UNIVERSITY OF CALGARY Sedimentation of oil sands tailings...

UNIVERSITY OF CALGARY

Sedimentation of oil sands tailings via microbial treatments

by

Jeremy James Robert Kooyman

A THESIS SUBMITTED TO THE SCHULICH SCHOOL OF ENGINEERING

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

BIOMEDICAL ENGINEERING SPECIALISATION

DEPARTMENT OF MECHANICAL & MANUFACTURING ENGINEERING

CALGARY, ALBERTA, CANADA

APRIL, 2011

© Jeremy Kooyman 2011

1

UNIVERSITY OF CALGARY SCHULICH SCHOOL OF ENGINEERING

SPECIALISATION IN BIOMEDICAL ENGINEERING

The undersigned certify to have read, and recommend to the Schulich School of Engineering for acceptance, a thesis entitled Sedimentation of oil sands tailings via microbial treatments submitted by Jeremy Kooyman in partial fulfilment of the requirements for the Specialisation in Biomedical Engineering.

________________________________________________ Supervisor, Dr-Ing. Robert Martinuzzi. Mechanical and Manufacturing Engineering

________________________________________________

External Examiner, Dr. David Wood. Mechanical and Manufacturing Engineering

_______________________ Date

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Abstract:

Bioremediation, the use of biological processes to overcome environmental problems, is

being explored as a possible method addressing the problems associated with tailings ponds

such as toxicity and process water consumption. Preliminary experiments that treated

mature fine tailings with BioTiger, a multispecies consortium with established

bioremediation abilities, yielded results that suggested a large potential for reducing the

environmental impact and energy requirements of oil sands development.

This research project examined the individual effects of the BioTiger species on the

sedimentation of mature fine tailings and found no notable difference between the BioTiger

species and experimental controls. However, a significant reduction in optical density was

observed in mixed samples treated with BioTiger, suggesting that oil sands operators could

use BioTiger to achieve the fines reduction goals outlined by the ERCB in Directive 074.

Future experiments will need to be conducted to determine the large-scale feasibility of this

process.

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Acknowledgments:

First and foremost, I would like to thank Dr. Martinuzzi for granting me the opportunity to

conduct several months of research in his laboratory. The experience has brought me closer

to achieving my educational goals.

I would also like to thank Dr. Kostenko for her patience and guidance throughout the

course of my research. Her expertise in many things microbial proved to be very valuable

while performing experiments.

Additionally, I would like to thank Mehdi Salek for his assistance with the unfortunately

omitted CFD portion of my research. I cannot fathom preparing a PhD defence while still

finding time to assist an undergraduate student.

Finally, I would like to thank everyone in the Biofilm Engineering Research Group for

tolerating my presence in the lab, and for the use of their facility.

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Table of Contents:

Abstract: ................................................................................................................................. 2

Acknowledgments: ................................................................................................................. 3

Table of Contents: .................................................................................................................. 4

List of Figures ......................................................................................................................... 8

List of Tables ........................................................................................................................ 11

List of Acronyms .................................................................................................................. 12

1.0 Significance .................................................................................................................... 13

2.0 Background ..................................................................................................................... 14

2.1 Overview .................................................................................................................... 14

2.2 Environmental Problems ............................................................................................ 15

2.2.1 Toxicity ................................................................................................................ 15

2.2.2 Water Consumption ............................................................................................. 16

2.3 Reclamation of Tailings Ponds ................................................................................... 16

2.3.1 Dry Landscape Approach .................................................................................... 16

2.3.2 Wet Landscape Approach .................................................................................... 17

2.4 Primary Options for MFTs Treatment ........................................................................ 18

2.4.1 Composite Tailings (CT) ..................................................................................... 18

5

2.4.2 Freeze-Thaw Dewatering of MFTs ..................................................................... 18

2.5 Microbial Activity in Oil Sands Tailings Ponds ......................................................... 19

2.6 Biofilms ...................................................................................................................... 20

3.0 Overview of Chapters ..................................................................................................... 21

4.0 Hypothesis ...................................................................................................................... 21

5.0 Specific Aims ................................................................................................................. 21

5.1 Evaluation of sedimentation capabilities of BioTiger bacteria .................................. 21

5.2 Evaluation of BioTiger bioremediation capability via alternative parameters ........... 22

6.0 Materials and Methods ................................................................................................... 22

6.1 MFT Samples ............................................................................................................. 22

6.2 Microbial Techniques ................................................................................................. 22

6.3 Sedimentation Measurements and Calculation........................................................... 23

6.4 BioTiger Sedimentation Experiments ........................................................................ 24

6.4.1 Materials .............................................................................................................. 24

6.4.2 Methods – General .............................................................................................. 25

6.4.3 Methods – 10 Day Experiments ......................................................................... 26

6.4.4 Methods – 3 Day Experiments ............................................................................ 27

6.4.5 Methods – 4 Hour Experiments ........................................................................... 29

6.4.6 Methods – Optical Density Analysis ................................................................... 30

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6.4.7 Methods – Full Spectrum Absorbance Analysis ................................................. 30

6.5 Shell Tailings Experiments ......................................................................................... 31

6.5.1 Materials .............................................................................................................. 31

6.5.2 Methods ............................................................................................................... 32

7.0 Results ............................................................................................................................ 33

7.1 BioTiger Sedimentation Experiments ........................................................................ 34

7.1.1 10 Day Sedimentation + Centrifugation Experiment .......................................... 34

7.1.2 4 Hour + 3 Day Control Sedimentation +/- Antibiotics Experiment ................... 37


7.3 Optical Density Analysis Results ............................................................................... 49

7.4 Full Spectrum Absorbance Analysis Results ............................................................. 56

8.0 Discussion ....................................................................................................................... 58

8.1 BioTiger Experiments ................................................................................................ 58

8.1.1 10 Day Sedimentation + Centrifugation Experiment .......................................... 58

8.1.2 4 Hour + 3 Day Control Sedimentation +/- Antibiotics Experiment ................... 59


8.3 UV-Vis Spectrometer Experiments ............................................................................ 61

8.3.1 Optical Density Analysis ..................................................................................... 62

8.3.2 Full Spectrum Analysis ....................................................................................... 63

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9.0 Conclusions + Future Work ............................................................................................ 64

10.0 References .................................................................................................................... 67

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List of Figures

Figure 1: Visual explaining the two parameters measured for sedimentation. .................... 23

Figure 2: Results of a 10 day MFT sedimentation experiment comparing the effects of the

individual strains of BioTiger bacteria against the water and media controls.Mixed samples.

.............................................................................................................................................. 34


individual strains of BioTiger bacteria against the water and media controls.Capped

samples. ................................................................................................................................ 35

Figure 4: Results of a 4 hour MFT sedimentation experiment comparing the effects of MFT

treated with water and R2A media against plain tailings.. ................................................... 37

Figure 5: Results of a 3 day MFT sedimentation experiment comparing the effects of

temperature, treatment with R2A media or water, and the presence of antibiotics, against

plain tailings. ........................................................................................................................ 38

Figure 6: The samples that experienced the greatest levels of sedimentation in Figure 5 are

re-plotted here, with standard deviation error bars. .............................................................. 39

Figure 7: Results of a 3 day MFT sedimentation experiment comparing the effects of

temperature, treatment with R2A media or water, and the presence of antibiotics, against

plain tailings.. ....................................................................................................................... 40

Figure 8: The samples that experienced the greatest levels of sedimentation in Figure 7 are

re-plotted here, with standard deviation error bars. .............................................................. 40

F ur 9: Tr p t 24 C Media-Cap + Antibiotics samples from Figure 7 and 8 illustrating

extremely high levels of sedimentation after centrifugation. ............................................... 41

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F ur 10: 36 C Water Control-Mix samples from Figure 5 showing bitumen smearing at

the free surface...................................................................................................................... 42


individual strains of native Shell MFT bacteria against the water and media controls.Mixed

samples. ................................................................................................................................ 44

Figure 12: The samples that experienced the greatest levels of sedimentation in Figure 11

are re-plotted here, with standard deviation error bars. ........................................................ 45


individual strains of native Shell tailings bacteria against the water and media controls.

Capped samples. ................................................................................................................... 46

Figure 14: The samples that experienced the greatest levels of sedimentation in Figure 13

are re-plotted here, with standard deviation error bars. ........................................................ 47

Figure 15: Optical density analysis of mixed samples vs. water control from Section 7.1.1..

.............................................................................................................................................. 49

Figure 16: Optical density analysis of mixed samples vs. media control from Section 7.1.1..

.............................................................................................................................................. 50

Figure 17: Optical density analysis of capped samples vs. water control from Section 7.1.1..

.............................................................................................................................................. 52

Figure 18: Optical density analysis of capped samples vs. media control from Section

7.1.1.. .................................................................................................................................... 53

Figure 19: Results of a full spectrum absorbance analysis on mixed samples detailed in

Section 7.1.1. ........................................................................................................................ 56

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Figure 20: Results of a full spectrum analysis on capped samples detailed in Section 7.1.1.

