. Govern- ment and Public Affairs Department. Fort McMurray, Alta. Mikula, R.K., Kasperski, K.L., Burns, R., and MacKinnon, M. 1996. The nature and fate of oil sands fine tailings. In Suspensions:

Fundamentals and applications in the petroleum industry. Edited by L.L. Schramm. Washington, D.C., ACS. 251: 677–723. Miller, T.L. 1991. Biogenic sources of methane. In Microbial pro- duction and consumption of greenhouse gases: Methane, nitro- gen oxides, and halomethanes. Editied by J.E. Rogers and W.B. Whitman. ASM Press. Washington, D.C. pp. 175–199. Miller, T.L., and Wolin, M.J. 1974. A serum bottle modification of the Hungate technique for cultivating obligate anaerobes. Appl. Microbiol. 27: 985–987. Raskin, L., Rittmann, B., and Stahl. D. 1996. Competition and co- existence of sulfate-reducing and methanogenic populations in anaerobic biofilms. Appl. Environ. Microbiol. 62: 3847–3857. Schramm, L.L., Stasiuk, E.N., and MacKinnon, M. 2000. Surfac- tants in Athabasca oil sands slurry condition, flotation recovery, and tailings processes. In Surfactants, fundamentals and applica- tions in the petroleum industry. Edited by L.L. Schramm. Cam- bridge University Press, U.K. pp. 365–430. Sinke, A.J.C., Cottaar, F.H.M., Buis, K., and Keizer, P. 1992. Methane oxidation by methanotrophs and its effects on phos- phate flux over the sediment-water interface in a eutrophic lake. Microbiol. Ecol. 24: 259–269. Skoog, D.A., and West, D.M. 1976. Fundamentals of Analytical Chem- istry. 3rd ed. Holt, Rinehart and Winston. New York. pp. 71–75.

937

Sobolewski, A. 1992. The microbial characteristics of oil sands tailings sludge. Consultant’s report submitted to Alberta Oil

Sands Technology Research Authority. Calgary, Alberta. Sobolewski, A. 1997. Microbial distributions in process-affected aquatic ecosystems. Consultant’s report submitted to Syncrude Research Ltd., November 1997. Standard methods for the examination of water and wastewater.

1985. 16th Edition. American Public Health Association. New

York, N.Y. pp. 880–882.

Steel, R.G.D., and Torrie, J.H. 1980. Principles and Procedures of Statistics: A biometrical approach. 2nd ed. McGraw-Hill Book Company. New York. pp. 60–64. Syncrude Canada Ltd. 1995. Syncrude Analytical Methods (SAM) Manual. 4th ed. Syncrude Canada Ltd. Research Department. Edmonton, Alta. Updegraff, K., Bridgham, S.D., Pastor, J., and Weishampel. P.

1998. Hysteresis in the temperature response of carbon dioxide

and methane production in peat soils. Biogeochem. Dord. 43:

253–272.

Wang, Z., Zeng, D., and Patrick, W.H., Jr. 1996. Methane emissions from natural wetlands. Environ. Monitor. Assess. 42: 143–161. Winfrey, M.R., and Zeikus, J.G. 1977. Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwa-

ter sediments. Appl. Environ. Microbiol. 33: 275–281. Zeikus, J.G., and Winfrey, M.R. 1976. Temperature limitation of methanogenesis in aquatic sediments. Appl. Environ. Microbiol. 31: 99–107. Zinder, S.H. 1993. Physiological ecology of methanogens. In Methanogenesis. Edited by J.G. Ferry. Champman & Hall, Inc. New York. pp. 128–206.

© 2000 NRC Canada