Manual 21384884

Manual 21384884
A STEADY-STATE MODEL FOR HEXAVALENT CHROMIUM
REDUCTION IN SIMULATED BIOLOGICAL REACTIVE BARRIER:
MICROCOSM ANALYSIS
PHALAZANE JOHANNA MTIMUNYE
A dissertation submitted in partial fulfilment of the requirement for the degree of
MASTER OF SCIENCE: APPLIED SCIENCE
WATER UTILIZATION
In the
Faculty of Engineering, Built Environment and Information Technology
Department of Chemical Engineering University of Pretoria, Pretoria, South Africa
April 2011
© University of Pretoria
Declaration
I, PHALAZANE JOHANNA MTIMUNYE, hereby declare that the work provided in this
dissertation is to the best of my knowledge original (except where cited) and that this work
has never been submitted for another degree at this or any other tertiary education institution.
Signature of candidate
This ………….. day of ………………………. 2011
ii
Dedication
This dissertation is dedicated to
My family
My late father who always believed in me and encouraged me to pursue my studies
My mother for her ongoing support, endless love, understanding, for believing in me always and
for always telling me that the will of God will never take me where the grace of the Lord will not
guide me
My brothers and sisters who supported me every step of the way both emotionally and financially
My friends for their presence in my life, who were always there when I needed to talk
iii
Acknowledgements
I would like to express my sincere gratitude to the following persons without who this
dissertation would not be possible:
Professor Evans Chirwa my study leader for the guidance, mentorship, motivation and advice he
gave me throughout the study. May the good Lord bless him.
Professor Fanus Venter from Department of Microbiology for assistance with the
characterization of bacterial isolates.
National Research Foundation of South Africa (NRF) for financial assistance throughout the
study.
Colleagues and friends who provided invaluable advice that contributed to this study.
My family and friends for helping me keep my feet on the ground by teaching me never to forget
that from my Lord cometh my help and through whom all my blessings flow.
I would like to thank God almighty and my Saviour Jesus Christ for the many blessings that he
has bestowed upon me since birth and without him I can not achieve anything.
iv
ABSTRACT Biological remediation of Cr(VI) contaminated soil and groundwater is an emerging field. In this
study, the in situ bioremediation technology for treating Cr(VI) contaminated groundwater
aquifers was evaluated using a laboratory microcosm system. The study was conducted using
columns with five equally spaced intermediate sampling ports along the length to facilitate finite
difference modelling of the Cr(VI) concentration profile within the column. Cr(VI) concentration
was continuously measured in the influent, in five equally spaced intermediate ports within the
column and in the effluent port. The change or the shift in microbial community within the
inoculated column was also monitored due to exposure to toxic conditions after seven weeks of
operation using the 16S rRNA genotype fingerprinting method.
The effect of introducing a natural carbon source (sawdust) in inoculated columns in comparison
with the performance of sterile controls under various loading conditions was also evaluated.
Near complete Cr(VI) removal was achieved in an inoculated carbon source reactor, whereas
only 69.5% of Cr(VI) removal was achieved in an inoculated column without an added carbon
source after 4 days of operation at 20 mg/L. In a sterile control reactor less than 2% of Cr(VI)
was removed after 4 days of operation at 20 mg/L. Experimental cores demonstrated a successful
Cr(VI) reduction process in the simulated microbial barrier system that was evaluated internally.
The model that simulates Cr(VI) removal and transport in the subsoil environment was
developed. The Cr(VI) mass balance model across the reactor that accounts for the flow
characteristics and biological removal mechanism successfully captured the trends of Cr(VI)
response profiles under quasi-steady state conditions for different loading conditions. This study
demonstrate the potential of applying effective Cr(VI) reducers in the reactive barrier systems to
contain or attenuate the spread of Cr(VI) contaminant in groundwater aquifer systems. The finite
difference model developed in this study to evaluate the behaviour of Cr(VI) in the reactor could
contribute towards improved designs of future in situ bioremediation systems that can be
implemented for remediation of Cr(VI) on site.
v
Table of Contents
Title…………………………………………………………………………….......................page
Declaration…………………………………………………………………..…...........................ii
Dedication…………………………………………………………………..…...........................iii
Acknowledgement……………………………………………..……………………...................iv
Abstract……………………………………………………….…………………….....................v
List of Figures……………………………………..……………………………………………..x
List of Tables………………………………………………………………………………........xii
List of Abbreviations.…………………………………………..……………………..…..........xiii
Symbol Nomenclature…………………………………………………….................................xiv
CHAPTER 1:INTRODUCTION................................................................................................1
1.1
Background…………………………….…………………………........…...……….........1
1.2
Methodology and Objectives of the Study………….....………………….……..............3
1.3
Outline of Dissertation.........……..........….......……………………………………….....4
1.4
Significance of Research........................….......………………………………………….4
CHAPTER 2:LITERATURE REVIEW………………………...….........................................5
2.1
Chromium Occurrences in the Environment…..................……...........…………...…......5
2.1.1
Chromium in Water…………..….........................……………....…..…………………...5
2.1.2
Chromium in Soil……………...............…………………………....…………………….6
2.1.3
Chromium in Air……………...…............………………………..…………………........6
2.2
Production of Chromium and Its Use…………………...................………………..........7
2.3
Potential Health Effects of Exposure to Chromium……………………………...............9
2.3.1
Nutrition and Toxicity: Risk to Human and Animal Health......…….………....................9
2.3.2
Toxicity to Microorganisms..............................................................................................10
2.4
Remediation Strategy…………....………….…………………………………………...11
2.4.1
Physicochemical Methods…………............………………………………………….....11
2.4.2
Bioremediation Method..............………………..........………………….……………...13
vi
2.4.3
Biological System Engineering........................................................................................14
2.5
Cr(VI) Reducing Organisms.............................................................................................15
2.6
Cr(VI) Reduction Pathways……………....................……………………………...…..18
2.6.1
Intracellular Processes...........…………....................…………………………………...19
2.6.2
Extracellular Processes……….....…......………………………………………………..19
2.6.3
Membrane-bound Processes……......………….............………………………………..20
2.7
Carbon Source……………..........……......…………………………………………..….20
2.8
Summary...........................................................................................................................21
CHAPTER3: MATERIALS AND METHODS..……………...........……………………......22
3.1
Bacterial Culture…………....……………...............………………….………………...22
3.1.1
Sources of Cr (VI) Reducing Bacteria……………..................………….……………...22
3.1.2
Culture Isolation.........….......………………………………………..............………….22
3.2
Growth Media…………………..........….....…………………………………………....23
3.2.1
Basal Mineral Media.............….............………………………………………………...23
3.3.2
Commercial Broth and Agar........................................................................………….....23
3.3
Reagents……..….....……………………………………………………………….……23
3.3.1
Standard Solutions and Chemicals…….........….........……………………………..........23
3.3.2
DPC Solution……………..........………………………………………………………..23
3.3.3
Chemicals……………..................…….....……………………………….……………..24
3.4
Culture Characterization...................................................................................................24
3.4.1
General Characterization of Aerobic Cultures using 16S rRNA ID’s…...........…..….....24
3.4.2
Culture Storage……..............……………………………........…………………………25
3.4.3
General Classification of Anaerobic Cultures using 16S rRNA ID’s…….............……..25
3.5
Batch Reactor Studies……………..........…..............…………………………………....27
3.5.1
Aerobic Cr(VI) Reduction Experiments............................................................................27
3.5.2
Anaerobic Cr(VI) Reduction Experiments……….……..................…………………….27
3.5.3
Abiotic Experiments……..............……........…………………………………………....28
3.6
Biomass Analysis……...............…………………………………………………….…..28
3.6.1
Total Biomass…......................……………………………………………………….….28
3.6.2
Viable Biomass…………........….....……..................……………………………...…....28
vii
3.7
Microcosm Studies...........................................................................................................29
3.7.1
Mineral Composition of Aquifer Media...........................................................................29
3.7.2
Reactor Setup....................................................................................................................29
3.7.3
Start up culture.................................................................................................................30
3.7.4
Reactor Start up................................................................................................................30
3.7.5
Reactors Operation...........................................................................................................31
3.8
Analytic Methods.............................................................................................................31
3.8.1
Cr(VI) Analysis................................................................................................................31
CHAPTER 4:CR(VI) REDUCTION BATCH KINETICS STUDIES.................................32
4.1
Preliminary Studies..........................................................................................................32
4.2
Performance Validation in Cultures.................................................................................32
4.2.1
Individual Pure Cultures versus Reconstituted Consortium Culture...............................32
4.2.2
Reconstituted Culture versus Natural/Original Consortium Culture...............................32
4.3
Cr(VI) Reduction Kinetics...............................................................................................34
4.3.1
Cr(VI) Reduction Kinetics Under Aerobic Conditions...................................................34
4.3.2
Cr(VI) Reduction Under Anaerobic Conditions..............................................................36
4.4
Total Biomass Evaluation................................................................................................38
4.4.1
Evaluation of Total Biomass at the initial Cr(VI) concentration of 100mg/L.................38
4.4.2
Evaluation of Total Biomass in the initial Cr(VI) concentration of 400 mg/L................38
4.5
Kinetic Modelling Theory................................................................................................39
4.5.1
Enzyme Kinetics..............................................................................................................39
4.5.2
Anaerobic Batch Cultures Modelling...............................................................................41
4.6
Parameter Estimation.......................................................................................................42
4.6.1
Kinetic Parameter Estimation under Anaerobic Conditions............................................43
4.7
Sensitivity Analysis..........................................................................................................44
4.7.1
Sensitivity Analysis of Anaerobic Batch Culture Kinetics..............................................44
4.8
Summary...........................................................................................................................45
viii
CHAPTER 5: MICROCOSM Cr(VI) REDUCTION KINETIC STUDIES……................46
5.1
Conceptual Basis of Microcosm Studies..........................................................................46
5.2
Performance Evaluation....................................................................................................46
5.2.1
Reconstituted Consortium Culture versus Native Soil Culture........................................46
5.3
Microcosm Kinetic Studies...............................................................................................47
5.3.1
Cr(VI) Removal Kinetics at Various Time Intervals........................................................47
5.3.1
Cr(VI) Removal Kinetics at Various Lengths..................................................................49
5.4
Microbial Culture Dynamics in the Aquifer System........................................................52
5.4.1
Characteristic of Initial Inoculated Consortium Culture..................................................52
5.4.2
Characterization of Inoculated Columns after Operation.................................................52
5.5
Kinetic Modelling of Cr(VI) Reduction in the Microcosm Columns...............................55
5.5.1
Model Description............................................................................................................55
5.5.2
Model Validation..............................................................................................................59
5.5.3
Parameter Optimization....................................................................................................59
5.5.4
Cr(VI) Removal Kinetics at Lower and Higher Concentrations......................................59
5.5.5
Summary of Parameters....................................................................................................60
5.6
Steady State Performance Model.....................................................................................65
5.6.1
Model Formulation...........................................................................................................65
5.6.2
Steady State Spatial Simulation........................................................................................67
5.6.3
Summary of Steady State Kinetic Parameters…....…….........………………………….71
5.7
Summary……………………….............………………………..................…………....71
CHAPTER 6: SUMMARY AND CONCLUSION………………….…....................……….73
APPENDIX A:
AQUASIM Version 2.0………............……………….....……………...75
APPENDIX B:
Octave Version 3.0………………..........…………………….....….…....87
APPENDIX C:
Target Site………………………….....…………………………............88
REFERENCES: ………...………………………………..……………………………………89
ix
List of Figures
Figure2-1
Cr cycle in environment..........................................................................
7
Figure2-2
World chrome ore reserves......................................................................
8
Figure2-3
World chromium ferroalloy production..................................................
8
Figure2-4
World production of chrome ore.............................................................
8
Figure2-5
Industrial usage of chromium ……….....................................................
9
Figure3-1
Laboratory set up microcosm columns……............................................
30
Figure4-1
Cr(VI) reduction between individual potential pure isolates and
reconstituted consortium culture at the initial Cr(VI) concentration of
100 mg/L under aerobic conditions……………..……...........................
Figure4-2
33
Comparison between reconstituted and natural consortium culture at
the initial Cr(VI) concentrations of 100 mg/L under aerobic
conditions……………………………………………………………….
33
Figure4-3
Aerobic Cr(VI) reduction in pure isolates at 50 mg/L.............................
35
Figure4-4
Aerobic Cr(VI) reduction in pure isolates at 100 mg/L...........................
35
Figure4-5
Cr(VI) reduction in aerobic reconstituted consortium culture at (50400 mg/L)………………………………………....................................
36
Figure4-6
Anaerobic Cr(VI) reduction in pure isolates at 50 mg/L.........................
37
Figure4-7
Cr(VI) reduction in anaerobic reconstituted consortium (50-200
mg/L).......................................................................................................
Figure4-8
37
Total biomass of aerobic reconstituted consortium culture at 100 and
400 mg/L..................................................................................................
38
Figure4-9
Anaerobic batch culture model validation at (50-200 mg/L)..................
43
Figure4-10
Sensitivity test for the initial Cr(VI) concentration of 100 mg/L with
respect to optimized parameters in anaerobic batch culture...............
Figure5-1
44
Performance of comparison between inoculated reactors with carbon
source and without carbon source at the initial Cr(VI) concentration of
20 mg/L....................................................................................................
Figure5-2
Performance of inoculated column without carbon source in
comparison with sterile-control column reactor at 30 mg/L …………..
48
48
x
Figure5-3
Performance of inoculated reactor amended with carbon source at 40
mg/L…………………………………………………………………….
Figure5-4
Performance of a sterile control reactor in removing Cr(VI) feed
concentration of 20 mg/L across the column...........................................
Figure5-5
50
Performance of native soil culture column in removing Cr(VI) feed
concentration of 20mg/L across the column............................................
Figure5-6
49
50
Performance of inoculated reactors in removing Cr(VI) feed
concentration of 20 mg/L in (A) C-source reactor, and (B) non Csource reactor…………………………………………………………...
Figure5-7
51
Phylogenetic tree of persistent bacterial cells in inoculated reactor
columns after operation derived from the 16S rRNA gene sequence,
Bacillus species........................................................................................
Figure5-8
53
Phylogenetic tree of persistent bacterial cells in inoculated reactor
columns after operation derived from the 16S rRNA gene sequence,
Enterococcus species...............................................................................
Figure5-9
54
Phylogenetic tree of persistent bacterial cells in inoculated reactor
columns after operation derived from the 16S rRNA gene sequence.
Possible Cr(VI) reducing species were detectable including,
Enterobacter species, and E. coli............................................................
Figure5-10
Simulation of Cr(VI) effluent at 20 mg/L in a (A) sterile control
column, (B)carbon source and non-carbon source reactor......................
Figure5-11
64
Simulation of Cr(VI) effluent in a carbon source reactor at various
lengths......................................................................................................
Figure5-13
63
Simulation of Cr(VI) effluent at (A) 30 mg/L in a non carbon source
reactor, (B) 40 mg/L in a carbon source reactor......................................
Figure5-12
55
69
Simulation of Cr(VI) effluent in a non-carbon source reactor over
length. The experimental data is the average values of the last three
sampling times.........................................................................................
70
xi
List of Tables
Table 2-1
Cr(VI) Reducing bacteria reported in literature.....................................
Table 3-1
Characterization of Cr(VI) reducing bacteria under aerobic
conditions……....................................................................................
Table 3-2
17
25
Characterization of Cr(VI) reducing bacteria under anaerobic
conditions…...........................................................................................
26
Table 3-3
Mineral composition of aquifer soil media…………………................
29
Table 4-1
Optimum kinetic parameter in anaerobic batch cultures…...................
44
Table5-1
Summary of performance of Cr(VI) reduction after
column
operation…….…...................................................................................
Table5-2
Optimum kinetic parameter values obtained for the biofilm in a
carbon source reactor...…......................................................................
Table5-3
62
Optimum kinetic parameter values for the biofilm at steady state in a
carbon source and a non-carbon source reactor.....................................
61
Optimum kinetic parameter values obtained for the biofilm in a noncarbon source reactor…………………...………………......................
Table5-4
47
68
xii
List of Abbreviations
AAS
Atomic adsorption spectrophotometer
APHA
American public health agency
BLAST
Basic Logical Alignment Search Tool
ChrR
Cr(VI) reductase
Cr
Chromium
Cr(VI)
Hexavalent chromium
Cr(III)
Trivalent chromium
CRB
Cr(VI) reducing bacteria
+CS
With carbon source
-CS
Without carbon source
CRL
Control
CFU
Colony forming units
DNA
Deoxyribonucleic acid
ETC
Electron transport chain
INC
Inoculated
MSM
Mineral salt medium
NADH
Nicotinamide adenine dinucleotide
NADPH
Nicotinamide adenine dinucleotide phosphate
NTV
Native soil culture
pH
Potential hydrogen
ppm
Parts per million
PVC
Polyvinyl chloride
RT-PCR
Reverse transcriptase- Polymerase chain reaction
rDNA
Ribosomal deoxyribonucleic acid
rRNA
Ribosomal Ribonucleic acid
rpm
Rotation per minute
TCA
Tricarboxylic acid
US EPA
United States Environmental Protection Agency
UV
Ultraviolet
WHO
World Health Organization
xiii
Symbol Nomenclature
Af
A
C
C
C
biofilm surface area (L2)
cross-sectional area of a reactor column (L2)
Cr(VI) concentration at time, t (ML-3)
state variable (ML-3)
Cr(VI) concentration at the surface (ML-3)
Cb
Cr
C eq
Dw
j
jc
k
K
Cr(VI) concentration in the bulk flow (ML-3)
Cr(VI) toxicity threshold concentration (ML-3)
equilibrium concentration at surface area (ML-3)
dispersion coefficient (L2T-1)
mass transport rate (LT-1)
Cr(VI) flux rate (ML-2T-1)
reaction rate coefficient (LM-3T-1)
inhibition coefficient (ML-3)
Kc
half velocity constant (ML-3)
kad
kd
k
adsorption rate coefficient (T-1)
cell death rate (T-1)
maximum specific Cr(VI) reduction rate(T-1)
Lw
L
ρc
Q
qc
rc
Rc
t
u
V
V
stagnant film thickness (L)
length of the reactor (L)
soil particle density (ML-3)
inflow rate (L-3T-1)
adsorption rate (ML-3T-1)
Cr(VI) reduction rate (ML-3T-1)
Cr(VI) reduction capacity coefficient (MM-1)
time (T)
flow velocity (LT-1)
volume of the reactor (L3)
differential volume (L3)
Xo
X
initial biomass concentration (ML )
biomass concentration at time, t (ML-3)
S
i
m
-3
Subscripts
C
chromium
f
in biofilm
in
influent
w
in water
o
initial
xiv
CHAPTER 1
INTRODUCTION
1.1 Background
Groundwater is usually of excellent quality, being naturally filtered in its passing through the
ground. Unfortunately, a threat is now posed by an ever-increasing number of soluble chemicals
from industrial activities. These chemicals are not completely removed by filtration as
groundwater passes through the aquifer. The principal pathway by which these metal ions may
enter groundwater systems includes leakage from the storage ponds, storm water run-on/off and
uncontrolled leaching from landfills (Moncur et al., 2005). The rate at which these metaleffluents enter the environment alters the natural flow of materials in the environment and also
cause potential hazard to the health of human and other life forms.
Chromium [Cr] is one of the most important chemical contaminant of concern which has been
classified as a priority pollutant by the United States Environmental Protection Agency (USEPA)
(Smith et al., 2002). It is the seventh most abundant element in the earth’s crust, which was
initially discovered by Nicolasa-Louis Vauquelin in 1797. Cr is detectable in the earth crust in
small quantities associated with other metals, particularly iron (Fe). The average concentration of
Cr in the continental crust has been reported as 125 mg/kg (National Academy of Science
(NAS), 1974). Cr exists in a series of oxidation states ranging from (-II) to (+VI), (Fendorf,
1995; Smith et al., 2002). However, only trivalent chromium [Cr(III)] and hexavalent chromium
[Cr(VI)] are of environmental significance as a result of their most stable oxidation state in the
natural environment.
Cr(III) and Cr(VI) display contrasting toxicity, mobility and bioavailability in the environment.
Cr(VI) is a potential soil, surface and groundwater contaminant that readily spreads beyond the
site of initial concentration through aquatic and groundwater systems (Cervantes et al., 2001;
Kamaludeen et al., 2003). It is rated as the third most abundant pollutant from anthropogenic
sources only superseded by organic pollutant species. Cr(VI) is also known to be mutagenic to
most organisms and carcinogenic to humans (Francisco et al., 2002; Caglieri et al., 2006). Due to
Cr(VI) toxicity, stringent regulations are imposed on the discharge of Cr(VI) to surface water to
1
below 0.05 mg/L by the U.S. EPA (Kiilunen, 1994; Baral et al., 2002; Kobya, 2004), while the
total Cr, including Cr(III), Cr(VI) and its other forms are regulated to below 2 mg/L (Zayed and
Terry, 2003).
On the other hand, the reduced form of chromium, Cr(III), is less toxic, less soluble, and forms
insoluble precipitates at higher pH (5.5-10). Cr(III) is also essential (in low concentrations) for
human and animal nutrition (Zayed and Terry, 2003; Viamajala et al., 2004). Therefore the
strong impact of Cr(VI) on the environment and also to human and animal health has increased
the demand for suitable technologies to neutralize the hazardous Cr(VI) to the less toxic Cr(III).
Currently, most of the Cr(VI) contaminated sites around the world are conventionally treated
using the pump-and-treat or dig-and-treat method which involves pumping or digging out the
contaminated material, adding of chemical reductants, precipitation followed by the
sedimentation or adsorption steps (Nyer, 1992; Eid and Zahir, 1996; Watts, 1998). These
methods, however, are not suitable for large scale wastewater treatment especially in developing
countries as they may be cost intensive and environmentally unfavorable. Additionally, chemical
products used for treatment generate harmful residuals and by-products that are difficult to treat.
Among the most recent alternative technologies for remediation of Cr(VI), microbial reduction
of Cr(VI) to Cr(III) as a normal function of their metabolism offers promise as a technology that
could play an important role in the decontamination of polluted sites. A wide array of bacterial
strains are capable of reducing Cr(VI) to Cr(III) under both aerobic and anaerobic conditions
(Guha et al., 2001; Zouboulis et al., 2004; Dermou et al., 2005; Zakaria et al., 2007;
Congeevaram et al., 2007; Zahoor and Rehman, 2009; Ahmad et al., 2010; Tekerlekopoulou et
al., 2010). However, most of the studies considering the effectiveness of microbial Cr(VI)
reduction for the treatment of wastewater under various environmental conditions were
performed in the laboratory using suspended cell systems (Chen and Hao, 1998; Shakoori et al.,
2000; Megharaj et al., 2003). In situ bioremediation technology using permeable reactive
barriers is a relatively new application which has been tested and sometimes implemented for
organic pollutants but not for toxic metals detoxification/removal (Liu et al., 2006). Only
recently, a detailed analysis on in situ Cr(VI) biological treatment focusing on the remediation of
spillage of Cr(VI) waste on the ground was conducted at the laboratory level (Molokwane and
Chirwa, 2009). However, in this study smaller laboratory scale columns (22-30 cm long) with
experimental data available only for the inlet and the outlet ports were used for this purpose.
2
The current study focuses on using larger laboratory scale columns to internally evaluate barrier
system. In this study the experimental data was collected from equally space longitudinal
sampling ports across each reactor to facilitate the finite difference modelling of Cr(VI)
concentration profiles along the column and to completely understand Cr(VI) reduction kinetics
within the reactor system. Fundamental knowledge and understanding of kinetic processes within
the reactor system responsible for Cr(VI) transformation will be valuable in developing the
appropriate biological systems that could be used to effectively treat Cr(VI) at contaminated sites
as well as predicting the microbial impact on the long term stewardship of the contaminated
sites.
1.2 Methodology and Objectives of the Study
The initial step towards the methodology of this study was to collect as much information as
possible related to the impacts of Cr(VI) pollution and current treatment practices from literature.
The primary objective of the research was to evaluate the prospect of pollution control in
groundwater aquifers using Cr(VI) reducing bacteria isolated from the local environments. In
order to achieve the primary objective, different experimental tasks were conducted on the
Cr(VI) reduction process, viz:

Investigation of Cr(VI) reduction kinetics in indigenous Cr(VI) reducing bacteria
grown both aerobically and anaerobically in batch reactors over a wide range of
initial Cr(VI) concentrations.

Evaluation of Cr(VI) reduction in aquifer microcosm reactors over a range of Cr(VI)
feed concentrations.

Investigation of the microbial culture shift in a microcosm system after operation.

Development of a mathematical model that simulates the contaminant movement
across the microcosm reactor at a transient state.

Development of the mathematical model that simulates the contaminant movement
across the reactor at a steady-state.
3
1.3 Outline of Dissertation
The outline of this dissertation is subdivided into three main parts:
Literature Review– contains the background information of the study and the records of recent
developments on the Cr(VI) bioremediation process. The information is focused on the
occurrence of chromium in the environment, impact of Cr on human health, animals and
microorganisms, remediation strategies, Cr(VI) reducing microorganisms, and biological Cr(VI)
reduction pathways.
Materials and Methods– illustrate all the materials and methods used during the study.
Cr(VI) Reduction Kinetic Studies– contains the performance evaluation studies and the kinetic
modelling of the batch system and continuous-flow bioreactor system.
1.4 Significance of Research
The introduction of Cr(VI) reducing bacterial species isolated from the sand drying beds could
be used in the formulation of biological permeable barriers for protection against the spread of
Cr(VI) contamination in groundwater systems. The model developed in this study under both
transient and steady-state is suitable for simulation of the contaminant movement in the porous
aquifer media under a range of Cr(VI) feed concentrations and it can be easily modified for
application in engineered biological systems for treating wastewater with higher concentrations
of toxic metals.
4
CHAPTER 2
LITERATURE REVIEW
2.1 Chromium Occurrences in the Environment
2.1.1 Chromium in Water
Trivalent chromium (Cr(III)) in water originates from natural sources, such as the weathering
of rock constituents, wet precipitation and dry fallout from the atmosphere, and run-off from
the terrestrial systems. Cr(III) can form both anionic (Cr(OH)4-, CrCl63-) or cationic
(Cr(H2O)63+, Cr(OH)2+) compounds, which are considered to be non-labile, inert species in
the environment. The main aqueous Cr(III) species are Cr3+, Cr(OH)2+, Cr(OH)3 and
Cr(OH)4–. Cationic Cr(III) is regarded as relatively nontoxic, and above pH 5.5 it precipitates
virtually as insoluble oxides and hydroxides, Cr(OH)3 in soil and water systems (McGrath
and Smith, 1990).
Hexavalent chromium (Cr(VI)) on the other hand is rarely naturally occurring in the
environment. Only 0.001% is attributed to natural geological processes (Merian, 1984).
Cr(VI) enters environmental water almost exclusively as a result of anthropogenic activities.
Cr(VI) compounds are highly soluble in water and forms chromates (CrO42-, HCrO4-) and
dichromate (Cr2O72-) which are thermodynamically stable over a large pH range in the
environment (Uyguner and Bekbolet, 2003). The equilibria of the Cr(VI) oxygenated species
favours extremely high solubility and is pH dependent. The following equations describe the
distribution of Cr(VI) species in aqueous solution:
H2CrO4 HCrO4- + H+
(2-1)
2HCrO4- Cr2O72- + H2O
(2-2)
HCrO4- CrO42- + H+
(2-3)
The Cr2O72- anion is dominant in acidic solution while the CrO42- prevails in basic or slightly
acidic Cr(VI) aqueous solution (Jain et al., 2005).
5
2.1.2 Chromium in Soil
The main source of Cr in natural soils is the weathering of the parent materials. The
concentration of Cr(III) and Cr(VI), in natural soil ranges from 7-220 mg/kg (McBride,
1994). However, in most soils Cr exists as Cr(III) and occurs within mineral structures or as
mixed Cr(III) and Fe(III) oxide. The compounds of Cr(III) in soil are considered to be stable
due to their slight mobility in acid media and their precipitation at pH 5.5.
In neutral to alkaline soils, Cr(VI) exists mostly in moderately to sparingly soluble chromates
(e.g. CaCrO4, BaCrO4, and PbCrO4) (James, 1996). In more acidic soils, (pH<6), HCrO4becomes a dominant form. The CrO42− and HCrO4− ions are the most mobile forms of Cr in
soils that can be easily taken up by plants into the deeper soil layers, causing ground and
surface water pollution (James et al., 1983b). Oxidation and reduction reactions in soil can
convert Cr(III) to Cr(VI) and Cr(VI) to Cr(III) (Makino et al., 1998). However these
oxidation and reduction processes are highly depend on pH, oxygen concentration, the
presence of appropriate reducers, and mediators acting as ligands or catalysts.
2.1.3 Chromium in Air
About 60% -70% of Chromium (Cr) present in the atmosphere originates from anthropogenic
sources and the remaining 30% - 40% is from the natural sources (Seigneur et al., 1995). The
main human activities contributing to the increase of Cr in the atmosphere are: ferrochrome
production, electroplating, pigment production, and tanning plus burning of fossil fuels,
stainless steel welding and waste incineration, while the natural sources of air-chromium are
forest fires and, volcanic eruptions, sea salt particles, erosion of soils and rocks (Pacyna et al.,
1988).
Chromium compounds in the air are present mainly as fine dust particles that eventually settle
over the land and water. Cr compounds can also occur in the air of non-industrialized areas in
concentrations of less than (0.1 µg/m3). The chemical forms of chromium in the air are not
known, but it is assumed that part of the air-chromium exists in the hexavalent form,
especially that is derived from high-temperature combustion. Chromium trioxide (CrO3) may
be the most important compound of Cr in the air (Sullivan, 1969).
6
atm O2
anthropogenic activities
duischarge and pollution
MnO2
Mn2+
leaching
plant uptake
adsorption/precipitation
red
OH
ox
Cr-citrate
R
HCrO4-
CO2
H 2O
Sun
ox
citrate
O
citrate
OH
red
Cr (III)
Cr 3+
precipitates & polymers
O
OH, organics
Figure 2-1: Cr cycle in environment (Bartlett, 1991; Yassi and Nieboer, 1988)
2.2 Production of Chromium and Its Use
Elemental Cr does not occur in nature, but is present in ores. Cr occurs in more than fifty
different ores such as barbertonite, brezinaite, chromite, chromitite and nichromite in nature.
Among the above mentioned Cr ores, chromite is the most important economical form of ore
and the two main products of the refined chromite ore are ferrochromium and metallic
chromium.
Cr production in the world is in the order of 10,000,000 tons per year (Cervantes et al., 2001).
About 72% of the mined chromium is used for metallurgical purposes, 12% for refractory
purposes and 17% for chemical purposes (Figure 2.5) (Papp, 1999). South Africa has
produced (since the 1940’s) 72% of the worlds Cr ore, with majority of the ore being mined
in the North Eastern region of the country (Mintek, 2004). South Africa is also the largest
exporter of chromite ore to the U.S., where chromite is not mined (Barnhart, 1997). Other
countries with exploitable Cr ore reserves include Philippines, Southern Zimbabwe, and
Turkey (Armitage, 2002) (Figure 2.2). Most of these chrome reserves come from the
bushveld igneous complex (BIC) ores and represent 44% of the world’s chromium ore
(Figure 2.3) and 47% of the world’s ferrochrome (Figure 2.4).
7
Figure 2-2: World chrome ore reserves (Armitage, 2002)
Figure 2-3: World chromium ferroalloy production (Armitage, 2002)
Figure 2-4: World production of chrome ore (Armitage, 2002)
8
Figure 2-5: Industrial usage of chromium (Papp, 1999)
Cr is used in metallurgy to manufacture ferrous and non-ferrous alloys. It is used in chemical
industries for pigment production, electroplating, leather tanneries, fungicides production and
wood preservation (Ryan et al., 2002; Middleton et al., 2003). Also, Cr serves as a catalyst in
the synthesis of many organic chemicals. The manufacture of chromite and chrome magnetite
bricks accounts for its use in the refractory (Palmer and Wittbrodt, 1991; Opperman and van
Heerden, 2007). As a result of its high corrosion, resistance and hardness, Cr can be
extensively utilized in manufacturing stainless steel.
The widespread use of chromium by metal and chemical industries (Kotas and Stasicka,
2000; Das and Mishra, 2008; Shai et al., 2009) produces wastes which are often very difficult
to treat. The Cr(VI)-bearing wastes may enter groundwater through improper disposal of
industrial effluent or through leakage due to improper handling and faulty storage containers
(accidental spills). Cr(VI), being highly mobile transport quickly into groundwater aquifers,
any of which may serve as direct water supply source for animal and human consumption in
communities that can not afford advance treatment of water (Krishna and Philip, 2005).
2.3 Potential Health Effects of Exposure to Chromium
2.3.1 Nutrition and Toxicity: Risk to Human and Animal Health
Chromium can enter the human body through ingestion or dermal contact. Relying on the
chemical, toxicological, and epidemiological evidence, regulation of Cr(VI) concentration is
different from that of Cr(III). Cr(III) is nutritionally required in trace amounts for normal
9
carbohydrates and lipid metabolism (Viamajala et al., 2004). When Cr(III) is at least taken up
through food and drinking water it may also even improve health and cure neuropathy and
encephalopathy. Deficiency to Cr(III) may increase the risk factors associated with diabetes
and cardiovascular diseases including elevated circulating insulin, glucose, and total
cholesterol (Zayed and Terry, 2003; Viamajala et al., 2004). However, long term exposure to
high concentration of Cr(III) may also lead to health problems such as cancer (Zhitkovich et
al., 1996).
Cr(VI) compounds on the other hand, have been found to be carcinogenic, mutagenic and
teratogenic to mammals (Flores et al., 1999; Francisco et al., 2002; Caglieri et al., 2006). The
toxicity of Cr(VI) on living organisms is associated to easy diffusion of Cr(VI) compounds
specifically chromate across the barrier of the cells via sulphate transport pathways as it bears
structural similarity with SO42- (Pattanapipitpaisal et al., 2002). Once in the cell, chromate
can oxidatively damage the DNA via the production of free radicals and cause illness such as
cancer within the living cell.
Short-term inhalation of high levels of Cr(VI) can cause adverse effects on human including
ulcers, irritation of nosal mucosa, allergic and asthmatic reactions, and nasal septum
perforation. Long-term exposure to high levels of Cr(VI) can cause kidney and liver damage,
stomach ulcers, irritation of the gastrointestinal tract, diarrhea, stomach and intestinal
bleeding, and death. Apart from its contact toxicity and carcinogenicity, Cr(VI) also causes
birth defects and decrease reproduction health in mammals (Losi et al.,1994b). The resulting
complications may result in death of the organism (Zayed and Terry, 2003). As a result of
these and other toxic effects, the World Health Organization (WHO) has set the maximum
acceptable chromium concentration in drinking water at 0.05 mg/L (Kiilunen, 1994; Lu and
Yang, 1995; Baral and Engelken, 2002).
2.3.2 Toxicity to Microorganisms
Cr(VI) is toxic to most microorganisms even at low concentrations, due to its ability to inhibit
enzyme activity, ‘poison’ cells non-specifically by blocking essential functional groups,
displacing essential metal ions and modifying the conformations of the biological molecules,
or induce mutations (Ehrlich, 1986). Cr(VI) ions are known to have inhibitory and mutagenic
effects on most microorganisms such as Escherichia coli, Bacillus subtilis, and Salmonella
10
typhimurium (Venitt and Levy, 1974; Petrilli and DeFlora, 1977; Ross et al., 1981; Zibilske
and Wanger, 1982; Aislabie and Loutit, 1984; Ajmal et al., 1984). The visible mutagenic
effects reported in bacterial species include cell elongations, cell enlargement, and inhibited
cell division, which eventually lead to cell growth inhibition (Coleman and Paran, 1983).
These mutagenic effects were reported to be only effective when Cr(VI) ions diffuse across
the cell membrane of a bacterial species. The subsequent reduction of these Cr(VI) ions
within the cell may alternatively result in the formation of free radicals which may generate
DNA alterations as well as toxic effects (Arslan et al., 1987; Kadiiska et al., 1994; Lui et
al.,1995; Molokwane, 2010). The genotoxic effects of bacterial cells include frame shift
mutations and base pair substitutions (Petrilli and DeFlora, 1977). Changes in morphologies
of gram-positive and gram-negative bacteria were also observed (Bondarenko et al., 1981).
2.4 Remediation Strategy
2.4.1 Physico-chemical Methods
Cr(VI) is currently extracted and treated from the contaminated environment using the
conventional methods including pump and treat, iron exchange, and electrochemical
precipitation method.
Pump and Treat Remediation
It is one of the most common approaches for contaminated groundwater remediation. This
method relies on pumps to bring polluted groundwater to the surface where it can be treated
efficiently and released back or reintroduced into the groundwater environment. The pump
and treat method may be considered as a best option in cases where the contaminant have
seeped into the groundwater. It can also be used to help to keep polluted groundwater from
spreading to drinking water wells. However this technique fails to attend to source of the
contamination in vadose zone and also create the problem of lowering the water table,
leaving behind residual contamination in new vadose zone. Also in new areas of low
permeability, residual levels of Cr will be missed, thereby creating future sources of
contamination (Bayer and Finkel, 2006).
Electrochemical Precipitation
This method utilizes an electrical potential to maximize the removal of heavy metal from
contaminated wastewater over the conventional chemical precipitation method (Kurniawan et
11
al., 2006). It is the most common method for removing toxic heavy metals up to parts per
million (ppm) levels from water. Electrochemical Cr(VI) reduction process is often employed
in combination with the pump and treat method and uses consumable iron electrodes and
electrical current to generate ferrous ions that react with Cr(VI) to Cr(III) as given:
3Fe2++ CrO42-+ 4H2O → 3Fe 3+ + Cr 3++ 8OH-
(2-4)
The efficiency of this method is affected by low pH and the presence of other salts (ions).
Additionally, this process takes a long period of time to reach the regulatory level for
remediation of contaminated sites and also it result in increased quantity of toxic sludge.
Ion Exchange
Ion exchange is a unit process by which ions of given species are displaced from an insoluble
exchange material by ions of a different species in a solution. In the ion exchange equipment
Cr-containing solution enters one end of the column under pressure, passes through the resin
bed and then Cr is removed from the solution. When the resin capacity is exhausted, the
column is backwashed to remove the trapped solids and then regenerated. Commonly used
matrices for ion exchange are synthetic organic ion exchange resins. The disadvantage of an
ion exchange method for Cr removal is that ion exchange resins are very selective (Lin and
Kiang, 2003). Additionally, ion exchange equipment can be quite expensive. Incomplete
removal of Cr in the salt solution is likely in the ion exchange method (Cabatingan et al.,
2001; Camargo, 2003). Furthermore, ion exchange equipment can not handle concentrated
metal solution as the matrix gets easily fouled by organics and other solids in wastewater and
also it is highly sensitive to pH of the solution.
The major drawbacks of these existing conventional treatment methods for Cr(VI)
contaminated soil and groundwater includes high energy expenditure in the process, use of
expensive toxic chemical reductants that result in the production of large quantity of toxic
sludge which is also difficult to treat (Komori et al.,1990; Blowes, 2002; Gonzalez et al.,
2003). This indicates that physicochemical methods are less effective in addressing the final
waste disposal problem. Bioremediation on the other hand is more attractive opinion as it
offers a potential of treating the waste under near neutral conditions and produces a minimum
or no toxic sludge.
12
2.4.2 Bioremediation Method
The term bioremediation has been used to describe the process of using living organisms;
primarily microorganisms to detoxify, degrade or destroy hazardous pollutants from the
environment (Glazer and Nikaido, 1995). Microbial Cr(VI) reduction have appeared to be
ubiquitous in nature as the consortia culture isolated from both Cr(VI) contaminated and
uncontaminated sites were able to reduce Cr(VI) (Turick et al., 1996; Chen and Hao, 1998;
Schmieman et al., 1998; Sani et al., 2002; Camargo et al., 2003). However the indigenous
microorganisms were more preferred over foreign isolates, as they displayed the best
characteristics for the remediation process (Vadali, 2001) and also their release in the
environment did not result into microbial diversity shift and yielding of new dominant
species in the environment.
The emerging technologies for bioremediation of Cr(VI) includes an in situ and an ex situ
technology. The in situ techniques are defined as those that are applied to soil and
groundwater at the site with minimal disturbance to the surrounding environment (Krishna
and Philip, 2005). Conversely ex situ techniques are defined as those that are applied to soil
and groundwater at an alternative site, in which case the contaminant is removed from the
actual site via excavation and pumping. In situ bioremediation technology is considered as
the most advantageous technique over the ex situ bioremediation technique as a result of its
low installation cost its potential to minimize the risk associated with waste transportation. In
situ bioremediation technology can therefore be applied to circumvent the limitation of
physicochemical methods.
Biological processes include (i) biotransformation (Shashidar et al., 2007; Molokwane et al.,
2008) which is the transformation of contaminated molecule into less or non-hazardous
molecules, (ii) biosorption (Juwarkar and Jambulkar, 2008) which involves the
detoxification/removal of hazardous substance instead of transferring them from one medium
to another by means of microbes and plants. It also defined as a metabolic passive process
(i.e. It does not require energy), and (iii) bioaccumulation is process similar to biosorption
process, but it differs in a way that it is an active metabolic process driven by energy from a
living organism and requires respiration (Velasquez and Dussan, 2009). In practise the
application of biological Cr(VI) reduction processes may be limited by high initial
concentrations of Cr(VI) which can cause a significant deactivation of the introduced
13
microorganisms; the presence of other metals and/or toxic organic compounds in the growth
medium which may severely inhibit the reduction activity of Cr(VI); the electron donor,
redox potential, pH and temperature (Shen and Wang, 1994a; Fulladosa et al., 2006; Wu et
al., 2010; Ye et al., 2010). Therefore efforts has been made by several authors to circumvent
the problem of limited biological Cr(VI) reduction capacity in contaminated environments by
isolating potential Cr(VI) reducing organisms that can survive the contaminated environment
and also by developing an appropriate biological reactor system that can effectively detoxify
Cr(VI) wastes both aerobically and anaerobically in the presence of other toxic compounds
(Mazerski et al., 1994; Shen and Wang, 1995; Chirwa and Wang, 2001).
2.4.3 Biological Systems Engineering
The principal biological systems used for environmental treatment can be divided into two
main categories: suspended and attached growth systems. The successful design and
operation of these systems requires full understanding of the types of microorganisms’
involved, specific reaction they perform, their nutritional needs and their reaction kinetics.
Suspended Growth System
In a suspended culture system the microorganisms which are responsible for the remediation
process are maintained in liquid suspension by appropriate mixing methods. The remediation
process in a suspended culture system may be operated under both aerobic and anaerobic
conditions with sufficient contact time provided for mixing the waste effluent with the