.............................................................................................................................................. 57

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List of Tables

Table 1: Summary of data presented in Figure 4.................................................................. 37

Table 2: Summary of 3Day sedimentation data presented in Figure 5. + Sign indicates

presence of antibiotics. ......................................................................................................... 39

Table 3: Summary of 3Day sedimentation data shown in Figure 7. + Sign indicates

presence of antibiotics. ......................................................................................................... 41

Table 4: Summary of 10 Day sedimentation data presented in Figure 11. .......................... 45

Table 5: Summary of 10 Day sedimentation data presented in Figure 13. .......................... 47

Table 6: Optical Density summary of data presented in Figures 15 and 16. ........................ 51

Table 7: Summary of statistical analysis of data presented in Table 6................................. 51

Table 8: Optical density summary of data presented in Figures 17 and 18. ........................ 54

Table 9: Summary of statistical analysis of data presented in Table 8................................. 54

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List of Acronyms

bbl: Oil barrel, 42 US gallons (158.9873 L)

CAPP: Canadian Association of Petroleum Producers

CT: Composite Tailings

EPS: Extracellular polymeric substance

ERCB: Energy Resources Conservation Board

FTFC: Fine Tailings Fundamentals Consortium

GHG: Greenhouse gas(es)

MFT: Mature fine tailings

MLSB: Mildred Lake Settling Basin

NA: Naphthenic acid

PTAC: Petroleum Technology Alliance Canada

SD: Standard deviation

SRNL: Savannah River National Laboratory

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1.0 Significance

Tailings ponds, a toxic by-produ t of A b rt ’s oil sands mining developments, are a

mixture of sand, clay, water, and an extremely viscous hydrocarbon called bitumen. At

present, these ponds occupy more than 130 km2 of Alberta. Studies indicate that oil sands

developments are releasing toxic elements into the Athabasca River and point to tailings

ponds as a contributing factor (Kelly et al., 2010). With bitumen production projected to

double by 2020 (CAPP 2009), major changes to current extraction and tailings remediation

techniques need to be developed and implemented in order to prevent an environmental

disaster.

Recent research with bacterial biofilms has shown that residual bitumen separation and clay

settling properties can be significantly enhanced when hydrodynamically conditioned.

These results suggest a large potential for reducing the energy requirements and

environmental impact of the oil sands.

This research project seeks to distinguish between the influence of viscosity and the

influence of shear rate on the formation, response, and structure of biofilms. Decoupling

these two influences will enable future research to maximize the beneficial effects of

biofilms for bioremediation applications through specific hydrodynamic conditioning.

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2.0 Background

2.1 Overview

Oil sands are a type of unconventional petroleum deposit consisting of a mixture of sand,

water, and a heavy, viscous hydrocarbon called bitumen, which can be converted to oil.

While these deposits are found across the world, the largest deposits are found in Alberta

and Venezuela (Tenenbaum 2009).

These deposits are found in four major areas in Alberta: Athabasca (the largest), Wabasca,

Peace River, and Cold Lake. It is estimated that there are 175 billion barrels of recoverable

oil in these reserves, ranking Alberta second behind Saudi Arabia for recoverable oil

(Tenenbaum 2009).

Shallow deposits, which are thought to contain 8-20% of A b rt ’s tot o s nds, r

surface mined using some of the largest trucks and shovels in the world. The oil sands

taken from these open pit mines are subjected to variations of the Clark Hot Water

Extraction Process (Clarke 1980), which consists of three major steps:

1. Oil sand is agitated in hot water with a small amount of caustic (often NaOH) to

maintain the pH in the range of 8.0-8.5.

2. Sand grains that have settled on the bottom of the settling tank are removed and the

oil froth that floats to the surface is recovered by skimming. Fine particulate matter,

dominated by clay minerals, remain in what is called the middling stream.

3. The stream is then processed, which provides incremental recovery of suspended

bitumen through conventional froth floatation.

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The slurry remaining after the Clark Hot Water Extraction Process is a mixture of sand, fine

clay particles, water, and residual bitumen. When these tailings are sent to a pond, the large

sand particles fall out of suspension quickly, forming a beach, while the remaining fine

tailings settle to the bottom of the pond. Over a few years, these fine tailings will settle to a

30 wt% solid (86% by volume water), stable slurry structure called mature fine tails (MFT).

The MFTs will remain in this stable slurry for decades due to its slow consolidation rate

(Chalaturnyk et al., 2002).

2.2 Environmental Problems

MFTs are a growing problem, being produced at a rate of 0.25 m3/barrel of bitumen.

Current estimates place the volume of MFTs at ~650 000 000 m3 (based on 2.6 billion bbl

of bitumen mined s n 1968) (PTAC 2007). Th A b rt ov rnm nt st t d th t ―Tox ty

and the loss of bitumen and process water are among the most serious problems associated

with the build up of these y t n s.‖ (Kot y r t ., 1995).

2.2.1 Toxicity

A ord n to th r port ―11 M on L tr s D y: Th T r S nds’ L k n L y‖ from

the activist group Environmental Defence, with consultation from the Pembina Institute, 11

million litres of toxic process-affected water escape from tailings ponds each day. The

principal acute toxicant in the affected water is a group of polycyclic aromatic

hydrocarbons known as naphthenic acids (NAs). Process affected waters can contain 20-

120 mg/L naphthenic acid; these acids are the most toxic components to aquatic organisms

(Price 2008). Studies have explicitly stated the toxic effects of naphthenic acids on fish,

insect, and phytoplankton communities (Kelly et al., 2010). Although some process-

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affected waters are not acutely toxic to fish, there are documented sub lethal effects for fish

exposed to process-affected waters.

2.2.2 Water Consumption

Taking into account current water recycling practices, approximately three barrels of water

are used in the production of one barrel of bitumen. Process water is often trapped within

the MFTs or declines in water quality, due to scaling, fouling, increased corrosivity, or

interference with extraction chemistry. This can lead to bitumen recovery becoming

impossible with the existing water and fresh water must be used instead.

Many oil sands developments in north-eastern Alberta tap into the Athabasca River for

their fresh water requirements. It is thought that unless future water demand is reduced, the

o s nds d v opm nts ―w r qu r ~45 m3s

-1 of w t r supp y by 2020‖, n r y h f of th

Ath b s R v r’s f ow dur n th w nt r months (S h nd r & Don hu , 2006).

2.3 Reclamation of Tailings Ponds

Current bioremediation techniques used for tailing ponds reclamation require the alteration

of the site environment in ways that encourage manipulation and fast metabolism of the

microorganisms that can degrade/detoxify the environmental contaminants. These

techniques take one of two paths: dry and wet landscapes. (Yang 2009)

2.3.1 Dry Landscape Approach

Dry landscape approaches aim to reduce the water content of the fine tails, forming a solid

deposit which is capable of being reclaimed as a land surface or wetland. Current

techniques include reliance evaporation, freeze-thaw, and the addition of calcium sulphate

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to extraction tailings to form consolidated tailings. Dry landscape approaches typically fail

to deal with the NA contaminated water that may leech from the reclamation area. (FTFC

1995)

2.3.2 Wet Landscape Approach

Wet landscape approaches typically take one of two forms: water capping or constructed

wetlands.

The water capping technique was originally used in the mining industry to prevent oxygen

interaction with toxic mining tailings. When applied to oil sands developments, the tailings

are transferred into an abandoned, mined-out pit, over which a layer of water is placed to

establish a cap over the tailings base. Computer simulations have estimated that two years

must elapse before concentrations of NAs and other organic compounds in the surface

water will be reduced below levels acutely toxic to aquatic biota. Given that the end goal is

to r s th ―r m d‖ w t r b k nto th nv ronm nt, th re is a need for increased

efficiency and biodegradation rate of the NAs. (Quagraine et al., 2005)

Constructed wetlands, after many years of use in municipal settings, are gaining acceptance

for use in the petroleum industry because of their ability to treat a wide range of pollutants.

Although a feasibility report on the Suncor constructed wetlands in 1991 concluded that

they had the potential to be a suitable low-cost, passive, and self-sustaining treatment

approach, preliminary results in 1992-1993 indicated that a very long hydraulic retention

time would be required and future results would be negatively impacted by the environment

during the winter months. (Allen 2008)

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2.4 Primary Options for MFTs Treatment

Over the past two decades, a large volume of work has been carried out to find effective

methods to densify the MFTs, reducing their volume and water retention (Guo 2009). The

two major methods in current oil sands tailings practice are explained below.

2.4.1 Composite Tailings (CT)

Composite Tailings are a mixture of MFTs, tailings sand, and gypsum (Syncrude Canada

Ltd. 2010). The introduction of the sand particles into the MFTs applies an internal stress to

the structure, causing a significant increase in the densification rate of the tailings (FTFC

1995). Gypsum acts as a chemical coagulant, causing the dewatering rate of the MFTs to

rapidly increase.

Further, the addition of chemical additives in the CT process can increase the salinity,

corrosion potential, and affect bitumen recovery at the plant (Mackinnon et al., 2000).

2.4.2 Freeze-Thaw Dewatering of MFTs

Recognized for several decades, the dewatering of slurries originated with research into

segregated ice-soil structures occurring in natural permafrost soils (Mackay 1974). When

water-saturated, fine-grained slurries are subjected to sub-freezing temperatures, negative

pore pressures develop between ice and unfrozen liquid pore water, causing water to

migrate to growing ice crystals, ultimately resulting in segregated reticulate ice structures

(Dawson et al., 1999).

Laboratory tests suggest that Freeze-thaw is an effective method to accelerate the

dewatering rate of MFTs. It has been shown that freeze-thaw increased the solids content of

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MFTs from 35% to approximately 44-48% (FTFC 1995). Chemically pre-treating the

MFTs prior to freeze-thaw has been shown to significantly improve the dewatering rate of

the MFTs, with combined treatment processes increasing the solids content from 35% to

70% (FTFC 1995).

Despite of these beneficial outcomes, freeze-thaw is not necessarily feasible on an

industrial scale because of the large surface area required to treat the large volume of MFTs

accumulated to date (Guo 2009)

2.5 Microbial Activity in Oil Sands Tailings Ponds

In 1985, Foght et al. examined the microbial content of tailings samples from the Mildred

Lake Settling Basin (MLSB), the primary tailings pond for Syncrude Canada Ltd. Methane

production was observed in samples that were supplemented with acetate or glucose and

n ub t d t 37 C, however no meth n w s d t t d n th s mp s n ub t d t 15 C, the

native temperature of the tailings. Coincidentally, in 1999 the MLSB was producing 12 g

CH4 m-2

d-1

over 40-60% of the 12 km2 pond surface area. The microbial metabolism of the

resident hydrocarbons was causing a total daily emission of 108 L of greenhouse gases

(GHG) (Holowenko et al., 2000).