microbial suspension. Studies on suspended culture systems have been investigated by
several authors (Mazerski et al., 1994; Shen and Wang, 1994a; Wang et al., 2000). It was
observed that suspended culture were more susceptible to Cr(VI) toxicity. It was also
observed that shock loadings of Cr(VI) in a suspended culture reactor leads to excessive loss
in biomass (Wang et al., 2000; Molokwane, 2010). This implies that for effectives Cr(VI)
reduction in a suspended culture reactor cells re-inoculation is required. The other drawback
of suspended growth process is that during the actual treatment of highly Cr(VI) concentrated
influent stream dilution of highly Cr(VI) concentrated influent to lower Cr(VI) concentration
is required before treatment in the suspended culture reactor. This relatively indicates that
larger reactor volume are required for treatment of relatively low concentrations of Cr(VI).
14
Attached Growth (Biofilm) System
In a biofilm system the microorganisms responsible for the treatment process are attached to
an inert packing material. The packing material used in attached growth processes includes
gravel, soil, rocks and a wide range of plastic and other synthetic materials. Attached growth
processes can be operated as an open or a closed system. In the open system aeration occurs
while in the closed system no air penetration is allowed in or out of the vessel. In these
processes the microorganisms responsible for the treatment process forms a biofilm on the
packing material. The pollutants are removed by passing the waste effluent through the
biofilm at an optimum flow rate and hydraulic retention time to allow sufficient contact time
between the attached cells and the distributed contaminant over the packed material. Studies
on biofilm systems have been investigated by (Wanner et al., 1995; Nelson et al., 1996;
Beaudoin et al., 1998; Chirwa and Wang, 2001; Molokwane, 2010). Biofilm systems are
preferred over suspended culture systems as they enable biomass to be retained in the reactor
at flow rates greater than the washout flow rates during the operation. Higher removal
kinetics of Cr(VI) were also observed in the biofilm system than in the suspended one as a
result of culture acclimatization and mass transport resistance across the biofilm layer on cell
exposure to toxicity (Wang and Chirwa, 2001). This indicates that the exposure of Cr(VI)
toxicity to bacterial cells decreases with the increasing biofilm depth.
2.5 Cr(VI) Reducing Organisms
Cr(VI) is toxic to biological systems as a result to its strong oxidizing potential that can
damage cells (Kotas and Stasicka, 2000). However some microorganisms are able to reduce
toxic Cr(VI) to less toxic Cr(III) in the presence or absence of oxygen (Francisco et al., 2002;
Polti et al., 2007). The microorganisms that are able to reduce Cr(VI) to Cr(III) are known as
chromium reducing bacteria (CRB). Although Cr(VI) can be reduced by algae and other
plants in soil, bacteria has been demonstrated to be the most efficient microorganism in
Cr(VI) reducing process (Basu et al., 1997; Cervantes et al., 2001; Ganguli and Tripathi,
2002; Francisco et al., 2002). Bacteria can reduce Cr(VI) to Cr(III) either aerobically or
anaerobically and in each case the process of reduction differs (Ackerley et al., 2004;
Molokwane, 2010).
There are multiple reports of mesophilic bacteria capable of reducing Cr(VI) under various
conditions. These include both gram-positive and gram-negative bacterial species (Pal and
15
Paul, 2005; Horton et al., 2006). Several authors have reported that gram-positive bacteria are
more chromate tolerant than gram-negative bacteria (Ross et al., 1981; Baldi et al., 1990;
Francis et al., 2000; Viti and Giovannetti, 2001; Viti and Giovannetti, 2005, Molokwane,
2010). These bacterial strains have been promoted for Cr(VI) contaminated environment as
they are able to protect themselves from toxic substances in the environment by transforming
toxic compounds through oxidation, reduction or methyliation into more volatile, less toxic or
readily precipitating form (Dermou et al., 2005). The sensitivity of gram-negative bacterial
species to Cr(VI) toxicity may be associated with their lack of true cell wall (Ross et al.,
1981).
The reduction of Cr(VI) by bacterial consortia culture isolated from the natural environment
have also been observed (Chirwa and Wang, 2000; Stasinanakis et al., 2004; Dermou et al.,
2005; Chen and Gu, 2005; Chang and Kim, 2007; Molokwane et al., 2008). The consortium
culture from the natural environment have been utilized for Cr(VI) reduction process in order
to obtain a close or a clear picture of what really happens in the environment when the
microorganisms do not live in pure cultures. The consortia culture have been found to be very
much effective in degrading and detoxifying a wide variety of pollutants in the environment
due to their diversity of metabolic pathways in the community (Sharma, 2002).
Microbial Cr(VI) reduction often results in the elevation of pH background. The increased pH
facilitates the precipitation of the reduced form of chromium as chromite oxide Cr(OH)3.In
general, microbial reduction of Cr(VI) and consequence precipitation of Cr(III) can be
illustrated as follows (Brock and Madigan, 1991; Zakaria et al, 2007):
3+
+
neutralpH
CrO42- +8H+ +3 e CRB
 Cr +4H2O 
  Cr(OH)3 +3H +H2O
(2-5)
where: CRB represent Cr(VI) reducing bacteria or enzyme. It can be seen in Equation (2-5)
that CrO42- needs to accept three electrons to be converted to Cr(III).
In the case of electron donor being acetate, microbial Cr(VI) reduction under anaerobic
conditions can be expressed as:
3CH3COO- + 8CrO42- + 17H2O CRB
 8Cr(OH)3 (s) + 6HCO3 + 13OH
(2-6)
16
Table 2- 1: Cr(VI) Reducing bacteria reported in literature
Name of a species
Isolation condition or system/Carbon source
References
Achromobacter
sp.CH1
Anaerobic/LB broth medium, glucose, acetate
Zhu et al., 2008
Acinetobacter
haemolyticus
Packed bed bioreactor/Liquid pineapple
wastewater
Ahmad et al., 2010
Activated sludge
Batch/Cheese whey, lactose, glucose, acetate,
citrate
Ferro Orozco et al., 2010
Agrobacterium
radiobacter EPS916
Aerobic-Anaerobic/glucose mineral salt
medium
Llovera et al., 1993
Aspergillus sp.
Batch/Potato dextrose broth and nutrient broth
Congeevaram et al., 2007
Bacillus sp.
Aerobic/sodium acetate
Zahoor and Rehman,
2009
Bacillus sp.E29
Aerobic/LB broth medium
Camargo et al., 2003
Bacillus subtilis
Cell suspension (Aerobic-Anaerobic)/glucose
Garbisu et al., 1998
Candida lipolytica
Batch/glucose
Ye et al.,2010
E. coli ATCC 33
456
Aerobic-Anaerobic/Nutrient broth medium,
glucose, acetate, glycerol and propionate
Shen and Wang, 1994b
Enterobacter
cloacae HO1
Anaerobic/KSC medium,sadium acetate
Wang et al.,1989
Ocherobacterium
Aerobic/glucose
Zhigou et al., 2009
Pentoea
agglomerans SP1
Anaerobic/acetate
Francis et al., 2000
Pseudomanas
putida MK1
Anaerobic/LB broth-citric acid
Park et al., 2000
Pseudomonas sp.
Batch/Anaerobic/VB broth medium, D-glucose,
Lactate or dextrose
McLean and Beveridge,
2001
Pseudomonas
aeruginosa
Aerobic/Nutrient broth medium, LB broth
medium
Auguilera et al.,2004
17
2.6 Cr(VI) Reduction Pathways
Heterogeneous organisms obtain their energy for metabolism by participating in several
oxidation-reduction reactions. In the environment where the photosynthesis does not occur
the transfer of electron is a driving force that governs all the microbial process. Depending on
the environment, microorganisms have adapted and evolved the ability to be able to mediate
various oxidation-reduction couples to conserve energy.
Depending on the microbial species the reduction of Cr(VI) can be explained by two
prevalent models: (i) direct enzymatic reduction, and (ii) indirect reduction. Direct enzymatic
reduction refers to the reduction by the metal reductase system whereas indirect mechanism
refers to Cr(VI) reduction mainly by conditions provided by bacterial source such as the
redox potential, or the bacterial metabolites (H2S).
Direct Enzymatic Reduction
It has been shown in various instances that Cr(VI) is fortuitously reduced by enzymes and
other primary physiological functions (Garbisu et al. 1998; Opperman and van Heerden,
2007). Direct enzymatic Cr(VI) reduction by bacterial species has been documented by
several researchers under both aerobic and anaerobic conditions (Fujie et al., 1996; Garbisu
et al., 1998; Guha et al., 2000; Yang et al., 2009; Molokwane, 2010). Microbial Cr(VI)
reduction under aerobic conditions has been reported to be generally associated with soluble
proteins using NADH as an electron donor or either as a requirement or for enhance activity
(Suzuki et al., 1992; Shen and Wang, 1993; Garbisu et al., 1998). In the absence of electron
donor, Cr(VI) reducing organisms may utilize endogenous reserves for reduction of Cr(VI)
through the activity of soluble reductase. Under anaerobic condition Cr(VI) reduction can be
carried out through energy yielding dissimilatory respiratory process in which Cr(VI) serves
as a terminal electron acceptor. The reduction of Cr(VI) under anaerobic conditions has been
reported to be generally associated with soluble reductase, a membrane-bound or both
reductase with the possibility of involving hydrogenase or cytochrome C3 (Tebo and
Obraztsova, 1998; Michel et al., 2001).
Indirect Enzymatic Reduction
Sulfate and iron-reducing bacteria are the two well known bacterial species which are able to
reduce Cr(VI) indirectly via their anaerobic metabolic end products, hydrogen sulphide (HS-)
18
and Fe (II), respectively (Pettine et al., 1994, 1998; Sedlak and Chan, 1997; Patterson et al.,
1997). The metabolic end products produced by these bacterial species act as a reducing
agent for the Cr(VI) in the medium.
2.6.1 Intracellular Processes
Transport of the metal across the cell membrane yields intracellular accumulation, which is
depended on the microbial activity (Asku et al., 1991). Earlier studies on microbial Cr(VI)
reduction indicate that bacteria such as P.putida PRS200, P. ambigua G-1 and E.coli
ATCC33456, produced a soluble reductase enzyme capable of catalyzing the reduction of
Cr(VI) to Cr(III). Experimentation with supernatant samples of cell extract and the intact
cells has been reported to show almost the same Cr(VI) reduction activity, indicating a
largely soluble reductase activity as a result of co-metabolism in cells (Shen and Wang,
1993).
In intracellular processes, Cr(VI) is reduced in the cytosol using cytoplasmic soluble
reductase enzymes. These enzymes play an intermediate role between associated biological
electron donors. The electron donors implicated with Cr(VI) reduction are NADH and/or
NADPH, which are active within a wide range of temperature (40-70C) and pH (6-9).
According to Suzuki et al. (1992), NADH in the cell protoplasm donates an electron to
Cr(VI) and generates Cr(V) that accepts two electrons from two molecules of the same coenzyme to produce Cr(III).
Cr6+ + e  Cr5+
(2-7)
Cr5++2 e  Cr3+
(2-8)
2.6.2 Extracellular Processes
Attached growth systems can influence the removal of metal species through adsorption or
extracellular polymeric substances and cellular excretions, which indicates that extracellular
processes are facilitated by viable microorganisms. In this process Cr(VI) reducing enzyme
are deliberately released into the media from the cell cytoplasm when Cr(VI) is detected in
the media to reduce Cr(VI). The evidence of extracellular Cr(VI) reduction has been
presented by few researchers (Shen and Wang, 1993; Chirwa and Wang, 1997b; Molokwane
19
et al., 2008) through a mass balance of Cr(VI) and the reduced Cr species in the supernatant
and cells. Extracellular Cr(VI) reduction is beneficial to the cell as the cell does not require
transport mechanism transfer chromate and dichromate into the cell and export Cr(III) into
the media. As a result, such reduction mechanism protect the cell from DNA damaging and
from Cr(VI) toxicity.
2.6.3 Membrane-bound Processes
The membrane-bound process may be facilitated by using dead or viable microorganisms.
Membrane bound Cr(VI) reductase has been revealed with P. fluorescens LB300, E. cloacae
HO1 (Bopp and Ehrlich, 1988; Wang et al., 1989). Mechanism of membrane-bound
reductases may ensure the occurrence of Cr(VI) reduction on the cell surface, forming
insoluble Cr(OH)3 in the external medium. As a result, such a reduction mechanism protects
cells from Cr(VI) toxicity.
2.7 Carbon Source
Chromium reducing bacteria may utilize a number of organic compounds to serve as electron
donors for the Cr(VI) reducing process. Although Cr(VI) reducing bacteria may utilize a
variety of organic compounds as electron donors for Cr(VI) reduction. Early studies have
shown that the majority of organic compounds which serves as electron donors are generally
limited to natural aliphatic, mainly low molecular weight carbohydrates, amino acids and
fatty acids (Wang and Shen, 1995). The selection of these organic compounds was dependent
on the growth condition and the type of Cr(VI) reducing microorganism. Addition of organic
compounds which are widely available and easily biodegradable (glucose, sodium acetate)
may support the growth of certain species under varies conditions, cause a dramatic increase
in the rate of Cr(VI) reduction of some species or protect the reducing enzyme from
inactivating. The effective role of brown sugar, in particular the glucose component, to
reduce Cr(VI) was also demonstrated by (Chirwa and Wang, 1997) as follows:
C6H12O6 + 8CrO42- + 34H+→8Cr3+ +6HCO3- + 20H2O
(2-9)
This equation indicates that complete brake down of 1 mol glucose would yield sufficient
energy to reduce 8 mol of Cr(VI). The effectiveness of glucose as a carbon source for the
reduction of Cr(VI) may be associated to the fact that glucose directly enters into glycolysis,
20
TCA cycle and ETC mechanism to donate energy/electrons for Cr(VI) reduction, whereas
other carbon sources need intermediate conversion process to form glucose.
2.8 Summary
Literature survey of this study illustrates that both physico-chemical and biological methods
have been utilized for the remediation of contaminated environments. The latter method
appeared to be of great interest compare to the traditional physico-chemical methods. The
microbial Cr(VI) reduction process has been investigated further by several authors under
various conditions using different systems. Although much research has been conducted on
Cr(VI) reduction processes, the problem of Cr(VI) pollution still remain a matter of concern
strongly affecting soil and groundwater systems. As an endeavor to solve the problem of soil
and groundwater contamination, this study evaluates the in situ bioremediation process as a
strategy for effective Cr(VI) pollution control in soil and groundwater systems.
21
CHAPTER 3
MATERIALS AND METHODS
3.1 Bacterial Culture
3.1.1 Sources of Cr (VI) Reducing Bacteria
The microbial culture consortium was collected from the sand drying beds at the Brits
Wastewater Treatment Works (North West Province, South Africa). The samples
were collected in sterile containers and stored at 4C in the refrigerator until used.
3.1.2 Culture Isolation
Bacteria cultures were isolated from the samples collected from sand drying beds at
Wastewater treatment plant using the enrichment culture technique. A grain (0.2 g) of
sludge was inoculated in the sterilized media (100 mL, Luria-Bettani (LB) broth
amended with 75 mg/L of Cr(VI)) for culturing. The inoculum was incubated for 24
hours at 30  2C under shaking at 120 rpm in a Rotary Environmental Shaker
(Labotec, Gauteng, South Africa). Aerobic cultures were grown in cotton plugged 250
mL Erlenmeyer flasks whereas anaerobic cultures were grown in 100 mL serum
bottles purged with pure (nitrogen) N2 gas (99% pure grade) and sealed with silicon
rubber stoppers and aluminium seals prior to incubation. After 24 hours enriched
bacteria strains were isolated by serial dilution of the cultivated culture.
Pure cultures were prepared by depositing 1 mL of serially diluted sample from the 7th
to the 10th tube in the petri dishes containing LB agar using the spread method. The
plates were then incubated for 24-48 hours at 30  2C to develop separate
identifiable colonies. Individual colonies based on their colour and morphology were
transferred into 100 mL sterile LB broth amended with 150 mg/L of Cr(VI) using a
heat sterile wire loop. Cells were allowed to grow for 24 hours and then again 1mL of
24 hours grown culture was serially diluted and 1mL from the 7th to the 10th tube was
deposited into a LB agar plate and incubated for 24-48 hours at 30  2C. The
persistent colonies from the third isolation of 200 mg/L Cr(VI) were used for detailed
Cr(VI) reduction experiments.
22
3.2 Growth Media
3.2.1 Basal Mineral Media
Basal Mineral Medium (BMM) was prepared by dissolving: 10 mM NH4Cl, 30 mM
Na2HPO4, 20 mM KH2PO4, 0.8 mM Na2SO4, 0.2 mM MgSO4, 50 µM CaCl2, 25 µM
FeSO4, 0.1 µM ZnCl2, 0.2 µM CuCl2, 0.1 µM NaBr, 0.05 µM Na2MoO2, 0.1 µM
MnCl2, 0.1 µM KI, 0.2 µM H3BO3, 0.1 µM CoCl2, and 0.1 µM NiCl2 into 1 L of
distilled water and then amended with 5 g of glucose to act as a carbon and energy
source for the bacteria. The prepared medium was sterilized before use by autoclaving
at 121°C at 115 kg/cm2 for 15 minutes.
3.2.2 Commercial Broth and Agar
The first three media, Luria-Bettani (LB) broth, Luria-Bettani (LB) agar, and Plate
count (PC) agar (Merck, Johannesburg, South Africa) was prepared by respectively
dissolving 25 g, 45 g, and 23 g in 1000 mL of distilled water. The LB and PC agar
media were cooled at room temperature after sterilization at 121°C at 115 kg/cm2 for
15 minutes and then dispensed into petri dishes to form agar plates for colony
development.
3.3 Reagents
3.3.1 Standard Solutions and Chemicals
Cr(VI) stock solution (1000 mg/L) was prepared by dissolving 3.74 g of 99% pure
K2CrO4 (Analytical grade) in 1 L deionised water. This stock solution was used
through out the experiments to serve as Cr(VI) source. The standard solutions of
Cr(VI) were prepared from the Cr(VI) stock solutions in a 10 mL volumetric flask by
diluting certain volume of Cr(VI) stock solution with distilled water to give desirable
final concentrations ranging from 0-8 mg/L. From these data points (absorbance
against concentration) a linear graph/calibration curve with the regression of 99% was
obtained.
3.3.2 DPC Solution
Diphenyl carbozide (Merck, South Africa) solution was prepared for Cr(VI) reduction
analysis by dissolving 0.5 g of 1,5 diphenylcarbozide in 100 mL of HPCL grade
acetone and was stored in a brown bottle covered with a foil.
23
3.3.3 Chemicals
Sodium chloride solution (0.85% NaCl) was prepared by dissolving 1.85 g of sodium
chloride salt in 100 mL distilled water and sterilized by autoclaving at 121C for 15
minutes. All chemicals used were of analytical grade obtained from Sigma Aldrich,
Johannesburg, South Africa.
3.4 Culture Characterization
The phylogenetic characterization of cells was performed on isolated individual
colonies of bacteria from the 7th to the 10th tube in the serial dilution preparation. In
preparation for the 16S rRNA (16 Svedburg unit ribosomal Ribo-Nucleic-Acid)
fingerprint method which is used to obtain DNA sequences of pure isolated cultures,
the colonies were first classified based on morphology. Seven different morphologies
were identified for the aerobic cultures. These cultures were streaked on LB agar
plates followed by incubating at 30 ± 7C for 18 hours.
Genomic DNA was extracted from the pure cultures using a DNeasy tissue kit
(QIAGEN Ltd, West Sussex, UK). The 16S rRNA genes of isolates were amplified by
a reverse transcriptase-polymerase chain reaction (RT-PCR) using primers pA and
pH1 (Primer pA corresponds to position 8-27; Primer pH to position 1541-1522 of the
16S gene. An internal primer pD was used for sequencing (corresponding to position
519 - 536 of the 16S gene). The resulting sequences were deposited in the GenBank to
be compared to known bacteria using a basic BLAST tool search of the National
Centre for Biotechnology Information (NCBI, Bethesda, MD).
3.4.1 General Characterization of Aerobic Cultures using 16S rRNA ID’s
In preparation to 16S rRNA sequence identification, the colonies were first classified
based on their morphology. The 16S rRNA resulted in a total of seven aerobic
isolates. At 99% identity results indicated the predominance of four aerobic
phenotypes. Partial sequences of 16S rRNA matched the following bacterial species:

Bacillus cereus ATCC 10987, Bacillus cereus 213 16S,

Bacillus thuringiensis (serovar finitimus), Bacillus mycoides

Microbacterium foliorum and Microbacterium sp. S15-M4.
24
Table 3- 1: Characterization of Cr(VI) reducing bacteria under aerobic conditions
(Molokwane et al., 2008; Molokwane, 2010)
Pure Culture
Species Identified
% Identity
X1
Bacillus cereus strain 213 16S, Bacillus
99
thuringiensis 16S
X2
Bacillus sp. ZZ2 16S, Bacillus cereus ATCC 10987,
99
Bacillus thuringiensis strain Al Hakam
X3
Bacillus sp. 32-661 16S, Bacillus cereus 16S
99
X4
Bacillus mycoides strain BGSC 6A13 16S, Bacillus
99
thuringiensis serovar finitimus strain BGSC 4B2
16S
X5
Bacillus mycoides strain BGSC 6A13 16S, Bacillus
99
thuringiensis serovar finitimus strain BGSC 4B2
16S
X6
Bacillus mycoides strain BGSC 6A13 16S, Bacillus
99
thuringiensis serovar finitimus strain BGSC 4B2
16S
X7
Microbacterium sp. S15-M4, Micribacterium
99
foliorum
3.4.2 Culture Storage
To 80 mL of bacterial culture, 20 mL of sterile glycerol was added (final glycerol
concentration: 20%, v/v). The culture was then vortexed to ensure that the glycerol
was evenly dispersed and then transferred into a screw cap tube, labelled and stored at
–70C. In order to utilize the pure stored bacterial isolates, the frozen cultures were
allowed to melt at room temperature for approximately 10-15 minutes. The cultures
were then streaked onto the surface of an LB agar plate using a sterile inoculating
loop. The labelled LB plates were then incubated for 18-24 hours at 30  2C.
3.4.3 General Classification of Anaerobic Cultures using 16S rRNA ID’s
A total of eighteen different morphologies were identified in anaerobically grown
cultures using the method, with only ten colonies partially identified by the BLAST
results. The results obtained indicated the predominance of seven facultative
anaerobic phenotypes.
25
Table 3- 2: Characterization of Cr(VI) reducing bacteria under anaerobic conditions
(Molokwane et al., 2008; Molokwane, 2010)
Pure culture
Colour on plate
Blast results
X1
Light brown/cream
Could not subculture/amplify
X2
Off-white
Enterococcus avium,
Enterococcus pseudoavium
99
X3
Cream
Uncultured bacterium clone Y2,
Acinetobacter sp. ANT9054
97
X4
Coral
Could not subculture/amplify
X5
Yellow
Could not subculture/amplify
X6a
Yellow
Arthrobacter sp. Sphe3,
uncultured soil bacterium clone
TA12
Arthrobacter sp. AK-1
X6b
% Identity
93,94
99
X7
Cream and yellow
rings
Bacillus drentensis, B.
drentensis
96,97
X8
Light brown
Could not subculture/amplify
X9
Light brown
Could not subculture/amplify
X10
Light brown
Oceanobacillus sp. JPLAk1,
Virgibacillus necropolis
99,98
X11
Off-white
Enterococcus faecium strain
R0026, Rumen bacterium R4-4
99
X12
Coral
Paenibacillus pabuli,
Paenibacillus xylanilyticus
strain XIL14
99
X13
Yellow
Could not subculture/amplify
X14
Orange
Could not subculture/amplify
X15
Cream
[Brevibacterium]
frigoritolerans, Bacillus sp.
R21S
X16
Yellow
Could not subculture/amplify
X17
Cream
Uncultured bacterium, Bacillus
sp. BS19
99
93
26
3.5 Batch Reactor Studies
3.5.1 Aerobic Cr(VI) Reduction Experiments
The pure cultures were grown aerobically in a 1 L Erlenmeyer flask containing 400
mL LB broth for a period of 24 hours. Cells were then collected by centrifuging at
6000 rpm (2820 g) at 4C for 10 minutes. The supernatant was decanted and the
remaining pellet was washed three times in a sterile saline solution (0.85% NaCl)
while centrifuging. Aerobic Cr(VI) reduction experiments were conducted in 250 mL
Erlenmeyer flasks by adding Cr(VI) stock solution into 100 mL BMM to give the
desirable effective final Cr(VI) concentration ranging between 50-400 mg/L. Prior
inoculating the flask with harvested cells, 1 mL of a sample was initially withdrawn
from the Erlenmeyer flask to determine the absorbance of Cr(VI) before introducing
the cells in each flask. The flasks containing the suspended cells were then plugged
with cotton wool to allow aeration while filtering away microorganisms from the air
and then incubated at 30  2C with continuous shaking on a lateral shaker (Labotec,
Gauteng, South Africa) at 120 rpm. All experiments were duplicated and performed at
a stationary phase. To monitor Cr(VI) reduction 1 mL of the samples were withdrawn
at regular time intervals. The withdrawn samples were then centrifuged using a 2 mL
eppendorf tubes at 6000 rpm (2820 g) for 10 minutes and the supernatant was used for
Cr(VI) concentration analysis.
3.5.2 Anaerobic Cr(VI) Reduction Experiments
The pure cultures were grown anaerobically in a 1 L Erlenmeyer flask containing 400
mL LB broth for a period of 24 hours. Cells were then collected under anaerobic
conditions by centrifuging at 6000 rpm (2820 g) at 4C for 10 minutes. The
supernatant was decanted and the remaining pellet was washed three times in a sterile
saline solution (0.85% NaCl) under an anaerobic glove bag purged with 99% N2 gas.
Anaerobic Cr(VI) reduction experiment were conducted in 100 mL serum bottles by
adding Cr(VI) stock solution into the BMM to give the desirable effective final
Cr(VI) concentration ranging between 50-200 mg/L. Prior inoculating the serum
bottles with the harvested cells under anaerobic conditions, 1 mL of a sample was
withdrawn from each serum bottle at various Cr(VI) concentration to determining the
absorbance of Cr(VI) before inoculating the bottles with viable cells. The cells were
then transferred into 100 mL serum bottles under an anaerobic glove bag purged with
27
99% N2 gas. The samples in the bottles were then directly purged with 99% N2 gas for
about 10 minutes to expel any oxygen gas before sealing with silicon rubber stopper
and aluminium seals. The samples were then incubated at 30  2C with continuous
shaking on a lateral shaker (Labotec, Gauteng, South Africa) at 120 rpm. Cr(VI)
reduction was monitored by withdrawing 1 mL of the sample at regular time intervals
using a sterile syringe. The withdrawn samples were then centrifuged using a 2 mL
Eppendorf tube at 6000 rpm (2820 g) for 10 minutes in a Minispin® Microcentrifuge
(Eppendorf, Hamburg, Germany) before Cr(VI) analysis to remove suspended cells.
3.5.3 Abiotic Experiments
Heat killed cultures were used to determine the abiotic Cr(VI) reduction in the batch
experiments. Overnight grown cells were heat killed by autoclaving at 121C for 20
minutes. Cultures were collected by centrifuging at 6000 rpm (2820 g) for 10 minutes
and then washed three times in a sterile saline solution (0.85% NaCl), while
centrifuging. The pellet collected from centrifuge were then used for Cr(VI) reduction
processes.
3.6 Biomass Analysis
3.6.1 Total Biomass
Samples 5 mL were withdrawn at regular time intervals 0-24 hours, centrifuged for 10
minutes at 6000 rpm (2820 g). The supernatant was used to analyse Cr(VI)
concentration and the settled pellet was used for biomass analysis. The pellet was
resuspended in 1 mL distilled water and filtered through a pre-weight Whatman filter
paper No.1. The filter with the microorganism was dried in the oven at 75-80C to get
a constant weight. The difference between the dried filter paper with cells and the
empty filter paper was considered as a biomass.
3.6.2 Viable Biomass
Samples (1 mL) were withdrawn from experimental batches at regular time intervals
of 0-24 hours for the analysis of viable cell concentration. Samples were then serially
diluted into a 9 mL sterile 0.85% NaCl solution and from the 7th to the 10th tube 0.l
mL of the diluted sample were transferred into a PC agar plate using the spread
method. The PC agar plates were then incubated for 18-24 hours at 30 ± 2◦C.
28
Colonies were counted after incubation and multiplied by a dilution factor. The
bacterial count was reported as colony forming units (CFU) per mL of sample.
3.7 Microcosm Studies
3.7.1 Mineral Composition of Aquifer Media
Samples were collected the aquifer zone at the depth of 3m below ground surface and
200-300 m from the hot spots. The mineral composition of the aquifer media was
quantitatively measured by inductively-coupled plasma mass spectrometry (ICP-MS).
This was done to reveal the source of possible interference and levels of background
of chromium in the aquifer media. The presence of other elements such as iron,
manganese and nitrates in the soil which can act as electron sinks and accept electron
from reactive organic and inorganic sources in the reduction process of Cr(VI) are
expected to course interference in the Cr(VI) reduction process and elements such as
calcium and magnesium are expected to course interference in the spectrometric
analysis of Cr(VI). The background Cr concentration in the soil sample was detected
as 50 g/kg. Table (3-3) shows elementary soil composition of significant presence.
Table 3- 3: Mineral composition of aquifer soil media (Molokwane, 2010)
Element
Symbol
Mass concentration (g/kg)
Aluminium
Al
4003
Calcium
Ca
2868
Magnesium
Mg
542
Sodium
Na
248
Iron
Fe
15145
Manganese
Mn
543
Zinc
Zn
367
3.7.2 Reactor Setup
Four columns constructed from a Plexiglas (PVC glass), (60 cm long, 5 cm internal
diameter) were installed in a laboratory as continuous flow columns as shown in
Figure (3-1). Each column consisted of an influent port, five intermediate ports and an
effluent port. The columns were then packed with aquifer media from the target site.
Prior closing the columns on both ends with PVC caps, one of the packed columns
was sterilized by autoclaving at 121C for 20 minutes and the other two columns were
29
amended with an organic electron donor (sawdust) to represent the heterogeneous
carbon source from decaying vegetation. The four packed columns were then capped
on both ends with PVC caps and installed vertically on the board by clapping. Each
column was vertically interconnected to a 500 mL reservoir that gravimetrically
transfers the contaminants into the packed bed reactor through the interconnecting
tubes at the vertical height of 58 cm. Reservoirs were also interconnected to one
another horizontally from the main feed container through out the re-circulation point
to maintain the continuous flow process.
Figure 3- 1: Laboratory set up microcosm columns
3.7.3 Start up culture
Reconstituted consortium culture isolated from the dried sludge was cultivated for 24
hours in the LB broth medium amended with 75 mg/L of Cr(VI). The cultivated cells
were then harvested by centrifuging at 6000 rpm (2820 g) for 10 minutes and then
thoroughly mixed with a BMM.
3.7.4 Reactor Start up
Prior experimental run, distilled water was fed through each column in order to
saturate pores with water. Flow rates were measured and adjusted to establish the
hydraulic retention time (HRT) of approximately 24 hours in each reactor. Two
30
reactor columns were then inoculated with viable cells for 24 - 48 hours, enough time
to allow uniform distribution and attachment of cells to soil particles in the reactor.
3.7.5 Reactors Operation
Cr(VI) solution from the main feed container was continuously fed into the receiving
feed containers (reservoirs) through the peristaltic pump to maintain the feed level in
the reservoirs. Cr(VI) feed from the reservoirs was then gravimetrically transferred to
the packed bed reactors as in the case of open aquifers at the actual contaminated site.
The microcosm reactors were operated as packed beds at different Cr(VI)
concentrations of (20, 30, 40 and 50 mg/L) respectively. The experiments were
conducted for seven weeks under micro-aerobic condition at an ambient pH and
temperature. Prior running new feed of Cr(VI) concentration in each column, distilled
water was passed through the aquifer media in each column to remove or to wash out
the traces or the residual Cr(VI) in the aquifer media from the previous run. Samples
were then withdrawn from each sampling port for analysis.
3.8 Analytic Methods
3.8.1 Cr(VI) Analysis
Cr(VI) reduction was determined colorimetrically using UV/vis spectrophotometer
(WPA, Light Wave II, and Labotech, South Africa). The measurement was carried out
using the DPC method according to the following procedure: In a 10 mL volumetric
flask, 0.2 mL of a sample was acidified with 1mL of 1N H2SO4, then followed by
distilled water and 0.2 mL of 1,5 DPC up to the mark (APHA, 2005). The mixture
was then agitated thoroughly for about 15-30 seconds and let to stand for about 3
minutes for full colour development. The red-violet purple colour formed was then
measured at wavelength of 540 nm (10 mm light path) using the calibrated
instrument. Total Cr was measured at a wavelength of 359.9 nm using a Varian AA–
1275 Series Flame Atomic Adsorption Spectrophotometer (AAS) (Varian, Palo Alto,
CA) equipped with a 3 mA chromium hollow cathode lamp. Samples were digested