Despite the undesirable GHG production, microbial activities have been shown to be

beneficial by catalyzing hydrocarbon biodegradation (Quagraine et al., 2005) (Siddique et

al., 2007) and improving tailings densification (Fedorak et al., 2003).

With the densification of MFTs estimated to take 150 years (Eckert et al., 1996), research

suggesting that methanogenic MFTs experience an accelerated densification rate when

20

compared with non-methanogenic MFTs (Fedorak et al., 2003) highlights the importance of

bacteria-based remediation techniques.

2.6 Biofilms

Bacterial biofilms are sessile colonies of synergetic microorganisms imbedded in self

generated extracellular polymeric substances (EPS), which contain chemically active

molecules including surfactants. The biofilm environment allows the expression of specific

phenotypes, which can be conditioned by the fluid environment. Hydrodynamic forces

have been shown to alter the structure of biofilms and consequently the response of bacteria

to environmental challenges, for example increasing the tolerance to antibiotics in higher

shear regions (Kostenko et al., 2010). Increased viscosity has been shown to reduce the

susceptibility of Staphylococcus aureus biofilms in two ways: (i) modified transport; and

(ii) stimulation of metabolic stress response resulting in increased EPS production and

tolerance (Kostenko et al., 2007).

Preliminary experiments have shown that using BioTiger, a multispecies consortium with

established bioremediation abilities, to treat oil sand tailings samples results in a 5-fold

increase in bitumen recovery over 24 hours when grown in biofilms and agitated as

compared to the traditional (planktonic) methods under similar flow conditions. (Kostenko

V. 2010)

These results suggest that the integration of biofilms into remediation technology will

reduce the energy requirements and environmental impact of oil sands developments.

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3.0 Overview of Chapters

With an overview of the significance and current state of bioremediation and how it

pertains to oil sands tailings detailed in Chapter 1 and 2, Chapter 4 introduces the

governing hypothesis of this research project.

Chapter 5 details the specific aims of this research project, expanding how the hypothesis

will be tested and explored.

Chapter 6 describes the materials and techniques used in the laboratory experiments.

Test results, including densification rates and bacteria colony counts, are presented in

Chapter 7, and discussed in Chapter 8.

Concluding remarks and suggestions for oil sands tailings bioremediation and future

research are presented in Chapter 9, with cited works appearing in Chapter 10.

4.0 Hypothesis

The hypothesis that is addressed in this paper is that the established bioremediation

capabilities of the BioTiger multispecies consortium can be adapted for the purpose of

increasing the rates of sedimentation experienced by MFTs.

5.0 Specific Aims

5.1 Evaluation of sedimentation capabilities of BioTiger bacteria

Conduct sedimentation experiments with different strains of BioTiger bacteria

Determine the effectiveness of each strain at 24 C nd 36 C incubation temperatures

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5.2 Evaluation of BioTiger bioremediation capability via alternative

parameters

Evaluate optical density of released water from samples treated with BioTiger

Determine the concentration of bituminous materials in released water

6.0 Materials and Methods

6.1 MFT Samples

The MFT samples used in this study were kindly provided by Shell Canada through an

informal agreement. All samples provided were stored in a sealed container at room

temperature.

6.2 Microbial Techniques

Two groups of bacteria were used for this experiment. The same bacteria preparation

technique was used for all procedures. Dr. Kostenko provided guidance regarding this

technique.

t r str ns w r r mov d from stor t 4 C and seeded in triplicate in individual

conical-bottomed tubes containing 20mL R2A media. Tubes were placed on a rocker at

room temperature and left to grow for 24 hours prior to experimentation.

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6.3 Sedimentation Measurements and Calculation

Figure 1: Two measurements were used to calculate the sedimentation of a sample. The first (H) was the distance

from the 10mL line on the tube to the free surface. The second (h) was the distance from the 10mL line on the tube

to the tailings/liquid interface.

Two measurements were taken for each sample with digital callipers, using the 10mL line

on the tube as a reference point.

The first measurement, H, was the distance from the 10mL line to the surface of the liquid.

The second measurement, h, was the distance from the 10mL line to the tailings/liquid

interface.

Sedimentation was calculated as a percentage of the total volume, with the two

measurements being converted from mm to mL, and compensated with the remaining

10mL of tube volume.

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6.4 BioTiger Sedimentation Experiments

6.4.1 Materials

BioTiger Multispecies Bacteria Consortium

o BPC

o BPB

o BP20

o BPE

o BPH

o BPK HG

o KN1

o KN3

o BPI

o BPJ

o BPF

o BPL

R2A Media (Per 500mL)

o 0.25g Casein Hydrolysate

o 0.25g Glucose

25

o 0.25g Peptone

o 0.25g Yeast Extract

o 0.15g K2HPO4

o 0.025g MgSO4

o 0.25g NH4Cl

o 0.025g CaCl2

o 0.15g Sodium Pyruvate

o 0.25g Starch (Soluble)

o pH 7.0 +/- 0.2

o Autoclave Liquid 20 cycle

Oil Sands Tailings

Antibiotics

o 2-methyl-5-nitro-1-imidazoleethanol

o Chloramphenicol

o Spectinomycin dihydrochloride pentahydrate

50mL Conical-bottomed tubes - VWR

Measurement Callipers

25ml Pipettes

6.4.2 Methods – General

MFT stock was stirred with a wooden stick to ensure that the consistency of the tailings did

not change as a function of depth and to avoid changing the properties of the tailings over

the course of several experiments. It was established that the tailings were suitably mixed

26

when one could pull tailings from the bottom of the pail and have them look the same as

those on top.

Three basic sets of tubes were prepared, from which the experimental controls and bacteria

samples would be made.

First, tubes treated with water were prepared by taking mixed MFT, adding 10% dH2O, and

mixing in a similar manner as described above. 20mL aliquots were placed in 50mL

centrifuge tubes and set aside for future use.

Second, tubes treated with media were prepared by taking mixed MFT, adding 10% 10X

R2A media, and mixing in a similar manner as described above. 20mL aliquots were placed

in 50mL centrifuge tubes and set aside for future use.

Third, tubes with plain tailings were prepared by placing 20mL aliquots of mixed MFT into

50mL centrifuge tubes.

6.4.3 Methods – 10 Day Experiments

These experiments took the tube types described in Section 6.4.2 and modified them to

accurately reflect the controls and samples required for this procedure.

The controls required for this type of experiment were titled Water Control – Mix, Water

Control – Cap, Media Control – Mix, and Media Control – Cap.

Water controls were prepared by taking 20mL aliquots of MFT + 10% dH2O and either

capping with 10ml dH2O to form the Water Control – Cap, or adding 10mL dH2O and

stirring until fully mixed to form the Water Control – Mix.

27

Media controls were prepared by taking 20mL aliquots of MFT + 10% 10X R2A media and

either capping with 10ml 1X R2A media to form the Media Control – Cap, or adding 10mL

1X R2A media and stirring until fully mixed to form the Media Control – Mix.

Test samples were prepared by taking the bacteria strains described in Section 6.2 and

listed in Section 6.4.1, adding 1mL 10X R2A media to each tube, then mixing completely.

10ml of each bacteria suspension was added to 20mL aliquots of MFT + 10% 10X R2A

media, and either mixed to form samples labelled BacteriaName – Mix, or capped to form

samples labelled BacteriaName – Cap.

S mp s ts w r th n stor d t th r room t mp r tur (24 C) or n ub t d t36 C.

Measurements, as described in Section 6.3 were taken at 2hours to establish a baseline, and

then every 24 hours for 10 days.

Once 10 days had elapsed, samples were centrifuged for 5 minutes at 1g, 5g, 10g, 25g, 50g,

100g, 250g, 500g, and 1000g. Measurements as described in Section 6.3 were taken in

between each segment of centrifugation.

6.4.4 Methods – 3 Day Experiments

These experiments took the tube types described in 6.4.2 and modified them to accurately

reflect the controls and samples required for this procedure.

The controls required for this type of experiment were titled Water Control – Mix +/-

Antibiotics, Water Control – Cap +/- Antibiotics, Media Control – Mix +/- Antibiotics, and

Media Control – Cap +/- Antibiotics.

28

The antibiotics used for this experiment were 2-methyl-5-nitro-1-imidazoleethanol,

chloramphenicol, and spectinomycin dihydrochloride pentahydrate, all at a concentration of

1000 μ /mL n so ut on of dH2O.


capping with 10ml dH2O to form the Water Control – Cap, capping with 10mL of the

antibiotics preparation to form the Water Control – Cap + Antibiotics, adding 10mL dH2O

and stirring until fully mixed to form the Water Control – Mix, or adding 10mL of the

antibiotics preparation and stirring until fully mixed to form the Water Control – Mix

+Antibiotics.


either capping with 10ml 1X R2A media to form the Media Control – Cap, capping with

10ml of the antibiotics preparation to form the Media Control – Cap + Antibiotics, adding

10mL 1X R2A media and stirring until fully mixed to form the Media Control – Mix, or

adding 10mL of the antibiotics preparation and stirring until fully mixed to form the Media

Control – Mix + Antibiotics.

An additional control, Plain Tailings, was used for this experiment. It was prepared by

taking the stirred MFT and placing 20mL into 50mL tubes.

S mp s ts w r th n stor d t th r room t mp r tur (24 C) or n ub t d t36 C.

Measurements, as described in Section 6.3 were taken at 2 hours to establish a baseline, and


29

Once 3 days had elapsed, samples were centrifuged for 5 minutes at 100RCF, 500RCF,

1000RCF, 2000RCF, and 3000RCF. Measurements as described in Section 6.3 were taken

in between each segment of centrifugation.