with concentrated nitric acid (HNO3) before analysis. Cr(III) was determined as the
difference between total Cr and Cr(VI) concentration. AAS was calibrated prior total
Cr analysis using 1-5 mg/L Cr(VI) concentration prepared from the Cr(VI) stock
solution and 2.5% nitric acid.
31
CHAPTER 4
CR(VI) REDUCTION BATCH KINETICS STUDIES
4.1 Preliminary Studies
Primarily, different cultures of bacteria from the dried sludge were tested for effectiveness in
reducing Cr(VI). This initial test was conducted at a low initial Cr(VI) concentration of 10
mg/L. It was observed from this test that four aerobic isolates (X2, X4, X5, X6) and three
anaerobic isolates (X6b, X12, X15) were able to remove Cr(VI) in the medium. Each of the
four aerobic isolates achieved complete Cr(VI) removal within 4 hours of incubation,
whereas the anaerobic isolates achieved near complete Cr(VI) removal at a longer period of
12 hours. The highest performing cultures were then used in future investigations that lead to
the development of a batch kinetic model and aquifer model.
4.2 Performance Validation in Cultures
4.2.1 Individual Pure Cultures versus Reconstituted Consortium Culture
Analysis under aerobic conditions were conducted to determine the rate of Cr(VI) reduction
in individual pure isolates and compare to performance with Cr(VI) removal by a
reconstituted consortium. Reconstituted consortium culture was obtained by culturing
specific colonies of the Cr(VI) reducing culture (X1+X2+X3+X4+X5+X6+X7). Results in
Figure (4-1) show that no species acting alone can achieve the same level of Cr(VI) reduction
rate as the reconstituted consortium culture. The reconstituted consortium culture out
performed the individual pure isolates acting alone. The two potential isolates acting alone
(X5 and X6) achieved 85% of Cr(VI) removal at 24 hours of incubation while reconstituted
consortium culture achieved a complete Cr(VI) removal within the same time of incubation
at the initial Cr(VI) concentration of 100 mg/L. These results indicate that CRB from the
dried sludge may provide a robust Cr(VI) reduction model.
4.2.2 Reconstituted Culture versus Natural/Original Consortium Culture
Figure (4-2) shows that the rate of Cr(VI) reduction in the reconstituted consortium was
much faster than the rate in the natural consortium culture. Hypothetically the rate of Cr(VI)
reduction was expected to be more faster in the natural consortium culture compared to the
reconstituted consortium culture. These unexpected results may be associated to higher
32
percentage of Cr(VI) reducing organisms in the reconstituted consortium culture due to preselection. The sterile control reactor on the other hand showed no Cr(VI) removal over time.
Cr(VI) concentration, mg/L
120
X5
X6
reconstituted consortium
culture(X1-X7)
100
80
60
40
20
0
0
20
40
60
80
100
Time, h
Figure 4- 1: Cr(VI) reduction between individual potential pure isolates and reconstituted
consortium culture at the initial Cr(VI) concentration of 100 mg/L under aerobic conditions
Cr(VI) concentration,mg/L
120
100
80
reconstituted consortium
culture (X1-X7)
60
original culture
(dried sludge)
cell-free (strerile-control)
40
20
0
0
10
20
30
40
Time, h
Figure 4- 2: Comparison between reconstituted and natural consortium culture at the initial
Cr(VI) concentrations of 100 mg/L under aerobic conditions
33
4.3 Cr(VI) Reduction Kinetics
4.3.1 Cr(VI) Reduction Kinetics Under Aerobic Conditions
Individual pure isolates X2, X4, X5, X6 acting alone achieved complete reduction of Cr(VI)
in batch with initial Cr(VI) concentration up 100 mg/L. Figure (4-3 and 4-4) shows that X6
was the highest performing isolate in reducing Cr(VI).
The reconstituted consortium, on the other hand, achieved a complete removal of Cr(VI) in
all batches under initial Cr(VI) concentrations up to 200 mg/L. For instance, 50 mg Cr(VI)/L
was completely removed within 4 hours of incubation, 100 mg Cr(VI)/L was completely
removed within 12 hours, and 200 mg Cr(VI)/L was completely removed within 50 hours of
incubation. The reduction activity of Cr(VI) at higher initial Cr(VI) concentrations of 300
and 400 mg/L was very slow compared to the reduction at relatively lower initial Cr(VI)
concentrations. Up to 82% of Cr(VI) was reduced after 120 hours of incubation at 300 mg/L
and further incubation to 200 hours increased the removal efficiency to 85%, approximately
60% of Cr(VI) was reduced within 200 hours of incubation at 400 mg Cr(VI)/L. Figure (4-5)
shows that at higher initial Cr(VI) concentrations (300 and 400 mg/L), the finite reduction
capacity of cells which is attributed to Cr(VI) toxicity on cells was reached after 60 hours of
exposure to Cr(VI). It also is observed in Figure (4-5) that once the finite reduction capacity
is reached, Cr(VI) reduction cease regardless of continued metabolic activity or cell
synthesis.
The aerobic experiments in this study clearly show that Cr(VI) reduction facilitated by cells
is inhibited by high initial Cr(VI) concentration in the medium. These observations are
consistent with early studies (Shen and Wang, 1994a; Shen and Wang, 1995; Wang and
Shen, 1997; Chirwa and Wang, 1997a) in which high levels of Cr(VI) inhibited both the
growth and Cr(VI) reduction capacity in both pure and mixed culture of bacteria. The
autoclave control on the other side exhibits no significant Cr(VI) reduction with time (Figure
4-5). Only 15% Cr(VI) removal efficiency was observed in heat-killed cells after the first 24
hours of incubation. The 15% removal in the heat-killed cells may be associated to the
reductase released into the medium from heat-lysed cells and regrwoth of cells that escaped
heat destruction. Therefore the insignificant Cr(VI) removal in the heat-killed cells over time
implies that the abiotic processes are negligible.
34
Cr(VI) concentration, mg/L
60
X2
X4
X5
X6
50
40
30
20
10
0
0
2
4
6
8
10
12
14
Time, h
Figure 4- 3: Aerobic Cr(VI) reduction in pure isolates at 50 mg/L
Cr(VI) concentration, mg/L
120
X2
X4
X5
X6
100
80
60
40
20
0
0
20
40
60
80
100
120
140
160
Time, h
Figure 4- 4: Aerobic Cr(VI) reduction in pure isolates at 100 mg/L
35
Cr(VI) concentration, mg/L
500
400
50 mg/L
100 mg/L
200 mg/L
300 mg/L
400 mg/L
heat killed cells(control)
300
200
100
0
0
50
100
150
200
Time, h
Figure 4- 5: Cr(VI) reduction in aerobic reconstituted consortium culture at (50-400 mg/L)
4.3.2 Cr(VI) Reduction Under Anaerobic Conditions
Cr(VI) reduction under anaerobic conditions was investigated due to its significant
engineering applications, since most of the environments where the in situ bioremediation
process could occur are closed systems such as sediment zones and under ground
environments whereby the direct contact with the atmosphere does not occurs. Additionally,
anaerobic biological processes are determined to be passive in nature with no excessive
energy input requirements for aeration. The experiments under anaerobic conditions were
conducted over initial Cr(VI) concentrations of (50–200 mg/L) at temperature of 30  2C
and pH 7 ± 0.2. Evaluation of Cr(VI) reduction at lower initial Cr(VI) concentration
compared to the aerobic Cr(VI) reduction experiments was associated to slower growth
observed in the anaerobic cultures. Figure (4-6) shows Cr(VI) reduction in individual pure
isolates X6b, X12 and X15. It is shown in this figure that the species (X6b, X12 and X15)
completely reduce 50 mg/L of Cr(VI) within 90 hours of incubation, whereas the
reconstituted anaerobic consortium culture achieved 78% removal efficiency at the initial
Cr(VI) concentration of 200 mg/L in 200 hours of incubation Figure (4-7). Results obtained
in anaerobic batch cultures showed a lower Cr(VI) reduction rate than in the aerobic culture.
36
Lower reduction rates of Cr(VI) in the anaerobic culture may be associated to slower
anaerobic bacterial activity.
Cr(VI) concentration, mg/L
60
X6b
X12
X15
50
40
30
20
10
0
0
20
40
60
80
100
120
Time, h
Figure 4- 6: Anaerobic Cr(VI) reduction in pure isolates at 50 mg/L
Cr(VI) concentration, mg/L
300
50 mg/L
100 mg/L
200 mg/L
250
200
150
100
50
0
0
50
100
150
200
Time, h
Figure 4- 7: Cr(VI) reduction in anaerobic reconstituted consortium (50-200 mg/L)
37
4.4 Total Biomass Evaluation
4.4.1 Evaluation of Total Biomass at the initial Cr(VI) concentration of 100 mg/L
The rapid increase in the biomass between 0–6 hours of exposure to Cr(VI) (Figure 4-8)
showed that the reconstituted consortium culture was not just surviving but also growing
rapidly at relatively high Cr(VI) concentration of 100 mg/L. A decline in biomass was
observed after 6 hours of incubation. The reason for this decline is not clear at the moment.
4.4.2 Evaluation of Total Biomass at the initial Cr(VI) concentration of 400 mg/L
The slight increase in biomass concentration between 0–12 hours was observed in the
bacterial consortium at 400 mg/L. The slow increase of suspended cell concentration within
the first 6 hours of exposure to high Cr(VI) concentration may be associated to cell
acclimatization to highly toxic environment. After 12 hours of incubation, the decline in
bacterial population was observed. These results suggest the inhibitive effect of Cr(VI) was
responsible for a slower growth rate.
14000
100 mg/L
Biomass, mg/L
12000
400 mg/L
10000
8000
6000
4000
0
5
10
15
20
25
Time, h
Figure 4- 8: Total biomass of aerobic reconstituted consortium culture at 100 and 400 mg/L
38
4.5 Kinetic Modelling Theory
4.5.1 Enzyme Kinetics
Cr(VI) reduction in living cells is assumed to be linked to cellular metabolism as it was
illustrated in the early studies (Wang et al., 1989; Ohtake et al., 1990; Shen and Wang, 1993
and others). Biochemical studies of enzymatic Cr(VI) reduction suggest that Cr(VI) reducing
mechanisms may be coupled to the membrane-electron transport system in Cr(VI) reducing
bacteria (Horitsu et al., 1989; Wang et al., 1989; Ishibashi et al., 1990; Srinath et al., 2002).
Following further research, Viamajala (2003) and others proposed that Cr(VI) reduction is
mediated by enzymes that are not substrate specific for Cr(VI) and that “chromate
reductases” may be serendipitous contributors to Cr(VI) reduction while engaged in other
primary physiological functions. The action of many different enzymes acting together can
have a net effect of one characteristic enzyme for a consortium culture, thus this effect can be
represented by one composite enzyme, ET.
The single enzyme kinetic model is based on the following reaction scheme:
Cr(VI) + E
k1


k2
k3
E*Cr(VI) 
E + Cr(III)
(4 -1)
where: E = enzyme, E*Cr(VI) = enzyme-Cr(VI) complex , k1 = rate constant for complex
formulation, k2 = rate constant for reverse complex formulation, k3 = rate constant for Cr(III)
formation.
Let Cr(VI) = C and E*Cr(VI) = E*
Therefore the rates of the above equation would be as follows:
dE *
 k1CET
dt k 1
(4-2)
dE *
 k2 E *
dt k  2
(4-3)
39

dE *
dCr ( III )

 k3 E *
dt k 3
dt
(4-4)
The rate formulation of E* can be represented as:
dE *
 k1CET  k 2 ( E*)  k 3 ( E*)
dt
(4-5)
where : ET (total complexe and un-complex enzyme) = E – E*
As soon as the enzyme and Cr(VI) are mixed ,the concentration of enzyme-Cr(VI) complex
will reach constant value so that a steady state can be applied as follows:
dE *
 0  k1C ( E  E*)  k 2 E * k 3 E *
dt
(4-6)
Solving Equation (4-6) for E* the following equation is obtained:
E* 
k1CE

k 1C  k 2  k 3
CE
k  k3
C 2
k1
(4-7)
Then the Cr(VI) reduction rate becomes:
 dC

dt
k 3CE
k  k3
C 2
k1
Analogous to Monod kinetics, k3 is analogous to maximum specific Cr(VI) reduction rate
(km), E is analogous to biomass concentration (X) and
k 2  k3
is analogous to half saturation
k1
constant (Kc).

kmC
 dC
X

dt
C  KC
(4-8)
This model was previously derived by several researchers (Shen and Wang, 1994b
Mazierski, 1995; Schmieman et al., 1998; Guha et al., 2001) where the soluble reductase
activity was the predominant mechanism of Cr(VI) reduction in bacterial systems. It was
40
observed as an appropriate expression to cope with both toxicity and mutation effect of
Cr(VI) on Cr(VI) reduction. It was also determined from this model that the rate and the
extend of Cr(VI) reduction in bacterial system depends on the number of cells in the reactor
and the capacity of reduction per cell represented by the term Rc. This indicates that the
amount of Cr(VI) reduced under resting cells conditions will be proportional to the amount of
cells inactivated by Cr(VI), thus the active biomass concentration is assumed to decrease
with the increasing amount of Cr(VI) reduced due to toxicity (Shen and Wang, 1994b).
Therefore X (biomass concentration) in Equation (4-8) can be represented as:
X  X0 
C0  C
Rc
(4-9)
Equation (4-8) can therefore be represented as follows:
k C
 dC
 m
dt
C  Kc