6.4.5 Methods – 4 Hour Experiments



The controls required for this type of experiment were titled Water Control – Mix, Media

Control – Mix, and Plain tailings.

Water controls were prepared by taking 20mL aliquots of MFT + 10% dH2O, adding 10mL

dH2O and stirring until fully mixed to form the Water Control – Mix.

Media controls were prepared by taking 20mL aliquots of MFT + 10% 10X R2A media,

adding 10mL 1X R2A media and stirring until fully mixed to form the Media Control –

Mix.

Plain tailings controls were prepared by taking 20mL of the mixed MFT and placing in into

50mL tubes.

Sample sets were then stor d t th r room t mp r tur (24 C) or n ub t d t36 C.

Measurements, as described in Section 6.3 were taken at 0 hours to establish a baseline, and

then at 4 hours.

30

Once 4 hours had elapsed, samples were centrifuged for 5 minutes at 100RCF, 500RCF,

1000RCF, 2000RCF, and 3000RCF. Measurements as described in Section 6.3 were taken

in between each segment of centrifugation.

6.4.6 Methods – Optical Density Analysis

Samples, as described in Section 6.4.3, that remained after the course of the experiment

were mixed for 10 seconds on a vortex machine to resuspend any material that may have

f n out of so ut on dur n stor t 4 C. Th y w r ow d to s t for 48 hours t 4 C.

200 μL of liquid was taken in triplicate from each sample tube, for a total of 9 samples per

species of BioTiger bacteria, and were placed into individual wells of a 96-well plate.

A UV-Vis spectrometer was used to analyze the optical density of the wells at a wavelength

of 590 nm.

Statistical analysis consisted of unpaired, two sample t-Tests assuming unequal variances,

with significance set at p<0.05, performed using Microsoft Excel 2007.

6.4.7 Methods – Full Spectrum Absorbance Analysis

Samples, as described in Section 6.4.3 and previously tested as described in Section 6.4.6,

w r r mov d from 4 C storage and 1.5 mL aliquots in triplicate were taken from each

sample tube, for a total of 9 samples per species of BioTiger, and placed into individual

2.0 mL Eppendorf tubes.

The Eppendorf tubes were centrifuged at 7000 RPM for 5 minutes to remove all biological

matter that may affect absorbance analysis.

31

200 μL of liquid was taken from each Eppendorf tube and placed into individual wells of a

96 well plate.

Plates were scanned using a UV-Vis spectrometer at wavelengths ranging from

200-1000 nm in steps of 10 nm.

6.5 Shell Tailings Experiments

6.5.1 Materials

Bacteria Strains isolated from Shell Tailings

o ST1

o ST2

o ST3

o ST4

o ST6

o ST7

o ST8

o ST9

o ST10

o ST11

o ST12

R2A Media (Per 500mL)

o As prepared in Section 6.4.1

Oil Sands Tailings

32

50mL Conical-bottomed tubes - VWR

Measurement Callipers

25ml Pipettes

6.5.2 Methods



The controls required for this type of experiment were titled Water Control – Mix, Water

Control – Cap, Media Control – Mix, and Media Control – Cap.


capping with 10ml dH2O to form the Water Control – Cap, or adding 10mL dH2O and

stirring until fully mixed to form the Water Control – Mix.


either capping with 10ml 1X R2A media to form the Media Control – Cap, or adding 10mL

1X R2A media and stirring until fully mixed to form the Media Control – Mix.

Test samples were prepared by taking the bacteria strains described in Section 6.2 and

listed in Section 6.5.1, adding 1mL 10X R2A media to each tube, then mixing completely.

10ml of each bacteria suspension was added to 20mL aliquots of MFT + 10% 10X R2A

media, and either mixed to form samples labelled BacteriaName – Mix, or capped to form

samples labelled BacteriaName – Cap.

Sample sets were then stored at room temperatur (24 C).

33

Measurements, as described in Section 6.3 were taken at 2hours to establish a baseline, and


Once 10 days had elapsed, samples were centrifuged for 5 minutes at 100RCF, 500RCF,

1000RCF, 2000RCF, and 3000RCF. Measurements as described in section 6.3 were taken

in between each batch of centrifugation.

7.0 Results

Unless otherwise indicated, graphed data displays the sedimentation of the tailings as a

function of time and centrifugal forces. Sedimentation has been established as the percent

ratio between the solid height of the tailings (h) and the total or clear liquid height of the

mixture (H). A lower percentage implies more sedimentation has occurred. Data is

expressed as the mean +/- SD.

Experiments are listed chronologically to facilitate discussion in later sections.

34

7.1 BioTiger Sedimentation Experiments

7.1.1 10 Day Sedimentation + Centrifugation Experiment

Figure 2: Results of a 10 day MFT sedimentation experiment comparing the e

-

- C and all samples were mixed. The sample that

experienced the greatest amount of sedimentation was the Media Control-Mix, with the least amount of

sedimentation experienced by the Water Control-Mix. The species of BioTiger were insignificantly distributed

between 35-40% sedimentation.

0

10

20

30

40

50

60

70

80

90

100

2 h

1 d

ay

2 d

ays

3 d

ays

4 d

ays

5 d

ays

6 d

ays

7 d

ays

8 d

ays

9 d

ays

10 d

ays

1 g

5 g

10 g

50 g

100 g

250 g

500 g

1000 g

Water Control-Mix

Media Control-Mix

BP20-Mix

BPB-Mix

BPC-Mix

BPE-Mix

BPF-Mix

BPH-Mix

BPI-Mix

BPJ-Mix

BPK HG-Mix

BPL-Mix

KN1-Mix

KN3-Mix

Sed

imen

tati

on

Time

BioTiger 10 Day + Centrifugation Sedimentation Mixed Samples

35

Figure 3: Results of a 10 day MFT sedimentation experiment comparing the effects of the individual strains of

BioTiger bacteria against the water and media controls. Sedimentation is given as a % on the Y-axis, with time and

centrifugati - C and all samples were capped. The sample

that experienced the greatest amount of sedimentation was the BPF-Cap, with the least amount of sedimentation

experienced by the Water Control-Cap. The species of BioTiger were insignificantly distributed between 35-45%

sedimentation.

The results of a 10 day experiment that examined the effects of the individual strains of

BioTiger bacteria, along with the water and media controls, on the sedimentation of MFTs

are shown in Figures 2 and 3. This data was analyzed by Dr. Victoria Kostenko (2010), and

is included in this section to illustrate several data trends.

The mixed samples, shown in Figure 2, experienced a noticeable amount of sedimentation

between 2 hours and 3 days at which point the sedimentation began to plateau until an

apparent decrease in sedimentation around the 6 day point that remained until

centrifugation. Once the samples were centrifuged, all samples except the

0

10

20

30

40

50

60

70

80

90

100

2 h

1 d

ay

2 d

ays

3 d

ays

4 d

ays

5 d

ays

6 d

ays

7 d

ays

8 d

ays

9 d

ays

10 d

ays

1 g

5 g

10 g

50 g

100 g

250 g

500 g

1000 g

Water Control-Cap

Media Control-Cap

BP20-Cap

BPB-Cap

BPC-Cap

BPE-Cap

BPF-Cap

BPH-Cap

BPI-Cap

BPJ-Cap

BPK HG-Cap

BPL-Cap

KN1-Cap

KN3-Cap

Sed

imen

tati

on

Time

BioTiger 10 Day + Centrifugation Sedimentation Capped Samples

36

Water Control-Mix experienced similar levels of sedimentation. Upon conclusion of the

experiment, the Media Control-Mix experienced the most sedimentation, and the Water

Control-Mix experienced the least.

Whereas the mixed samples experienced noticeable sedimentation, the capped samples,

shown in Figure 3, experienced a lower level of sedimentation in comparison during the 2

hour to 3 day interval. Similar to the mixed samples, the 6 day interval marks an apparent

reduction in sedimentation that remained until centrifugation. Once the capped samples

were centrifuged, they experienced lesser sedimentation than the mixed samples, but ended

up with an average sedimentation level similar to the mixed samples. Upon conclusion of

the experiment, the BPF-Cap sample experienced the most sedimentation, and the Water

Control-Cap experienced the least.

37

7.1.2 4 Hour + 3 Day Control Sedimentation +/- Antibiotics Experiment

Figure 4: Results of a 4 hour MFT sedimentation experiment comparing the effects of MFT

-

- C and all samples were mixed. The sample that experienced

the greatest amount of sedimentation was the C Media Control, with the least amount of sedimentation

experienced by the C Water Control-Cap. The plain tailings experienced greater amounts of sedimentation than

the Water Controls, but much less than the Media Controls.

Table 1: Summary of data presented in Figure 4.

Sedimentation

0 hr 4 hr 100RCF 500RCF 1000RCF 2000RCF 3000RCF

24C Media Cont. 97.82 92.76 75.03 61.46 49.81 46.39 43.47 24C H2O Cont. 100.00 100.00 98.41 97.74 98.32 98.50 98.03 24C Plain Tailings.

100.00 100.00 99.54 97.23 97.75 91.89 89.18

36C Media Cont. 95.87 89.65 70.63 56.53 49.72 46.16 42.82 36C H2O Cont. 100.00 100.00 96.30 96.51 96.30 95.80 96.43 36C Plain Tailings.

100.00 100.00 100.92 99.28 94.15 91.47 85.99

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

24C Media Cont.

24C H2O Cont.

24C Plain Tailings.

36C Media Cont.

36C H2O Cont.

36C Plain Tailings.Sed

imen

tati

on

Time

4 Hour Control + Centrifugation Sedimentation Mixed Samples

38

The results of a 4 hour experiment that examined the sedimentation effects of MFTs treated

with R2A media and dH2O, against plain MFTs, is shown in Figure 4 and summarized in

Table 1. Th xp r m nt w s ondu t d t 24 nd 36 C and all samples were mixed.