C C 
 X o  0

Rc 

(4-10)
where: km = maximum specific rate of Cr(VI) reduction (T-1); Kc = half-velocity
concentration (M.L-3); Xo = initial biomass concentration (ML-3); C = Cr(VI) concentration at
time, t, (ML-3); C0 = initial Cr(VI) concentration (ML-3); and Rc = Cr(VI) reduction capacity
of cells (MM-1).
4.5.2 Anaerobic Batch Cultures Modelling
The biotransformation of Cr(VI) to Cr(III) in batch culture result from the activity of cells.
Although the reduction of Cr(VI) to Cr(III) may be limited by reaction kinetics under
physiological conditions (Glaze, 1990). The kinetics of Cr(VI) reduction in the anaerobic
batch culture may be improved by coupling Cr(VI) reduction to the energy yielding
dissimilatory respiration process in which Cr(VI) serves as a terminal electron sink (Lovley
and Phillips, 1994). It was observed in the anaerobic batch cultures of this study that Cr(VI)
reduction (metabolic process) under low initial Cr(VI) concentration appeared to benefit
from the presence of Cr(VI). Therefore in such a case the toxicity threshold concentration
had to be reached before Cr(VI) inhibition become effective. The model developed in
Equation (4-10) based on enzymatic Cr(VI) reduction kinetics could not describe the kinetics
41
for Cr(VI) under anaerobic conditions as the kinetic process under anaerobic conditions was
more complex as a result of high biodiversity of anaerobic species and slow growing culture
that is susceptible to toxic loading of Cr(VI). Therefore the results obtained in Equation (410) suggested a Cr(VI) reduction kinetic equation that accounts for Cr(VI) toxicity threshold
concentration (Molokwane and Chirwa, 2009). The non-competitive inhibition model that
accounts for Cr(VI) toxicity threshold concentration is represented as follows:
 dC

dt
kmC
K
 Cr
 1
 C
o





K c  C 

C C 
 X o  o

Rc 

(4-11)
where: km = maximum specific rate of Cr(VI) reduction (T-1); Kc = half-velocity concentration
(M.L-3); Cr = Cr(VI) toxicity threshold concentration (ML-3); Xo = initial biomass
concentration (ML-3); K = limiting constant (ML-3); and Rc = maximum Cr(VI) reduction
capacity of cells (MM-1).
4.6 Parameter Estimation
The unknown kinetic parameters in Equation (4-11), km, Kc, Ki, K and Rc were determined by
performing a nonlinear regression analysis using the Computer Program for Identification
and Simulation of Aquatic Systems (AQUASIM 2.0), (Riechert, 1998). For each parameter, a
search was carried out through a range of values. Trial values of the unknown parameters
were initially guessed. Constrains were also enforced to set upper and lower limits for each
parameter so that nonsensical or invalid parameter values were omitted. Whenever
optimization converged at/or very close to a constraint, the constraint was relaxed until the
constraint no longer forced the model.
The process was repeated until unique values lying away from the constraints but between set
limits were found for each parameter. The best fit values were obtained by repetition of
parameter estimation of the unknowns. The objective function for parameter optimization
was defined as the least sum of the squares between the observed and the modelled
concentrations and was computed as follows:
1 i n
 
  yi  y 
n  q i 1
2
2
(4-12)
42
where:  = average deviation of model from the measured values; y i = observed variables; y
= simulated variables; n = number of observations; and q = degrees of freedom representing
the number of parameters being evaluated.
4.6.1 Kinetic Parameter Estimation under Anaerobic Conditions
Experimental data with initial Cr(VI) concentration of 100 mg/L was initially used to
estimate the kinetic parameters, km, Kc, Rc and K. The validation of this model was performed
and Figure (4-9) confirms that the kinetics parameter values obtained at 100 mg Cr(VI)/L
simulated Cr(VI) reduction data very well for a broader range of Cr(VI) concentrations under
anaerobic conditions (50 and 200 mg/L). However, the maximum Cr(VI) reduction capacity
was not experimentally observed which implies that a unique value of the model kinetic
parameter, Rc, can not be obtained. Cr(VI) reduction capacity of cells, Rc, in this model was
observed to increase with increasing initial Cr(VI) concentration Table 4-1.
Cr(VI) concentration, mg/L
250
model
exp. 50 mg/L
exp. 100 mg/L
exp. 200 mg/L
200
150
100
50
0
0
50
100
150
200
Time, h
Figure 4- 9: Anaerobic batch culture model validation at (50-200 mg/L)
This indicates that the more viable cells are exposed to higher doses of Cr(VI) the more the
population of bacterial species is decreased or the more the cell finite reduction capacity is
reached due to Cr(VI) toxicity within cells. Additionally Figure (4-9) shows that both the
model predictions and experimental data indicated that the rate of Cr(VI) reduction became
43
slower with continuous reduction of Cr(VI) and finally ceased when initial Cr(VI)
concentration exceeded 100 mg/L.
Table 4- 1: Optimum kinetic parameter in anaerobic batch cultures
2
Co (mg/L)
km (h-1)
Kc (mg/L)
50
0.131816
672.09135
10
0.089395
259.1872
100
0.131816
672.09135
10
0.090764
268.6806
200
0.131816
672.09135
10
0.124514
1306.9093
K (mg/L)
Rc (mg/mg)
4.7 Sensitivity Analysis
4.7.1 Sensitivity Analysis of Anaerobic Batch Culture Kinetics
The sensitivity functions of Cr(VI) concentration under anaerobic condition with respect to
Co,, Kc, km, and Rc were also analyzed to compare the effect of each parameter on the
reduction process.
30
km
K
Rc
Kc
Sensitivity of Cr(VI)
20
10
0
-10
-20
-30
0
20
40
60
80
100
120
140
Time,h
Figure 4- 10: Sensitivity test for the initial Cr(VI) concentration of 100 mg/L with respect to
optimized parameters in anaerobic batch culture
It is observed in Figure (4-10) that the anaerobic batch culture model is highly sensitive to
Kc, Rc, and km in the first 50 hours of incubation. The response was extremely high in the first
44
5 hours of incubation which indicates that the cell Cr(VI) reduction activity was high during
that period of incubation. The impact of the non-competitive parameter, K, was mild at 100
mg/L, since this was just a threshold value when the presence of Cr(VI) was not longer
beneficial to the metabolic process.
4.8 Summary
This chapter describes Cr(VI) reducing capability in different consortia of bacteria
reconstituted in our laboratory from previous isolated pure cultures or CRB’s. It is
demonstrated in this chapter that, for successful design and operation of suspended growth
biological system in wastewater treatment, it is essential to understand the types of
microorganisms involved. Batch studies conducted on different configurations indicate
enzymatically mediated Cr(VI) reduction in anaerobic cultures. The evaluation of the noncompetitive inhibition model with Cr(VI) toxicity threshold concentration for anaerobic
batch cultures was necessary as the original model based on pure culture kinetics (Equation
4-10) failed to fit experimental data. The non-competitive inhibition model with Cr(VI)
toxicity threshold best represented Cr(VI) reduction in anaerobic cultures with Cr(VI)
toxicity threshold concentration of approximately 100 mg/L following mechanism observed
by (Molokwane, 2010). The higher Cr(VI) toxicity threshold concentration in this study
compared to other studies may be associated to non-toxic carbon source (glucose) which was
used in this study. The model predicted well the experimental data at a wide range of Cr(VI)
concentrations (50, 100, and 200 mg/L). The Cr(VI) reduction kinetic parameters obtained in
this study however were slightly different from those found in earlier studies (Molokwane,
2010) using the same culture. This result may be associated to slightly different experimental
conditions such as: (i) the cell age, as time is a crucial parameter when using cultures and
thus implies that the duration of culture in the freezer may result into slight loss of cell
activity after keeping or storing the cell culture for several weeks prior using; (ii) initial
biomass concentration; and other factors which may also involve random errors. On the other
hand testing for sensitivity of each kinetic parameter in the model it has been observed that
the model is highly sensitive or affected by the change in kinetic parameters (km, Kc, and Rc),
similar results were also observed by (Molokwane, 2010). This indicates the reliability of the
non-competitive inhibitory model with Cr(VI) toxicity threshold concentration in evaluating
Cr(VI) reduction under anaerobic conditions. Mathematical representations determined from
the anaerobic batch modelling in this study would be used for simulation of Cr(VI) effluent
in aquifer environments in the next chapter.
45
CHAPTER 5
MICROCOSM Cr(VI) REDUCTION KINETIC STUDIES
5.1 Conceptual Basis of Microcosm Studies
Microbial barrier studies were conducted to simulate the behaviour of Cr(VI) across the soil
strata into the open aquifer system at the contaminated site. The aquifer soil samples were
collected at the depth of 3m below the ground surface of the contaminated site for microcosm
study purpose. In this study the impact of carbon source on Cr(VI) reduction and removal in
an aquifer system was evaluated, as the aquifer zone is characterized as the high pore volume
zone with lower organics content from the decaying vegetation. Microcosms were installed
in the laboratory and operated as packed-bed continuous flow bioreactor systems.
The influent loading in the columns was simulated by gravity feeding as in the case of open
aquifer at the site. The performance of each column was evaluated based on the influent and
the effluent Cr(VI) concentration under sustained hydraulic loading. In order to develop
appropriate biological systems that can effectively reduce Cr(VI) at the contaminated sites
the kinetic processes within the reactor system influencing Cr(VI) reduction and removal
were evaluated. The one dimensional dispersion-reaction model was evaluated in this study
to determine the optimum kinetic parameters for microbial barrier system at a transient state.
In order to evaluate the spatial modelling of Cr(VI) concentration profiles along the column
under quasi-steady state conditions which is referred to as steady-state in this study, the plugflow reaction model that account for flow characteristics and biological removal mechanism
was developed.
5.2 Performance Evaluation
5.2.1 Reconstituted Consortium Culture versus Native Soil Culture
The Cr(VI) reducing performance between the consortium culture reconstituted from the
potential pure isolates of the dried sludge and the native bacterial species in the aquifer
medium columns was compared. Table (5-1) summarise the overall performance of different
aquifer medium reactors. The results shows that the rate of Cr(VI) reduction was more
pronounced in the column inoculated with reconstituted consortium culture than in the non-
46
inoculated native soil culture column. The absence of Cr(VI) reduction in the native soil
culture column may be associated to the absence of Cr(VI) reducing culture in the soil. In
addition, majority of the microbial species found in the aquifer soil samples could not be
cultured using the conventional methods, this implies that most of the soil species in the soil
sample are not identifiable.
Table5- 1: Summary of performance of Cr(VI) reduction after column operation
Reactor no.
Flow rate Q(mL/min)
Cr(VI)concentration (mg/L)
Cr(VI) removal
efficiency, (%)
R1(NTV+CS)
0.233
20
<10
40
<8
50
<5
20
100
40
55
50
<10
20
<2
40
<2
50
<2
0.225
20
69.5
Average (Q)=0.23
30
55
R2 (INC+CS)
R3 (Control)
R4 (INC- CS)
0.218
0.248
5.3 Microcosm Kinetic Studies
5.3.1 Cr(VI) Removal Kinetics at Various Time Intervals
Experimentation on Cr(VI) reduction in packed-bed reactors was conducted at various
Cr(VI) concentrations ranging from 20-50 mg/L in carbon source and non-carbon source
reactors over time. Figure (5-1) shows that the inoculated soil column amended with sawdust
as a carbon source outperformed the inoculated soil column without carbon source.
Significant amounts of Cr(VI) were removed from the influent feed with maximum
observable rates of approximately 2 mg/L/h in the inoculated soil column amended with
sawdust. This indicates that the presence of carbon source in the inoculated column greatly
enhance the performance of Cr(VI) removal. Figure (5-2 and 5-3) also demonstrate that
47
Cr(VI) reduction performance is improved by the presence of the carbon source as Cr(VI)
removal efficiency in a non-carbon source reactor at 30 mg Cr(VI)/L was observed to be
equal to Cr(VI) removal efficiency at 40 mg Cr(VI)/L in a carbon source reactor after one
week of operation.
Cr(VI) concentration, mg/L
25
20
R1
R2
R3
R4
15
(NTV+CS)
(INC+CS)
(CRL)
(INC-CS)
10
5
0
0
20
40
60
Time, h
80
100
120
Figure5- 1: Performance of comparison between the inoculated reactors with carbon source
and without carbon source at the initial Cr(VI) concentration of 20 mg/L.
Cr(VI) concentration, mg/L
35
30
25
20
15
10
R2(INC-CS)
R1(NTV+CS)
R3(CRL)
5
0
0
20
40
60
80
100
120
140
160
180
Time, h
Figure5- 2: Performance of inoculated column without carbon source in comparison with
sterile-control column at 30 mg/L.
48
Cr(VI) concentration, mg/L
50
40
30
20
R1(NTV+CS)
R2(INC+CS)
R3 (CTRL)
10
0
0
20
40
60
80
100
120
140
160
Time, h
Figure5- 3: Performance of inoculated reactor amended with carbon source at 40 mg/L
5.3.2 Cr(VI) Removal Kinetics at Various Lengths
Cr(VI) Concentration Profile in a Sterile Control Column
The rate at which Cr(VI) was reduced within the sterile control reactor was shown to be
insignificant through out its operation Figure (5-4). This indicates that the abiotic processes
are negligible.
Cr(VI) Concentration Profile in Native Soil Culture Column
Figure (5-5) shows that the rate of Cr(VI) reduction increases insignificantly with increasing
reactor length over time in the native soil culture column amended with sawdust. It is also
observed in Figure (5-4 and 5-5) that performance of native soil culture column in Cr(VI)
removal is similar to that of a sterile control column. The low performance of the native soil
culture in reducing Cr(VI) in a column at both lower and higher initial Cr(VI) concentrations
may be associated to the absence of Cr(VI) reducing culture in the soil. Based on the
experimental data and the microbial culture dynamics obtained in the native soil culture
column at various Cr(VI) feed concentrations it can be postulated that the native species in
the soil samples are Cr(VI) resistors as they remained persistent in the column after long
period of exposure to Cr(VI) loadings but were unable to reduce Cr(VI) in the reactor.
49
Cr(VI) concentration, mg/L
25
20
15
53hours
75hours
102 hours
10
5
0
0
10
20
30
40
50
60
Distance, cm
Figure5- 4: Performance of a sterile-control reactor in removing Cr(VI) feed concentration of
20 mg/L across the column
Cr(VI) concentration, mg/L
25
20
15
53 hours
82 hours
102 hours
10
5
0
0
10
20
30
40
50
60
Distance, cm
Figure5- 5: Performance of native soil culture column in removing Cr(VI) feed concentration
of 20mg/L across the column
Cr(VI) Concentration Profile in the Inoculated Columns
Spatial variation of Cr(VI) removal in the inoculated reactors was evaluated over time. Data
collected from equally spaced longitudinal sampling ports over random hours of operation
50
were evaluated for Cr(VI) removal across the reactors. Figure (5-6) demonstrate that the rate
of Cr(VI) removal at non-steady state, thus for the first 50-75 hours of operation increases
significantly over length. Up to 70% of Cr(VI) removal efficiency was achieved during the
first 53 hours of operation across the reactor Figure (5-6A).
Cr(VI) concentration, mg/L
25
(A)
102 hours
82 hours
53 hours
20
15
10
5
0
0
10
20
30
Distance, cm
40
50
60
Cr(VI) concentration, mg/L
25
51 hours
75 hours
102 hours
(B)
20
15
10
5
0
0
10
20
30
40
50
60
Distance, cm
Figure5- 6: Performance of inoculated reactors in removing Cr(VI) feed concentration of 20
mg/L in (A) C-source reactor, and (B) non C-source reactor
51
Although the rate of Cr(VI) reduction was observed to increase with increasing length, near
complete Cr(VI) removal was achieved at the final effluent port over time. It is also observed
in Figure 5-6 (A-B) that the effluent Cr(VI) concentration did not stabilize until a quasisteady state was reached, thus after three to four days of operation depending on the column
experimental condition. The rate of Cr(VI) reduction at quasi-steady state became
insignificant over time ( less than 5%). The insignificant Cr(VI) removal over time may be
associated to various reasons which involves: (i) Cr(VI) inhibitory effects on the Cr(VI)
reducing bacteria; (ii) loss of Cr(VI) reducing capacity, and (iii) saturation of the physical
chemical processes-adsorption and biosorption in the reactors over time.
5.4 Microbial Culture Dynamics in the Aquifer System
5.4.1 Characteristic of Initial Inoculated Reconstituted Consortium Culture
The pure cultures isolated from the dried sludge were grown aerobically as a reconstituted
consortium culture. Table (3-1), from Chapter 3 shows the pure isolates which were initially
inoculated in the specific aquifer media reactors as a reconstituted consortium culture
(X1+X2+X3+X4+X5+X6+X7) prior contaminant loading.
5.4.2 Characterization of Inoculated Columns after Operation
After operating the columns under oxygen stressed condition, the microcosm columns were
opened, to analyse the microbial community shift in the dried sludge culture and native soil
culture column. Microbial shift analysis due to exposure to toxic conditions was monitored
by 16S rRNA fingerprinting method. The results presented by the Phylogenetic trees in
(Figure 5-7) confirms that after microcosm system operation (seven weeks) the well known
Cr(VI) reducers, Bacillus thuringiensis and Bacillus cereus remained persistent in different
reactors, R2 (inoculated column + CS) and R4 (inoculated column - CS). In the native soil
culture column (R1), the Bacillus anthracis remained persistent. Figure (5-8) confirms that
Enterococcus faecium remained persistent in both (R1) and (R2) and Enterococcus villorum
remained persistent in (R1).
This indicates that Bacillus thuringiensis and Bacillus cereus in different inoculated columns
(R1 and R4) are less sensitive to Cr(VI) toxicity or their resilience against Cr(VI) toxicity.
The presence of diverse bacterial species in the native culture column (R1) which are
52
Bacillus anthracis, Enterococcus faecium, Enterococcus villorum and several unidentified
species in the soil after operation indicate that the native bacterial species in the soil sample
are most certainly Cr(VI) resistors, but not Cr(VI) reducers as non of the cultured soil
bacteria were recognised or reported from the literature as Cr(VI) reducing species.
Figure5- 7: Phylogenetic tree of persistent bacterial cells in inoculated reactor columns after
operation derived from the 16S rRNA gene sequence, Bacillus species
53
Figure5- 8: Phylogenetic tree of persistent bacterial cells in inoculated reactor columns after
operation derived from the 16S rRNA gene sequence, Enterococcus species
54
Figure5- 9: Phylogenetic tree of persistent bacterial cells in inoculated reactor columns after
operation derived from the 16S rRNA gene sequence. Possible Cr(VI) reducing species were
detectable including, Enterobacter species and E. coli
5.5 Kinetic Modelling of Cr(VI) Reduction in the Microcosm Columns
5.5.1 Model Description
Cr(VI) transport through saturated porous media is a highly dynamic process that can not be
fully define through batch reactor experiments. Anaerobic batch studies were initially
55
conducted in this study at various initial Cr(VI) concentration prior continuous-flow studies
to evaluate the fundamentals of each biological process at various time intervals. The
evaluation of batch studies under anaerobic condition prior microcosm column studies is
associated to the oxygen stressed condition attained in the microcosm packed-bed reactor
after long period of operation. Advection and dispersion are the main modes of transport of
Cr(VI) in the groundwater. However, reaction-microbial reduction in this study also
significantly influence the fate and transport of Cr(VI) in a saturated porous media. A
detailed mathematical model that simulate microbial Cr(VI) removal in the fixed-media
reactor system includes a system of coupled differential equations which represent Cr(VI)
reduction rate (rc), mass transport rate (jc), adsorption rate (qc) and dispersion:
Advection
The transport of dissolved species of Cr(VI) along with bulk fluid flow which is represented
as:
 d (CV )
 Au (C in  C )
dt
(5-2)
where: C = effluent Cr(VI) concentration (ML-3); V = volume of the reactor (L3); Cin =
influent Cr(VI) concentration (ML-3); Q (Au) = influent flow rate (L3T-1); A = cross sectional
area of reactor (L2); u = velocity of the flow (LT-1); and t = time (T).
Flux through attached cell layers on soil particles
Mass transfer within the attached cell layer is described by Fick’s law for dispersion. The
contaminant flux across the stagnant layer to the biofilm is a function of the contaminant
dispersion coefficient and concentration and is represented as:
D
d CV 
dC
  Dw
  w A f Cb  Cs    jC . A f
dt
dx
Lw
(5-3)
where: Dw = dispersion coefficient of Cr(VI) in water (L2T-1); dC/dx = Cr(VI) concentration
gradient (ML-3L-1); Lw = thickness of stagnant layer (L); C b = bulk liquid Cr(VI)
56
concentration (ML-3); and C S = Cr(VI) surface concentration (ML-3). NB: In most mass
transfer-limited reactions C b  C S , therefore Cs is negligible.
Reduction due to reaction
Since the aquifers were operated under predominately anaerobic conditions, Cr(VI) reduction
model with toxicity threshold inhibition was chosen. The kinetic rate parameters obtained
from the batch anaerobic cultures were maintained in the continuous flow systems with
minor adjustments allowed due culture sensitivity to Cr(VI) toxicity after long a period of
operation under oxygen stressed conditions.
 dC

dt

C C 
kmC
 X 0  0
   rc
Rc 
  1 Cr   
K c  C  K  C0  


(5-4)
where: km = maximum specific rate of Cr(VI) reduction (T-1); Kc = half-velocity concentration
(M.L-3); Cr = Cr(VI) toxicity threshold concentration (ML-3); Xo = initial biomass
concentration (ML-3); K = limiting constant (ML-3); and Rc = Cr(VI) reduction capacity of
cells (MM-1).
Adsorption
Removal of Cr(VI) in the reactor depends at the rate at which the Cr(VI) is transported and
adsorbed in the biofilm of the reactor and also in the reaction taking place on the surface
area. The removal of Cr(VI) by adsorption is represented as:
 dC
 k ad ( C eq  C )  q c
dt
(5-5)
where: kad = adsorption rate coefficient (T-1); Ceq = equilibrium concentration at surface area
(ML-3); C = Cr(VI) concentration at any time (ML-3); and qc= rate of Cr(VI) removal by
adsorption (T-1).
57
Total mass balance of the reactor involving all the non-linear ordinary differential equations
for modelling the fate and transport of Cr(VI) in a packed-bed reactor at the transient state
can be represented as follows:
d ( CV )
 Au( C in  C )  rc V  jC .A f  q c V
dt
(5-6)
where: C = effluent Cr(VI) concentration (ML-3); V = volume of the reactor (L3); Cin =
influent Cr(VI) concentration (ML-3); Q = influent flow rate (L3T-1); rc = reduction rate
coefficient (ML-3T-1); A = cross sectional area of reactor column at time t (L2); Af = biofilm
surface area (L2); u =velocity of the flow (LT-1); and qc = the rate of Cr(VI) removal by
adsorption (T-1). N.B: Au = Q (inflow rate, L3T-1).
The coupled mass balance equations were simulated using a forth-order Runge-Kutta routine
for solution of simultaneous ordinary and partial differential equations in A Computer
Program for Identification and Simulation of Aquatic Systems (AQUASIM 2.0) (Reichert,
1998).
Basic assumptions made to formulate the model are summarised as follows:

The flow in the column is one-dimensional

The flow is turbulent and has no radial gradient in velocity (thus a plug flow
condition)

The porous medium is homogeneous

The rate of nutrient dissolution is greater than the rate of nutrient consumption

Some microbes are mobile and some are immobile

The contaminant is toxic and has inhibitory effect on microbial growth rate

Cr(III) generated due to biotransformation is either precipitated and retained or
adsorbed onto the soil matrix almost immediately

Temperature and pH are constant
58
5.5.2 Model Validation
The model for saturated packed column with dispersion was adapted from AQUASIM 2.0
and tested with a sterile packed-bed reactor (control). This model was used in combination
with Cr(VI) kinetic parameters adapted from the anaerobic batch culture studies due to
oxygen stressed condition attained in the reactor after long period of operation. The model
was initially tested with sterile-control column and then used to simulate time series data at
various Cr(VI) concentrations in the microcosm reactors as shown in Appendix A.
5.5.3 Parameter Optimization
Kinetic parameters were obtained by performing a nonlinear regression analysis using
AQUASIM 2.0. For each kinetic parameter, a search was carried out through a range of
values which were initialized by guessed values and values from batch studies. To ascertain
that the optimized parameters obtained using the mathematical model were dependable,
upper and lower constrains were set for each parameter to allow the omission of invalid
parameter values. Whenever optimization converged at or very close to a constraint, the
constraint was relaxed until the constraint no longer forced the model. This process was
repeated until unique values lying away from the constraints, but between the set limits were
found for each parameter.
5.5.4 Cr(VI) Removal Kinetics at Lower and Higher Concentrations
The optimum kinetic parameters summarized in Table (5-3 and 5-4), shows that the
dispersion coefficient in the inoculated column amended with sawdust as a carbon source is
much higher than the one observed in the inoculated column reactor without carbon source.
This indicates that the rate at which the contaminant disperses into the cell layer attached to
the aquifer soil particles influence the removal of Cr(VI) by biomass. Therefore higher rates
of Cr(VI) reduction in the inoculated carbon source reactor can be attributed to higher
dispersion rate in the column. It is also shown in Table (5-3) that the cell death rate is
relatively faster in the non-carbon source reactor than in the carbon source reactor; this
indicate that the organic carbon source in the reactor enhance the cell activity.
Results on Cr(VI) effluent simulation in a sterile control reactor, inoculated carbon source,
and non-carbon source reactor at various initial Cr(VI) concentrations are demonstrated in
Figure (5-10 and 5-11). It is observed in Figure (5-10 and 5-11) that in the order of 5-10
59
hours of operation, adsorption sites on the aquifer media particles is saturated. This implies
that the adsorptive process reaches the equilibrium state in the column, thus adsorption
coefficient (qc) approaches zero. Therefore the mechanisms which are responsible for Cr(VI)
removal in a long run are limited to reduction by kinetics adapted from the anaerobic batch
reaction, advection, and mass transport.
5.5.5 Summary of Parameters
The model for saturated soil column with dispersion was adapted from AQUSIM 2.0 for
simulation of soil columns. The kinetic parameters for reduction rate process were obtained
and optimised in a batch reactor system and were directly applied in continuous flow process.
However minor adjustments were applied in continuous-flow process as a result of low levels
of biomass in the continuous flow reactor system as compared to the batch reactor system.
Cultures grown under carbon source showed higher Cr(VI) reduction capacity than the
cultures grown on inorganic carbon source from the soil. Most physical parameters were
determined from known literature values of similar systems. Mass transport and adsorption
parameters were estimated from continuous-flow reactor data. The breakthrough
characteristics of the saturated soil column were observed to be typically of packed-bed
reactor systems with moderate dispersion depicting an exponential rise up to maximum point
and then followed by reduction in effluent as Cr(VI) reducing culture become more
established. The model for the saturated soil columns with dispersion adapted from
AQUASIM 2.0 successfully simulated the operation of microcosm used in this study.
60
Table5- 2: Optimum kinetic parameter values obtained for the biofilm in a carbon source
reactor
Parameter Symbol
Definition
Constrains
[ lower, upper]
Optimum value
State variable
Influent Cr(VI)
---
110-6
20-40
Half velocity concentration
[0, 15]
11.3
km (h )
Specific reduction rate
[0, 0.02]
0.0051
Kd (h-1)
Cell death rate coefficient
[0, 1000]
0.0025
 (h-1)
Biomass growth rate
[0, 1000]
0.023
Rc (mg/mg)
Cr(VI) reduction capacity
[0, 0.5]
0.283
Cr (mg/L)
Cr(VI) toxicity threshold
--
50
 (%)

porosity
alpha
---
0.4
0.5
rho_s (kg/m3)
Soil particle density
--
2300
Qin (L/h)
Influent flow rate
--
0.015
D (m2/s)
Dispersion coefficient
[0, 100]
6.02-95.4
A (m2)
Cross sectional area
--
0.00196
2
Biofilm surface area
--
0.000785
Biological parameters
C (mg/L)
Cin (mg/L)
concentration
Kc (mg/L)
-1
Physical parameters
Af (m )
-- Constant values
61
Table5- 3: Optimum kinetic parameter values obtained for the biofilm in a non carbon source
reactor
Parameter Symbol
Definition
Constrains
Optimum value
[ lower, upper]
Biological parameters
C (mg/L)
Cin (mg/L)
State variable
Influent Cr(VI) concentration
---
110-6
20-30
Kc (mg/L)
Half velocity concentration
[0, 15]
12.51
km (h-1)
Specific reduction rate
[0, 0.02]
0.0010
Kd (h-1)
Cell death rate coefficient
[0, 1000]
0.0031
 (h-1)
Biomass growth rate
[0, 1000]
0.018
Rc (mg/mg)
Cr(VI) reduction capacity
[0, 0.5]
0.099
Cr (mg/L)
Cr(VI) toxicity threshold
--
50
porosity
--
0.4
alpha
--
0.5
rho_s (kg/m )
Soil particle density
--
2300
Qin (L/h)
Influent flow rate
--
0.015
D (m2/s)
Dispersion coefficient
[0, 100]
1.52-11.7
A (m2)
Cross sectional area
--
0.00196
Af (m2)
Biofilm surface area
--
0.000785
Physical parameters
 (%)

3
-- Constant values
62
Cr(VI) concentration, mg/L
25
(A)
20
15
model simulation
measured
effluent
influent
10
5
0
0
20
40
60
Time, h
80
100
120
25
Cr(VI) conentration, mg/L
(B)
20
model simulation
exp. +CS effluent
exp. -CS effluent
exp. influent
15
10
5
0
0
20
40
60
80
100
120
Time, h
Figure5- 10: Simulation of Cr(VI) effluent at 20 mg/L in a (A) sterile control column, (B)
carbon source and non-carbon source reactor
63
Cr(VI) concentration, mg/L
35
(A)
30
25
20
15
model simulation
exp. effluent
exp. influent
10
5
0
0
20
40
60
80
100
120
140
160
Time, h
Cr(VI) concentration, mg/L
50
(B)
40
30
20
model simulation
exp. effluent
exp. influent
10
0
0
20
40
60
80
Time, h
100
120
140
160
Figure5- 11: Simulation of Cr(VI) effluent at (A) 30 mg/L in a non-carbon source reactor,
(B) 40 mg/L in a carbon source reactor
64
5.6 Steady State Performance Model
5.6.1Model Formulation
Data collected from equally space longitudinal sampling ports were used to facilitated the
spatial modelling of Cr(VI) concentration profiles across the reactor under different loading
conditions. Steady-state in a packed-bed system is mainly, among many other reasons
associated to the enlargement of the mass transfer boundary layer thickness at low velocity
which results into limitation of reaction by diffusion. The actual behaviour of Cr(VI) along
the reactor under quasi-steady state conditions was modelled as a plug flow reactor using the
finite difference model.
The generalized mole balance continuous equation on species (C) over a catalyst weight (W)
at a steady-state can represented as follows:
Fc (W) – Fc (W+∆W) + γc (∆W) = 0
(5-7)
where: Fc = molar flow rate of Cr(VI) (MT-1); W = mass of aquifer soil particles (M); and γc
= reaction rate (ML-3T-1).
Dividing equation (5-7) by (∆W) yield the following equation:
Fc W   Fc W  W 
 c
W
(5-8)
The basic limiting process for calculus states that: for any quantity Q which is a smooth
continuous function of L:
Q dQ
Q2  Q1
 lim

L2  L1 L2  L1
L  0 L
dL
lim
Therefore taking the limit as ∆W→0, in equation (5-8) we arrive at the differential form of
the mole balance for a plug flow reactor:
dFc
 c
dW
(5-9)
65
Expressing (Fc) in terms of concentration yield the following equation:
Fc (MT-1) = Qin C
(5-10)
where: Qin = inflow rate (L3T-1); and C = Cr(VI) concentration any time (ML-3). On the other
hand W (mass of aquifer soil particles) which is more important to the rate of reaction can be
represented as:
(5-11)
W (M) =  c A f L
Levenspiel (1999) has shown that the reaction rate of the microbial reactions that are
subjected to reactant toxicity can be represented as:
C 

  c = kC 1 

Cr 

n
(5-12)
where: γc = reaction rate (ML-3T-1); k = reaction rate coefficient (L.M-3.T-1); C = effluent
Cr(VI) concentration at any time (ML-3); Cr = Cr(VI) toxicity concentration, (ML-3); n =
empirical dimensionless variable (M-1M-1).
Therefore equation (5-9) can be represented as second order ordinary differential equation
(ODE) representing both the flow characteristics and the predominant removal mechanism as
follows:
 c A f
dC

 kC 
dL
 Qin

C 
1 


Cr 

n
(5-13)
where: C = effluent Cr(VI) concentration at any time (ML-3), L = height of a reactor (L), k =
reaction rate coefficient (L.M-3.T-1), ρc = density of aquifer soil particles (ML-3), Af = biofilm
surface area (L2), Qin = inflow rate (L3T-1), Cr = Cr(VI) toxicity concentration, (ML-3), n =
empirical dimensionless variable (M-1M-1). N.B: n varies with the reactor environment.
66
Assumptions Governing the Model:

There is no mixing in the axial direction, this implies that molecular and/or turbulent
mass dispersion is negligible in flow direction

Uniform properties in the direction perpendicular to the flow (flow is one
dimensional)

The net growth of bacteria is zero at this state, i.e., Cr(VI) reduction is by resting cells