Both temperatures of the Media Control-Mix samples experienced the greatest level of

sedimentation, whereas the plain tailings experienced a limited amount, and the Water

Control-Mix experienced the least. The change in temperature does not appear to have

influenced sedimentation.

Figure 5: Results of a 3 day MFT sedimentation experiment comparing the effects of temperature, treatment with

R2A media or water, and the presence of antibiotics, against plain tailings. Se -

- C and all samples

were mixed. The samples that experienced the greatest amount of sedimentation were C Media Control

C Water Control + Antibiotics C Water

Control-Mix. The plain tailings experienced greater amounts of sedimentation than the Water Control, but much

less than the Media Controls.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

24C Media Mix +

24C H2O Mix +

24C Plain Tailings

24C Media Mix

24C H2O Mix

36C Media Mix +

36C H2O Mix+

36C Plain Tailings

36C Media Mix

36C H2O Mix

Sed

imen

tati

on

Time

3 Day Control +/- Antibiotics + Centrifugation Sedimentation Mixed Samples

39

Figure 6: The samples that experienced the greatest levels of sedimentation in Figure 5 are re-plotted here, with

standard deviation error bars.

Table 2: Summary of 3Day sedimentation data presented in Figure 5. + Sign indicates presence of antibiotics.

Sedimentation

2 hr 1 day 2 days 3 days 100RCF 500RCF 1000RCF 2000RCF 3000RCF

24C Media Mix +

96.42 85.58 82.49 81.24 73.33 61.40 48.55 41.27 39.05

24C H2O Mix +

99.69 98.16 96.94 95.61 96.06 95.73 93.97 91.90 90.37

24C Plain Tailings

100.0 100.0 100.00 100.00 99.66 98.54 96.41 91.56 85.06

24C Media Mix

95.42 89.08 85.91 84.75 75.26 63.17 51.32 47.54 44.00

24C H2O Mix 100.0 100.0 100.00 100.00 99.54 99.08 98.53 98.29 99.71

36C Media Mix +

96.16 88.09 86.90 85.84 78.71 69.89 57.87 50.72 46.31

36C H2O Mix+ 100.0 97.58 95.87 94.27 92.78 89.96 87.53 86.17 80.96

36C Plain Tailings

100.0 100.0 100.00 100.00 97.96 95.83 92.63 89.03 84.26

36C Media Mix

91.64 85.67 84.37 83.76 69.51 53.62 45.96 42.93 39.28

36C H2O Mix 100.0 100.0 100.00 100.00 96.77 93.43 94.78 94.55 87.57

35.00

45.00

55.00

65.00

75.00

85.00

95.00

24C Media Mix +

24C Media Mix

36C Media Mix +

36C Media Mix

Sed

imen

tati

on

Time

3 Day Control +/- Antibiotics + Centrifugation Sedimentation Mixed Samples

40

Figure 7: Results of a 3 day MFT sedimentation experiment comparing the effects of temperature, treatment with

R2A media or water, and the presence of antibiotics, against plain tailings. Sedimentation is given as a % on the Y-

axis, with time and centrifugation speed on the X-axis. Experi C and all samples

were capped. The sample that experienced the greatest amount of sedimentation was CMedia + Antibiotics,

with the least amount of sedimentation experienc C Water Control-Cap.



0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

24C Media Cap +

24C H2O Cap +

24C Media Cap

24C H2O Cap

36C Media Cap +

36C H2O Cap +

36C Media Cap

36C H2O Cap

Sed

imen

tati

on

Time

3 Day Control +/- Antibiotics + Centrifugation Sedimentation Capped Samples

30.00

35.00

40.00

45.00

50.00

55.00

60.00

65.00

70.00

24C Media Cap +

24C Media Cap

36C Media Cap +

36C Media Cap

Sed

imen

tati

on

Time

3 Day Control +/- Antibiotics + Centrifugation Sedimentation Capped Samples

41

Table 3: Summary of 3Day sedimentation data shown in Figure 7. + Sign indicates presence of antibiotics.

Sedimentation 2 hr 1 day 2

days 3 days

100 RCF

500 RCF

1000 RCF

2000 RCF

3000 RCF

24C Media Cap + 67.01 65.83 65.15 64.73 56.89 45.40 38.79 35.91 33.17

24C H2O Cap + 72.83 71.38 71.05 70.53 70.23 67.27 66.60 65.05 62.96

24C Media Cap 63.38 65.90 66.26 65.83 59.66 53.20 44.77 42.50 37.71

24C H2O Cap 68.78 68.33 67.98 67.91 67.80 68.84 68.03 66.67 67.95

36C Media Cap + 66.26 66.50 66.30 66.20 62.98 55.27 48.76 45.24 42.51

36C H2O Cap + 70.78 70.51 70.04 70.17 65.54 66.22 64.75 63.66 61.39

36C Media Cap 64.92 66.66 65.86 65.34 64.02 57.39 48.03 45.19 41.67

36C H2O Cap 67.23 67.71 67.73 67.56 68.28 66.36 65.33 63.05 60.83

Figure 9 C Media-Cap + Antibiotics samples from Figure 7 and 8 illustrating extremely high levels of

sedimentation after centrifugation.

42

Figure 10 C Water Control-Mix samples from Figure 5 showing bitumen smearing at the free surface. This

collection of bitumen would smear against the sides of the tube making accurate measurements challenging during

the centrifugation portion of the experiment.

The results of a 3 day experiment that examined the effects of temperature, tailings

treatment, and presence of antibiotics on the sedimentation of MFTs is shown in Figures 5

and 6 (Mixed Samples), summarized in Table 2, and Figures 7 and 8 (Capped Samples),

and summarized in Table 3. Important observations are shown in Figures 9 and 10.

The mixed samples, shown in Figures 5 and 6, experienced a noticeable amount of

sedimentation in the first 24 hours, which levelled off prior to centrifugation. Consistent

with previous experiments, samples that had been treated with R2A media experienced the

greatest level of sedimentation upon centrifugation. While previous experiments showed

that the plain tailings would have greater sedimentation than the water treated samples,

F ur 5 shows th t th 36 C Water Control-Mix + Antibiotics had a h h r v of

43

sedimentation than the plain tailings samples at both temperatures. The remaining Water

Contro s mp s xp r n d s m r v s of s d m nt t on, w th th x pt on of th 24 C

Water Control-Mix sample, which had an apparent reduction in sedimentation. This

accuracy issue is illustrated in Figure 9, and is attributed to bitumen smearing at the free

surface. The bitumen would smear against the sides of the tube during centrifugation and

obscure the interface needed for an accurate measurement.

Figure 6 highlights an important observation. In previous experiments, samples incubated

t th mor m t bo y t v t mp r tur of 36 C xp r n d h h r v s of

s d m nt t on th n thos k pt t 24 C. Figure 6 shows th t M d Contro s mp tr t d

w th nt b ot s nd k pt t 24 C had th s m v of s d m nt t on s s mp k pt t

36 C without antibiotics. Additionally, th s mp k pt t 36 C with antibiotics experienced

less sedimentation than s mp k pt t 24 C without antibiotics. These differences are

seemingly insignificant and merely suggest a trend.

The capped samples, shown in Figures 7 and 8, did not experience any sedimentation prior

to centrifugation at the 3 day point. Upon centrifugation, samples treated with R2A media

experienced more sedimentation than samples treated with dH2O. The greatest amount of

sedimentation was experienced by the 24 C Media-Cap + Antibiotics sample; th st

mount of s d m nt t on w s xp r n d by th 24 C Water-Cap sample.

F ur 8 nd t s th t s mp s k pt t 24 C xp r n d mor s d m nt t on th n s mp s

k pt t th mor m t bo y t v 36 C.

44


It was thought that the sedimentation capabilities of the naturally occurring bacteria in the

MFTs could potentially rival those of the BioTiger, so 11 strains of naturally occurring

bacteria were isolated by Dr. Victoria Kostenko from the MFTs for testing.

Figure 11: Results of a 10 day MFT sedimentation experiment comparing the effects of the individual strains of

native Shell MFT bacteria against the water and media controls. Sedimentation is given as a % on the -

- C and all samples were mixed. The

sample that experienced the greatest amount of sedimentation was the Media Control-Mix, with the least amount

of sedimentation experienced by the Water Control-Mix. The species of native Shell tailings bacteria were

insignificantly distributed between 41-46% sedimentation.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

24C H2O Mix

24C Media Mix

ST1 Mix

ST2 Mix

ST3 Mix

ST4 Mix

ST6 Mix

ST7 Mix

ST8 Mix

ST9 Mix

ST10 Mix

ST11 Mix

ST12 Mix

Sed

imen

tati

on

Time

Shell Tailings 10 Day + Centrifugation Sedimentation Mixed Samples

45



Table 4: Summary of 10 Day sedimentation data presented in Figure 11.

Sedimentation

2 hr 1 day 2 days 3 days 4 days 5 days

6 days

7 days

8 days

9 days

10 days

100 RCF

500 RCF

1000 RCF

2000 RCF

3000 RCF

24C H2O Mix 100.00 100.00 100.00 100.00 100.00 98.34 97.36 94.87 93.39 94.26 94.36 99.48 99.34 94.70 95.08 94.79

24C Media Mix

96.01 88.34 82.69 82.47 82.01 81.92 81.96 81.05 81.40 80.94 80.48 67.35 53.25 45.46 39.68 36.87

ST1 Mix 96.68 90.50 87.86 86.43 85.49 85.59 85.19 84.95 85.01 84.64 84.77 78.18 63.79 49.38 47.16 42.58

ST2 Mix 93.00 86.76 85.70 84.16 83.59 83.51 83.00 83.05 82.84 81.96 82.16 74.45 58.27 48.66 45.20 41.05

ST3 Mix 94.67 88.22 85.80 85.81 84.58 84.89 84.48 84.37 83.96 83.17 83.20 78.73 65.27 55.80 47.27 44.02

ST4 Mix 94.84 90.55 88.55 87.53 87.12 86.60 86.07 86.14 85.52 84.83 85.42 78.61 66.37 52.48 47.97 44.84

ST6 Mix 96.53 91.17 88.86 87.16 86.18 86.16 85.74 85.26 85.32 85.35 84.97 77.10 63.88 49.71 46.57 43.39

ST7 Mix 94.97 88.32 85.48 83.75 82.35 82.00 81.30 81.31 80.51 80.17 79.85 75.74 63.50 50.37 46.39 42.44

ST8 Mix 93.07 87.12 86.06 84.76 83.92 83.40 83.31 83.32 83.06 82.87 82.50 76.63 60.49 50.29 46.57 43.07

ST9 Mix 95.84 90.68 88.69 87.18 85.15 85.28 85.67 85.04 84.57 84.89 84.63 81.21 63.72 52.49 49.54 46.30

ST10 Mix 97.91 94.02 91.89 89.66 88.84 89.46 88.34 88.25 87.74 87.27 87.02 82.61 66.59 53.65 50.15 46.89

ST11 Mix 93.61 89.43 87.23 86.22 85.75 85.53 85.27 84.92 84.29 83.71 83.63 78.76 66.74 52.29 48.05 45.10

ST12 Mix 96.39 91.67 88.47 85.84 84.42 84.41 82.69 83.28 82.27 81.92 81.94 79.90 65.80 51.72 48.36 44.04

35.00

45.00

55.00

65.00

75.00

85.00

95.00

24C Media Mix

ST1 Mix

ST2 Mix

ST7 Mix

Sed

imen

tati

on

Time

Shell Tailings 10 Day + Centrifugation Sedimentation Mixed Samples

46

Figure 13: Results of a 10 day MFT sedimentation experiment comparing the eff

-

- C and all samples were capped.

The greatest amount of sedimentation was experienced by ST1, ST2, ST3, and ST12 (insignificant difference). The

sample with the least amount of sedimentation was the Water Control-Cap. The native species of Shell tailings

bacteria were insignificantly distributed between 38-42% sedimentation.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

24C H2O Cap

24C Media Cap

ST1 Cap

ST2 Cap

ST3 Cap

ST4 Cap

ST6 Cap

ST7 Cap

ST8 Cap

ST9 Cap

ST10 Cap

ST11 Cap

ST12 Cap

Sed

imen

tati

on

Time

Shell Tailings 10 Day + Centrifugation Sedimentation Capped Samples

47



Table 5: Summary of 10 Day sedimentation data presented in Figure 13.

Sedimentation

2 hr 1 day 2 days

3 days

4 days

5 days

6 days

7 days

8 days

9 days

10 days

100 RCF

500 RCF

1000 RCF

2000 RCF

3000 RCF

24C H2O Cap 67.17 67.94 67.22 66.91 66.93 66.47 65.68 66.63 66.55 66.50 65.84 67.46 66.57 65.26 63.16 62.72

24C Media Cap

64.88 64.53 64.38 64.21 64.36 64.16 64.02 63.62 63.56 63.54 63.40 63.04 62.00 50.05 45.63 42.51

ST1 Cap 65.37 65.65 65.17 65.37 65.46 65.30 65.04 65.20 64.91 64.49 64.68 62.96 53.97 44.66 42.02 38.37

ST2 Cap 66.93 66.51 66.07 66.35 65.59 66.21 66.25 66.10 65.97 65.83 65.84 63.23 53.69 44.98 41.29 38.13

ST3 Cap 64.90 64.64 65.14 65.82 65.82 65.24 65.14 65.56 65.46 65.33 64.95 63.21 55.34 45.24 41.99 38.97

ST4 Cap 65.86 66.20 66.55 66.78 66.33 66.20 66.01 65.52 65.81 65.96 65.70 65.56 59.87 49.45 45.49 42.38

ST6 Cap 64.98 65.28 65.64 64.55 64.68 65.24 64.42 65.03 64.60 64.22 64.34 63.86 56.88 48.82 44.43 41.63

ST7 Cap 64.77 64.85 65.10 65.91 64.86 65.42 65.26 65.39 64.70 64.32 64.71 63.56 58.05 47.34 43.42 39.73

ST8 Cap 66.97 66.64 66.34 66.35 65.72 65.92 65.44 65.88 65.15 65.06 65.50 64.73 59.37 48.62 44.72 41.15

ST9 Cap 64.26 64.35 65.12 64.80 64.24 64.32 64.00 64.49 64.24 64.04 64.23 63.13 57.95 48.57 44.61 41.17

ST10 Cap 65.45 64.90 65.03 64.45 64.08 64.28 64.01 64.99 64.62 64.18 64.59 62.92 59.09 48.14 44.77 42.11

ST11 Cap 64.26 64.48 64.38 63.93 63.79 64.26 64.35 64.42 64.28 63.73 64.12 61.90 55.94 46.19 42.54 39.75

ST12 Cap 67.32 66.43 66.05 65.57 65.45 66.23 65.43 65.78 66.13 65.53 65.87 62.31 56.49 46.78 42.69 38.88

30.00

35.00

40.00

45.00

50.00

55.00

60.00

65.00

70.00

ST1 Cap

ST2 Cap

ST3 Cap

ST12 Cap

Sed

imen

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on

Time

Shell Tailings 10 Day + Centrifugation Sedimentation Capped Samples

48

The results of a 10 day experiment that examined the effects of the individual strains of

naturally occurring Shell tailings bacteria, along with the water and media controls, on the

sedimentation of MFTs is shown in Figures 11-14. Mixed samples are shown in Figures 11

and 12 and are summarized in Table 4. Capped samples are shown Figures 13 and 14 and

summarized in Table 5.

The mixed samples, shown in Figures 11 and 12, experienced a noticeable amount of

sedimentation in the first 2 days, which levelled off prior to centrifugation. Consistent with

previous experiments, upon centrifugation the Media Control sample experienced the

greatest amount of sedimentation (36.87%), the Shell tailings bacteria experienced between

41-46% sedimentation, and the Water Control experienced the least (94.79%).

Like previous experiments, accuracy problems with the Water Control samples, as shown

in Figure 10, were present and illustrated in Figure 11.

The capped samples, shown in Figures 13 and 14, did not experience any sedimentation

prior to centrifugation. Upon centrifugation, the Media Control-Cap experienced 42.51%

sedimentation, the Shell tailings bacteria experienced 38-42% sedimentation, and the Water

Control-Cap experienced 62.72% sedimentation.

49

7.3 Optical Density Analysis Results

Figure 15: Optical density analysis of mixed samples vs. water control from Section 7.1.1. All BioTiger samples

experienced significant (p<0.01, **) levels of optical density reduction when compared to the Water Control Mix

sample.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Water Cont Mix

BPE Mix BPB Mix BPC Mix BPF Mix BPH Mix BPI Mix BPJ Mix BPK Mix BPL Mix KN1 Mix KN3 Mix BP20 Mix

Ab

so

rban

ce

Sample

BioTiger 10 Day + Centrifugation Sedimentation Mixed Samples vs. Water Control Optical Density Analysis

**

50

Figure 16: Optical density analysis of mixed samples vs. media control from Section 7.1.1. Select BioTiger samples

experienced significant levels of optical density reduction when compared to the Media Control Mix sample, BPC

Mix (p<0.05, *), and BPL Mix (p<0.01, **).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Media Cont Mix

BPE Mix BPB Mix BPC Mix BPF Mix BPH Mix BPI Mix BPJ Mix BPK Mix BPL Mix KN1 Mix KN3 Mix BP20 Mix

Ab

so

rban

ce

Sample

BioTiger 10 Day + Centrifugation Sedimentation Mixed Samples vs. Media Control Optical Density Analysis

*

**

51

Table 6: Optical Density summary of data presented in Figures 15 and 16.

Sample Number (Absorbance)

Mean Std. Dev.

1 2 3 4 5 6 7 8 9

Water Cont Mix

3.7293 3.9153 3.7842 3.7049 3.4159 3.8215 0.5172 0.342 0.6547 2.653889 1.619448

Media Cont Mix

0.0894 0.0914 0.1364 0.1072 0.1312 0.143 0.1525 0.1615 0.1552 0.129756 0.027392

BPE Mix 0.0809 0.1 0.0806 0.1705 0.1634 0.1975 0.1155 0.1311 0.1331 0.130289 0.040749

BPB Mix 0.0865 0.2245 0.1328 0.0836 0.092 0.1001 0.1127 0.1432 0.1614 0.126311 0.045632

BPC Mix 0.1163 0.1001 0.1105 0.1281 0.1377 0.1206 0.0727 0.0819 0.0803 0.105356 0.022951

BPF Mix 0.2276 0.2047 0.2428 0.0899 0.09 0.0846 0.0508 0.0493 0.05 0.121078 0.080287

BPH Mix 0.2197 0.2331 0.2313 0.1077 0.091 0.0903 0.1164 0.1247 0.1068 0.146778 0.061998

BPI Mix 0.1107 0.1298 0.1256 0.1929 0.245 0.2457 0.2817 0.3025 0.2977 0.214622 0.076977

BPJ Mix 0.057 0.0559 0.0561 0.1568 0.1527 0.1856 0.2532 0.2545 0.2832 0.161667 0.090643

BPK Mix 0.1401 0.1399 0.257 0.1099 0.1047 0.1206 0.1021 0.1018 0.1016 0.130856 0.049793

BPL Mix 0.058 0.0558 0.0555 0.0904 0.1082 0.1151 0.0772 0.0752 0.0799 0.079478 0.021891

KN1 Mix 0.1675 0.1541 0.1723 0.2919 0.3493 0.3443 0.1546 0.1596 0.1601 0.217078 0.085257

KN3 Mix 0.2227 0.2275 0.2207 0.3523 0.2909 0.3459 0.2872 0.3117 0.3613 0.291133 0.056784

BP20 Mix 0.0796 0.0858 0.078 0.2082 0.2102 0.2079 0.0834 0.079 0.0842 0.124033 0.063604

Table 7: Summary of statistical analysis of data presented in Table 6.

Vs. Water Cont Mix Media Cont Mix p>0.05

Water Cont Mix - - p<0.05 Media Cont Mix 0.000795828 - p<0.01

BPE Mix 0.000797685 0.487232036

BPB Mix 0.000790491 0.424528073

BPC Mix 0.000750462 0.028651301

BPF Mix 0.000784579 0.382612837

BPH Mix 0.000832043 0.233506945

BPI Mix 0.000983217 0.005473539

BPJ Mix 0.000866803 0.169211645

BPK Mix 0.000799527 0.477325347

BPL Mix 0.000705556 0.000314909

KN1 Mix 0.000990639 0.00757977

KN3 Mix 0.001183437 2.85165E-06

BP20 Mix 0.000787969 0.404393098

52

Figure 17: Optical density analysis of capped samples vs. water control from Section 7.1.1. Select BioTiger samples

experienced significant levels of optical density reduction when compared to the Water Control Cap sample, BPE

Cap (p<0.05, *), BPB Cap (p<0.01, **), BPC Cap (p<0.01, **), and BPK Cap (p<0.05, *).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Water Cont Cap

BPE Cap BPB Cap BPC Cap BPF Cap BPH Cap BPI Cap BPJ Cap BPK Cap BPL Cap KN1 Cap KN3 Cap BP20 Cap

Ab

so

rban

ce

Sample

BioTiger 10 Day + Centrifugation Sedimentation Capped Samples vs. Water Control Optical Density Analysis

***

**

*

53

Figure 18: Optical density analysis of capped samples vs. media control from Section 7.1.1. One BioTiger sample

experienced significant levels of optical density reduction when compared to the Media Control Cap sample, BPB

Cap (p< 0.05, *).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Media Cont Cap

BPE Cap BPB Cap BPC Cap BPF Cap BPH Cap BPI Cap BPJ Cap BPK Cap BPL Cap KN1 Cap KN3 Cap BP20 Cap

Ab

so

rban

ce

Sample

BioTiger 10 Day + Centrifugation Sedimentation Capped Samples vs. Media Control Optical Density Analysis

*

54

Table 8: Optical density summary of data presented in Figures 17 and 18.

Sample Number (Absorbance)

Mean Std. Dev.

1 2 3 4 5 6 7 8 9

Water Cont Cap

0.1332 0.1642 0.1454 0.1878 0.2185 0.3788 0.1686 0.1748 0.1744 0.193967 0.073429

Media Cont Cap

0.0982 0.0963 0.0919 0.3059 0.2998 0.2992 0.1058 0.1216 0.1913 0.178889 0.096762

BPE Cap 0.1222 0.1047 0.1294 0.1091 0.1247 0.1493 0.1318 0.1417 0.1396 0.128056 0.014735

BPB Cap 0.0921 0.0913 0.0904 0.1335 0.134 0.1346 0.0877 0.0858 0.0854 0.103867 0.022741

BPC Cap 0.0978 0.0943 0.0981 0.1025 0.1032 0.1139 0.1136 0.1358 0.1976 0.117422 0.032617

BPF Cap 0.2986 0.2866 0.3098 0.13 0.1079 0.113 0.2799 0.4313 0.372 0.258789 0.116507

BPH Cap 0.2017 0.2011 0.2112 0.1705 0.2096 0.2268 0.2765 0.3753 0.3919 0.251622 0.080027

BPI Cap 0.1216 0.1244 0.1585 0.3896 0.3655 0.3895 0.2718 0.2483 0.1938 0.251444 0.109853

BPJ Cap 0.2664 0.2931 0.1994 0.1592 0.1928 0.1837 0.1869 0.1992 0.1977 0.208711 0.042652

BPK Cap 0.0669 0.0672 0.0687 0.145 0.1394 0.1484 0.1813 0.1811 0.198 0.132889 0.052591

BPL Cap 0.1718 0.1634 0.1505 0.2961 0.2615 0.3003 0.2382 0.265 0.297 0.2382 0.060851

KN1 Cap 0.1326 0.122 0.1278 0.1944 0.1658 0.1887 0.1666 0.1959 0.1987 0.165833 0.031217

KN3 Cap 0.3445 0.2894 0.2956 0.3351 0.3621 0.3046 0.2058 0.1757 0.1914 0.278244 0.069893

BP20 Cap

0.2671 0.291 0.2487 0.183 0.1859 0.1831 0.1819 0.1632 0.202 0.211767 0.045233

Table 9: Summary of statistical analysis of data presented in Table 8.

Vs. Water Cont Cap

Media Cont Cap

p>0.05

Water Cont Cap - - p<0.05 Media Cont Cap 0.357406845 - p<0.01

BPE Cap 0.013451954 0.07891606

BPB Cap 0.002786168 0.024912863

BPC Cap 0.007786003 0.050540383

BPF Cap 0.090706488 0.067169834

BPH Cap 0.065411152 0.051371041

BPI Cap 0.106469621 0.078243087

BPJ Cap 0.305598353 0.207773002

BPK Cap 0.030975275 0.117025357

BPL Cap 0.092185884 0.071777671

KN1 Cap 0.156414283 0.354069717

KN3 Cap 0.011979522 0.012322812

BP20 Cap 0.273247426 0.187798458

55

Optical density was analyzed for all strains of BioTiger used in the experiment detailed in

Section 7.1.1. Mixed samples are shown in Figure 15 (vs. Water Control) and Figure 16

(vs. Media Control), with data summarized in Table 6 and statistical analysis shown in

Table 7. Capped samples are shown in Figure 17 (vs. Water Control) and Figure 18 (vs.

Media Control), with data summarized in Table 8 and statistical analysis shown in Table 9.

When compared to the Water Control Mix, all mixed samples had a statistically significant

(p<0.01) reduction in optical density.

When compared to the Media Control Mix, the BPC Mix (p<0.05, *) and BPL Mix

(p<0.01, **) had statistically significant reductions in optical density. Several samples had

a statistically significant increase in optical density when compared to the Media Control,

specifically BPI Mix (p<0.01), KN1 Mix (p<0.01), and KN3 Mix (p<0.01).

When compared to the Water Control Cap, BPE Cap (p<0.05, *), BPB Cap (p<0.01, **),

BPC Cap (p<0.01, **), and BPK Cap (p<0.05, *) samples had a significant reduction in

optical density. The KN3 Cap sample (p<0.05) experienced a statistically significant

increase in optical density when compared to the Water Control Cap.

When compared to the Media Control Cap, BPB Cap (p<0.05, *) was the only sample to

have a statistically significant reduction in optical density. The KN3 Cap sample (p<0.05)

had a statistically significant increase in optical density when compared to the Media

Control Cap.

56

7.4 Full Spectrum Absorbance Analysis Results

Figure 19: Results of a full spectrum absorbance analysis on mixed samples detailed in Section 7.1.1.

0

0.5

1

1.5

2

2.5

3

3.5

4

200 300 400 500 600 700 800 900 1000

Ab

so

rban

ce

Wavelength (nm)

BioTiger 10 Day + Centrifugation Sedimentation Mixed Samples Full Spectrum Absorbance Analysis

Mean BPC - Mix

Mean BPL - Mix

Mean BPF - Mix

Mean KN1 - Mix

Mean Water Cont. Mix

Mean Media Cont. Mix

57

Figure 20: Results of a full spectrum analysis on capped samples detailed in Section 7.1.1.

The results of a full spectrum (200-1000 nm) absorbance analysis was performed on the 4

mixed (Figure 19) and capped samples (Figure 20) detailed in Section 7.1.1 that

experienced the most sedimentation at the end of the 10 day incubation period are shown

above. The number of samples was reduced in order to reduce the length of time the

UV-Vis Spectrometer would require for analysis.

Both the mixed and capped samples analyzed show intense peaks between 200-300 nm,

with nothing notable occurring beyond these wavelengths.

0

0.5

1

1.5

2

2.5

3

3.5

4

200 300 400 500 600 700 800 900 1000

Ab

so

rban

ce

Wavelength (nm)

BioTiger 10 Day + Centrifugation Sedimentation Capped Samples Full Spectrum Absorbance Analysis

Mean BPF - Cap

Mean BPI - Cap

Mean KN1 - Cap

Mean BPH - Cap

Mean Water Cont. Cap

Mean Media Cont. Cap

58

It should be noted that the plates were incorrectly blanked prior to analysis, so data

presented here is not normalized, and cannot be used to calculate the concentrations of

various compounds.

8.0 Discussion

8.1 BioTiger Experiments

8.1.1 10 Day Sedimentation + Centrifugation Experiment

It had recently been shown that using BioTiger, a patented biofilm with established

bioremediation abilities, to treat oil sand samples resulted in a 5-fold increase in bitumen

recovery and an equivalent increase in chelating heavy metal ions over 24 hours. Tailings

samples treated with P. aeruginosa bacteria showed a notable increase in bitumen recovery

from clay sludge after a 36 hour treatment due to bacterial activity that induced separation

of the oil sands components. This proves that the increased bitumen recovery is

proportional to densification of the tailings and release of water. However, the contributions

of the individual strains that comprise BioTiger to the bioremediation process remains to be

determined. Additionally, it remains unclear if the BioTiger consortium is more effective

than the naturally occurring bacteria in the tailings, as supplemented with R2A growth

media.

The results illustrated and summarized in Section 7.1.1 reflect that none of the individual

BioTiger strains were capable of outperforming the media controls, in both the mixed and

capped forms. They also show that samples treated with media outperform those treated

with water.

59

While the centrifugation data shows a negligible difference between the mixed and capped

groups, prior to centrifugation the capped samples experienced more sedimentation than the

mixed ones. The reason for this is unknown.

8.1.2 4 Hour + 3 Day Control Sedimentation +/- Antibiotics Experiment

During a conference call with the Savannah River Natural Laboratory (SRNL) it was

decided that in order to determine the differences between the media control and the

bacteria-added samples (Section 7.1.1), another experiment was needed to clarify whether

the differences were due to chemical or biological change.

The experiment detailed in Section 7.1.2 was done in two parts: a 4 hour and a 3 day

growth period. The governing hypothesis was that the artificially short growth period of the

4 hour experiment would limit the sedimentation effects of the naturally occurring tailings

bacteria, and the difference in sedimentation between the media and water treated samples

would be primarily caused by the chemical differences between the two substances.

As Figure 4 shows, the samples mixed with the media experienced far more sedimentation

than those treated with water, with differences manifesting themselves after a mere four

hours. This suggests that the previously observed performance of samples treated with just

media (Media Control, Section 7.1.1) is largely due to chemical or charge effects, rather

than the native tailings bacteria.

Figure 5 shows the results from the three day portion of the experiment with mixed

samples. Notably, Water Control samples treated with antibiotic outperformed those which

were not. Further, all but one (the 36°C H2O mix+) were outperformed by the plain

60

tailings, a finding consistent with Figure 4. Examining the media treated samples, the 24°C

Media Control Mix+ outperformed the 24°C Media Control Mix, whereas the opposite was

true for the 36°C samples. However, as shown in Figure 6, this could all be due to

experimental error. The general trend of samples treated with media experiencing more

sedimentation than samples treated with water described in Section 8.1.1 still stands (i.e.

Media Control Mix and Media Control Cap samples experienced more sedimentation than

Water Control Mix and Water Control Cap samples).

Figure 7 shows the results from the three day portion of the experiment with capped

samples. Samples capped with water, both with and without the antibiotic treatment,

performed similarly, especially at 36°C. The trend of media treatment increasing

sedimentation continues, with all media treated samples experiencing more sedimentation

than their water treated counterparts. As shown in Figure 7, the 24°C Media-Cap +

Antibiotics sample experienced far more sedimentation than all other samples, including

samples from previous experiments. A picture of the replicates is shown in Figure 9.

The sedimentation of the 24°C media+ sample in Figure 7 is perhaps the most important

observation from this experiment. Typically it has been observed that samples treated with

media at 36°C experience the most sedimentation, but these results show otherwise.

Overall the data presented in Sections 7.1.1 and 7.1.2 suggest that the differences between

the water treated samples and the media treated samples is most likely due to the

chemical/charge differences between the substances, rather than bacterial activity, when the

artificially short time-span of this experiment is considered. This trend was echoed during

61

the 3 day experiment involving antibiotics to eliminate the native bacteria strains in the

tailings.


The inconclusive results from testing of th oT r mu t sp s onsort um’s

sedimentation capacity required testing of the sedimentation capabilities of the naturally

occurring bacteria (those stimulated by the R2A media in the Media Control samples). 11

strains of bacteria were isolated from the tailings for testing.

The data from this experiment, shown in Section 7.2, is consistent with the trends

established in Section 7.1. On average, capped samples experienced greater levels of

sedimentation than the mixed samples. Media controls typically outperformed the samples

treated with a specific strain of bacteria, with the exception of Figure 14 where the media

control was outperformed by at least four species of the Shell tailings bacteria.

Overall, the results from this experiment suggest that the hypothesis that the naturally

occurring Shell Tailings bacteria could outperform the BioTiger bacteria in sedimentation

cannot be maintained.

8.3 UV-Vis Spectrometer Experiments

During a conference call with researchers at SRNL, it was decided that the inconclusive

results of the sedimentation experiments required expanding the BioTiger experiments to

include an analysis of the released water chemistry, with a particular interest in water

turbidity as it pertains to the required reduction in fine particles in fluid tailings under the

ERC ’s Directive 074 (ERCB 2009). An inventory of samples retained from previous

62

experiments was performed and it was decided that the most suitable samples for analysis

were those from experiments outlined in Sections 6.4.3 and 7.1.1, tailings samples treated

with th nd v du oT r str ns, s n th y h d b n stor d t 4 C and closely

mimicked what a potential future experiment might investigate.

8.3.1 Optical Density Analysis

The analysis of the optical density of samples treated with BioTiger shown in Section 7.3

supports the view microbial activity may be used to reduce the turbidity of process affected

water.

The most significant finding from this experiment is shown in Figure 15, where all mixed

samples experienced a statistically significant (p<0.01) reduction in optical density at

590 nm when compared to the Water Control Mix.

The results from the capped samples, shown in Figures 17 and 18, are not as notable as

those from the mixed samples. This is likely due to the experimental procedure, where

samples treated with BioTiger were capped with the bacteria-containing R2A mixture, and

the Water Control Cap was capped with dH2O. While several samples had a statistically

significant reduction in optical density, the magnitudes of the reduction were much less

than in the mixed samples, suggesting a more subtle effect in this regard.

In both the mixed and capped sample analysis, several strains of BioTiger had an apparent

increase in optical density when compared to the media control, rather than a reduction.

Shown in Table 7, the BPI-Mix, KN1-Mix, and KN3-Mix samples had a statistically

significant increase in optical density. Shown in Table 9, the KN3-Cap sample had a

63

statistically significant increase in optical density. These results suggest that there is an

effect of microbial activity on the optical density of the released water, be it beneficial or

detrimental. Furthermore, the significant increases or decreases in optical density imply that

these effects are not limited to the suggested charge/chemistry effects of media, as seen in

the sedimentation experiments.

Overall, the net reduction in optical density suggests that the BioTiger bacteria are reducing

the amount of suspended fines in the released water. Fines are mineral solids with particle

sizes equal to or less than 44 μm (ERCB 2009). In accordance with Directive 074: Tailings

Performance Criteria and Requirements for Oil Sands Mining Schemes, Operators must

reduce the amount of fines in dedicated disposal areas by 20% before June 30, 2011, by

30% before June 30, 2012, and by 50% before June 30, 2013 (ERCB 2009). While the

large-scale feasibility of treating oil sands tailings with BioTiger has not been investigated,

the results presented here suggest that future investigation would be worthwhile.

8.3.2 Full Spectrum Analysis

A full spectrum absorbance analysis was performed on the top 4 mixed and capped

samples, outlined in Sections 6.4.3 and 7.1.1, that experienced the most sedimentation at

the end of the 10 day incubation. Results are shown in Figures 19 and 20.

Both the mixed and capped samples feature similar peaks between 200-300 nm, suggesting

the presence of aromatic hydrocarbons in the released water. Unfortunately, procedural

errors prevented the plates from being properly blanked, so the amount of bituminous

material could not be calculated.

64

Had the blanking worked properly, the amounts of bituminous material in the released

water would have been calculated using an extinction coefficient of 400 L/mol cm (Ali &

Alghannam, 1979), and the equation:

Where A is the absorbance, ε is the extinction coefficient, and l is the path length.

9.0 Conclusions + Future Work

From Section 7.1.1 the figures show that at the end of centrifugation, the average

sedimentation caused by the BioTiger species is 35-40% for both the cap and mixed

samples, despite discrepancies prior to centrifugation (capped samples typically

outperformed mixed samples prior to this point). This suggests there is a limit to the

sedimentation effects of media/water chemistry and bacteria.

From Section 7.1.2, figures demonstrate that there is an immediate effect of media as

shown in Figure 4. This suggests that there is a chemical effect of the R2A media on the

MFTs, rather than a bacteria effect since the effects of their activity would be negligible in

4 hours, a finding that calls into question the governing hypothesis of this study.

Further adding to the confusion are the results shown in Figures 5 and 7 where samples

treated with antibiotics to inhibit bacterial activity outperformed those that were treated

with just media, suggesting that the naturally occurring bacteria in the tailings were not

suitable for sedimentation application, an observation in conflict with the results from

Section 7.1.1 where the media controls outperformed established bioremediation bacteria.

65

From Section 7.2, it is shown that at the end of centrifugation, the average sedimentation

caused by the Shell Tailings bacteria is not as much as that caused by the BioTiger bacteria.

This suggests that the BioTiger multispecies consortium is more suited to sedimentation

than the naturally occurring strains.

The inability of all samples to routinely outperform their controls, and the high levels of

sedimentation of capped controls treated with antibiotics, shown in Figures 7, 8, and 9,

suggest that the overall capacity of the BioTiger multispecies consortium for the

sedimentation of MFTs is limited, and their bioremediation capabilities should be examined

via other parameters.

Expanding the scope of the BioTiger bioremediation evaluation, the statistically significant

reduction in optical density of the released water in mixed samples, shown in Section 7.3

and Figure 15, was potentially the most significant finding of the experiment when put in

an industrial context.

Directive 074 requires oil sands operators to achieve drastic reductions in suspended fine

particles in tailings ponds on a yearly basis. While the ability of oil sands operators to meet

th s r du t ons won’t b known until the first deadline (June 30, 2011), the reduction in

released water optical density via treatment with BioTiger suggests that there exists

potential for passive tailings pond treatment to improve the quality of the process-affected

water.

Future steps should be taken to evaluate the oil sands tailings bioremediation potential of

BioTiger under parameters other than sedimentation, including optical density, pH, and

66

UV-Vis Spectrometry. Explicitly, steps should be taken to repeat the experiments detailed

in Section 6.4.6 and 6.4.7, as data presented in this th s s w s r ord d from s mp s stor d

t 4 C for an extended period of time.

67

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