The system is a homogenous catalytic system
5.6.2 Steady State Spatial Simulation
The slope of Cr(VI) concentration profiles across the reactor was defined by a second order
ODE (Equation 5-13). The plug flow model was initially tested with control column to
simulate length series data under various loading conditions in the reactor using the
Computer Program for Solving Numerical Problems (Octave 3.0) as shown in Appendix B.
Performance under Carbon Source
The rate of Cr(VI) removal along an inoculated carbon source reactor was evaluated using
the plug flow model in Equation (5-13). The model was initially tested with Cr(VI)
experimental run of 50 mg/L which is considered as control due to insignificant Cr(VI)
removal observed at 50mg/L in an inoculated carbon source reactor. The optimum kinetic
parameters obtained at 50 mg/L which are k (reaction rate coefficient) and n (empirical
variable) were then used to simulate Cr(VI) effluent under different loading conditions.
The experimental run at 20 mg/L in a carbon source reactor gives a clear representative
picture of bioreduction Figure (5-12). It also observed in Figure (5-12 B) that at the initial
Cr(VI) concentration of 40 mg/L, inhibition of Cr(VI) in Cr(VI) reducing culture occurred in
the reactor. The inhibition of Cr(VI) in the reactor may be associated to Cr(VI) toxicity
within the cells and the loss of Cr(VI) reduction capacity due to blockage of the media pores
with Cr(III) precipitate. The experimental data in Figure 5-12 (A-C) is the average of the last
three sampling times when the quasi-steady state was reached in the column. The Cr(VI)
67
toxicity threshold concentration was assumed to be 50 mg/L as it is observed in Figure 5-12
(C) that at the initial Cr(VI) concentration of 50 mg/L the reduction of Cr(VI) was
insignificant over time across the reactor. The optimum reaction rate coefficient, k, was
determined to be 5.210-8 (L/mg/h) and the empirical variable, n =2. The model depicted
well the trends of Cr(VI) concentration profiles under quasi-steady state conditions for
different loading conditions in a carbon source reactor with the R-squared value of 95%. The
experimental data points outside the model trend line were considered as outliers.
Performance under non Carbon Source
The plug flow model developed in this study under quasi-steady state condition for different
loading conditions Equation (5-13) was used to simulate Cr(VI) effluent concentration in an
inoculated non-carbon source reactor. Cr(VI) experimental run of 30 mg/L was initially
tested with the model. The optimum kinetic parameters obtained from the Cr(VI) feed
concentration of 30 mg/L were then used to simulate Cr(VI) effluent concentration at 20
mg/L. Figure (5-13) gives a clear representative picture that the inhibitory effects of Cr(VI)
in a non-carbon source reactor occurs at a relatively lower initial Cr(VI) feed concentration
compared to carbon source reactor. This is indicates that the carbon source in the reactor
greatly enhance the removal of Cr(VI) in the aquifer medium.
Compared to the kinetic parameters obtained in a carbon source reactor under quasi-steady
state conditions for different loading conditions the kinetic parameters in a non-carbon
source reactor were adjusted, thus k = 9.910-9 L/mg/h and n = 1. The model successfully
captured the trends of Cr(VI) response profiles under quasi-steady state conditions for
different loading conditions in the inoculated non-carbon source reactor with the R-squared
value of 94.7% . The experimental data points outside the model trend line were considered
as outliers.
Table5- 4: Optimum kinetic parameter values for the biofilm at steady-state in a carbon
source and a non carbon source reactor
Kinetic parameter
k (L/mg/h)
Description
Reaction rate
Carbon source
Non Carbon source
reactor
reactor
5.210-8
9.910-9
2
1
coefficient
n (mg/mg)
Empirical variable
68
Cr(VI) concentration, mg/L
25
(A)
model simulation
measured effluent
20
15
10
5
0
0
10
20
30
40
50
60
70
Distance,cm
Cr(VI) concentration, mg/L
50
(B)
model simulation
measured effluent
40
30
20
10
0
0
10
20
30
40
50
60
70
Distance,cm
60
Cr(VI) concentration, mg/L
(C)
50
model simulation
measured effluent
40
30
20
10
0
0
10
20
30
Distance,cm
40
50
60
70
Figure5- 12: Simulation of Cr(VI) effluent in a carbon source reactor at various lengths
69
25
Cr(VI) Concentration, mg/L
(A)
model simulation
measured effluent
20
15
10
5
0
0
10
20
30
40
50
60
70
Distance,cm
Cr(VI) concentration, mg/L
40
(B)
model simulation
measured effluent
30
20
10
0
0
10
20
30
40
50
60
70
Distance, cm
Figure5- 13: Simulation of Cr(VI) effluent in a non-carbon source reactor over length. The
experimental data is the average values of the last three sampling times where the quasisteady state was achieved in the column.
70
5.6.3 Summary of Steady State Kinetic Parameters
The model for saturated soil column at the steady state was determined by an ordinary
differential equation as shown in Equation (5-13). The parameter, n, which is defined as the
empirical dimensionless variable in this model various with the environmental conditions as
it is associated to the rate of Cr(VI) toxicity in each column experimental condition. The
n

C 
toxicity term in Equation (5-13), 1    0 as n . Table (5-5) shows that the value of,
Cr 

n, in an inoculated carbon source reactor is greater than the value of, n, in an inoculated non
carbon source reactor. This is indicates that the inhibitory effects of Cr(VI) on Cr(VI)
reducing cultures is approached at a faster rate in the non carbon-source reactor than in the
carbon source reactor. This implies that the higher the value of, n, the slower the rate of
Cr(VI) toxicity within the cells and thus also imply that the rate of cell inactivity within the
reactor will be slow. It is also observed in Table (5-5) that the reaction rate coefficient, k, also
varies with the reactor experimental conditions. Minor adjustments on, k, in the non-carbon
source reactor were determined in the model. The results obtained in a carbon source and
non-carbon source reactor indicates that the presence of the carbon source may greatly
influence the rate of Cr(VI) reduction in the reactor by improving the rate of cell activity.
5.7 Summary
Microcosm studies showed that the isolated bacterial species from the dried sludge can
effectively reduce Cr(VI) from the contaminated aquifer systems. The reduction of Cr(VI) in
the inoculated reactors was observed to be both time and length dependent. A one
dimensional dispersion-reaction model evaluated in this study was able to simulate
contaminant movement in the aquifer system for a broader range of Cr(VI) concentrations at
various time intervals. At a steady-state the removal of Cr(VI) along the reactor was
estimated using the plug flow model expressed as function of spatial variable, L, which was
derived based on component/mole balance continuity equation in Equation (5-13).
The results obtained in the kinetic modelling of Cr(VI) reduction at quasi-steady state
demonstrated that the inhibitory effects of Cr(VI) on Cr(VI) reducing microorganisms in the
inoculated reactor without carbon source are approached faster than in the inoculated reactor
with carbon source. The model developed in this study under quasi-steady state condition
was able to predict well the experimental data at various Cr(VI) feed concentrations. This
71
indicates that the developed predictive model in this study can be effective in facilitating the
final scale up and operation of the microbial barrier in field.
After seven weeks of operation under oxygen stressed conditions the microbial community
shift was expected. Microbial shift results showed that among all the bacterial species
isolated from the dried sludge (X1 to X7) which were initially inoculated in the column
reactors as a reconstituted consortium culture, Bacillus thuringiensis (X5 and X6) and the
Bacillus cereus (X2), the predominant well known Cr(VI) reducers remained persistent in all
inoculated reactor columns, (R2 and R4). The results obtain in this chapter correlates with
the results which were obtained in the previous chapter (Chapter 4) where X5 and X6 were
evaluated as potential isolates in Cr(VI) reduction process.
72
CHAPTER 6
SUMMARY AND CONCLUSION
The improper release of Cr(VI) solid and liquid waste from various industries in South Africa
and around the world is a subject of paramount concern. Therefore biotransformation of
hazardous Cr(VI) to less toxic Cr(III) is essential. Batch experimental studies were conducted
in this study under various Cr(VI) concentration to evaluate the effectiveness of indigenous
culture from the local environment in reducing Cr(VI) in the Cr(VI) contaminated
environments. The rate of Cr(VI) reduction in the anaerobic batch culture was observed to be
generally slower than that observed in the aerobic batch culture. Near complete Cr(VI)
reduction occurred in consortium culture reconstituted from the potential pure anaerobic
isolates with a lower initial Cr(VI) concentration of 100 mg/L after 65 hours of incubation.
Cr(VI) transport through saturated porous media is a highly dynamic process that can not be
fully defined through batch reactor systems, therefore further experimental studies in
continuous-flow system were necessary for this purpose. In order to evaluate the
fundamentals of each biological process over time prior continuous-flow system operation
anaerobic batch studies were initially conducted prior continuous-flow system operation as
batch reactors are easy to operate and to analyse compared to continuous-flow systems.
The feasibility of using Cr(VI) reducing cultures in the aquifer environment was
demonstrated by better performance of microcosm reactors inoculated with Cr(VI) reducing
consortium culture from the local environment. The impact of carbon source reactor in
Cr(VI) reduction was also evaluated in the study. Microcosm reactors supplemented with
sawdust as carbon source outperformed the one without carbon source with Cr(VI) removal
efficiency of 55% at a Cr(VI) feed concentration of 40 mg/L, which is the current highest
groundwater Cr(VI) concentration at the remediation wells at the studied site. The inhibitory
effects of Cr(VI) on the Cr(VI) reducing organisms in the reactor was demonstrated by a
steady-state operation which was achieved after three to four days of operation. The
microcosm reactor conditions favoured the well known Cr(VI) reducing species, Bacillus
cereus, Bacillus thuringiensis after seven weeks of operation.
73
Batch modeling results showed that the performance of Cr(VI) reducing culture fitted well
the non-competitive model associated with Cr(VI) toxicity threshold predicted under
anaerobic conditions. The model used for simulation of Cr(VI) effluent within the saturated
aquifer media over time in the microcosm system was adopted from AQUASIM 2.0 and used
in combination with kinetic parameters obtained from the anaerobic batch cultures. The
kinetic model with dispersion simulated well the soil column experimental data with a plug
flow regime.
At a steady-state the plug flow model that accounts for insignificant Cr(VI) removal along the
column over the last three sapling times of operation was derived in the study based on the
first principle of mass conservation. The Cr(VI) mass balance model under quasi-steady state
condition accounts for both flow characteristics and the predominant removal mechanism
(biological transformation) in the reactor. The model simulated well the experimental data at
various Cr(VI) concentration. The outcome of this study is a good basis for testing the
concept in pilot scale study on site.
Recommendations
In order to achieve optimum application of this technology future research will be needed in
the following areas:
 The experiment that accounts for Cr(III) removal in the reactor must be taken into
consideration as the Cr(OH)3 generated during Cr(VI) transformation process may
clog in the reactor and thus will reduce the performance of the system after a long
period of operation.
 The composition of sawdust must be analysed to evaluate organics which are
biodegradable and non-biodegradable in the sawdust.
 Develop a method that accounts for biomass concentration profile within the
column.
74
APPENDIX A
AQUASIM Version 2.0 ********************************************************************************** Variables ********************************************************************************** A : Description : Cross‐ sectional area Type : Constant variable Unit : m2 Value : 0.00196 Standard Deviation: 1 Minimum: 0 Maximum: 100 Sensitivity Analysis: active Parameter Estimation: active ‐‐‐ ‐‐‐‐‐‐ ‐‐‐ ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Af : Description : Biofilm surface area Type : Constant variable Unit : m2 Value : 0.000785 Standard Deviation: 1 Minimum: 0 Maximum: 100 Sensitivity Analysis: active Parameter Estimation: active ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
: Description : Alpha Type : Formula variable Expression : 0.5 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 75
C : Description : Dissolved Cr(VI) concentration Type : state variable Unit : mg/L Relative Accuracy: 1e‐006 Absolute Accuracy: 1e‐006 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
C2: Description: Cr(VI) toxicity threshold concentration Type: Constant Variable Unit: mg/L Value: 50 Standard Deviation: 1 Minimum: 0 Maximum: 60 Sensitivity Analysis: active Parameter Estimation: active ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Calcnum: Description: Type: Program Variable Unit: h Reference to: calculation number ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Cmeas: Description: Measured Cr(VI) Type: Real List Variable Unit: mg/L Argument: t Standard Deviations: global Rel. Stand. Deviation: 0 Abs. Stand. Deviation: 1 Minimum: 0 Maximum: 1e+009 76
Interpolation Method: linear interpolation Sensitivity Analysis: inactive Real Data Pairs (15 pairs): Argument Value 0 20 3 7 6 3.62 9 4.66 12 6.15 15 5.83 18 6.28 21 5.83 24 5.56 27 4.7 53 4.48 58 2.54 78 0.25 82 0.19 102 0 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Co: Description: Initial Cr(VI) concentration Type: Formula Variable Unit: mg/L Expression: 20 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
C_crit: Description: Type: Formula Variable Unit: mg/L Expression: 0.01 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 77
Cin: Description: measured Cr(VI) influent Type: Real List Variable Unit: mg/L Argument: t Standard Deviations: global Rel. Stand. Deviation: 0 Abs. Stand. Deviation: 1 Minimum: 0 Maximum: 1e+009 Interpolation Method: linear interpolation Sensitivity Analysis: inactive Real Data Pairs (15 pairs): Argument Value 0 18.5 18.5 6 18.5 9 18.7 12 18.7 15 18.7 18 18.1 21 18.8 24 18.9 27 18.1 53 18.4 58 18.9 78 18.9 82 18.8 102 20.2 3 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 78
C_in_1: Description: Type: Real List Variable Unit: mg/L Argument: t Standard Deviations: global Rel. Stand. Deviation: 0 Abs. Stand. Deviation: 1 Minimum: 0 Maximum: 1e+009 Interpolation Method: linear interpolation Sensitivity Analysis: inactive Real Data Pairs (4 pairs): Argument Value 0 0 0.01 1 1 0.51 0 0.5 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
D: Description: Dispersion coefficient Type: Constant Variable Unit: m2/h Value: 95.4 Standard Deviation: 1 Minimum: 0 Maximum: 100 Sensitivity Analysis: active Parameter Estimation: active ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ K: Description: Type: Formula Variable 79
Unit: mg/L Expression: 0.5 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
k: Description: Relaxation rate constant for sorption of B Type: Formula Variable Unit: 1/h Expression: 10000 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Kc: Description: Half velocity Cr(VI) concentration Type: Constant Variable Unit: mg/L Value: 11.272 Standard Deviation: 1 Minimum: 0 Maximum: 14 Sensitivity Analysis: active Parameter Estimation: active ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
km: Description: Maximum specific Cr(VI) reduction rate Type: Constant Variable Unit: 1/h Value: 0.0051 Standard Deviation: 1 Minimum: 0 Maximum: 0.02 Sensitivity Analysis: active Parameter Estimation: active ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
: Description: Specific biomass growth rate Type: Constant Variable 80
Unit: 1/h Value: 0.023 Standard Deviation: 1 Minimum: 0 Maximum: 1000 Sensitivity Analysis: inactive Parameter Estimation: inactive ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Kd: Description: Cell death rate Type: Constant Variable Unit: 1/h Value: 0.0025 Standard Deviation: 1 Minimum: 0 Maximum: 1000 Sensitivity Analysis: active Parameter Estimation: active ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Kf: Description: Type: Formula Variable Unit: Expression: 0.00025 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Qin: Description: Inflow rate Type: Formula Variable Unit: L/h Expression: 0.015 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Rc: Description: Cr(VI) reduction capacity coefficient Type: Constant Variable 81
Unit: mg/mg Value: 0.283 Standard Deviation: 1 Minimum: 0 Maximum: 0.5 Sensitivity Analysis: active Parameter Estimation: active ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
rho_s: Description: Density of solid material Type: Formula Variable Unit: kg/m3 Expression: 2300 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
S: Description: Adsorbed concentration Type: Dynamic Surface State Variable Unit: mg/kg Relative Accuracy: 1e‐006 Absolute Accuracy: 1e‐009 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Smax: Description: Type: Formula Variable Unit: mg/kg Expression: 0.00029 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
S_eq: Description: isotherm Type: Variable List Variable Unit: mg/kg Argument: calcnum Interpolation Method: linear interpolation List of data (1 pair): Argument Value 0 S_eq_0 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 82
‐S_eq_0: Description: Isotherm for no sorption Type: Formula Variable Unit: mg/kg Expression: kd*C ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
S_eq_Freundlich: Description: Freundlich isotherm Type: Formula Variable Unit: mg/kg Expression: if C>C_crit then Kf*C^alpha else Kf*C_crit^alpha*C/C_crit endif ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
S_eq_Langmiur: Description: Langmuir isotherm Type: Formula Variable Unit: mg/kg Expression: Smax*C/(K+C) ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
S_eq_lin: Description: Linear isotherm Type: Formula Variable Unit: mg/kg Expression: Kd*C ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
t: Description: time Type: Program Variable Unit: h Reference to: Time ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ theta: Description: Porosity Type: Formula Variable Unit: Expression: 0.4 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
X: Description: Biomass concentration 83
Type: Formula Variable Unit: mg/L Expression: Xo*exp‐(‐Kd)*t ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Xo: Description: Initial biomass concentration Type: Constant Variable Unit: mg/L Value: 180.8 Standard Deviation: 1 Minimum: 0 Maximum: 10000 Sensitivity Analysis: inactive Parameter Estimation: active ********************************************************************************** Processes ********************************************************************************** Reduction: Description: Cr(VI) reduction Type: Dynamic Process Rate: (K^(‐1*(Co‐C2)/Co))*km*C*(X‐(Co‐C)/Rc)/(Kc+C) Stoichiometry: Variable: Stoichiometric Coefficient C ‐1 Sorption: Description: Cr(VI) sorption Type: Dynamic Process Rate: k*(S_eq‐S) Stoichiometry: Variable: Stoichiometric Coefficient C ‐rho_s*(1‐theta)/theta S 1 84
********************************************************************************** Compartments ********************************************************************************** Column: Description: Saturated packed column Type: Mixed Reactor Compartment Compartment Index: 0 Start coordinate: 0 End coordinate: 1 Cross sectional area: A Mob.Vol.Frac: theta Dispersion: with dispersion Number of grit points: 52 Resolution: high Active Variables: C and S Active Processes: reduction and sorption Initial Conditions: Variable(Zone) : Initial Condition C(advection zone) : Cmeas Input type : inlet input Water flow: Qin Loading variable: C: Qin*Cin ********************************************************************************** Definitions of Calculations ********************************************************************************** Calc_0: Description: Calculation Number: 0 Initial Time: 0 Initial State: given, made consistent Step Size: 0.1 85
Num. Steps: 110 Status: active for simulation active for sensitivity analysis ********************************************************************************** Definitions of Parameter Estimation Calculations ********************************************************************************** fit1: Description: Calculation Number: 0 Initial Time: 0 Initial State: given, made consistent Status: active Active parameters: D, Kc, Kd, km, and  Method: simplex Maximum number of iterations: 100 ********************************************************************************** Plot Definitions ********************************************************************************** Concentration plot: Description: Cr(VI) concentration Abscissa: Time Title: Cr(VI) Concentration Abscissa Label: Time [h] Ordinate Label: Concentration [mg/L] Curves: Type : Variable [Calcnum, column advection zone, Time/Space] Value : C [0,column advection zone,1] Value : Cmeas [0,columnadvection zone,0] Value : Cin_meas [0, column advection zone,0] Value : Cin [0,column advection zone,0] 86
APPENDIX
B
Octave Version 3.0 ********************************************************************************** Lraw= [0,10, 20, 30, 40, 50, 60] ; Craw= [20.0, 18.4, 10.97, 6.75, 6.2, 5.9, 0.19] ; Cr= 50 ; Af= 7.85 ; rho_s= 2300000 ; Qin= 0.015 ; k = 0.000000052 ; n=2 ; L= linspace (0, 60, 100) ; Co= 20 ; dCdL= @(C, L) (‐k*C)*((Af*rho_s)/Qin)*((1‐(C/Cr))^n) ; Ci= lsode (dCdL, Co, L) ; plot(Lraw, Craw, ‘o’, L, Ci) data= [Ci] data= [L’] legend (‘experimental values’, ‘model simulation’) ; xlabel (‘distance, cm’) ; ylabel (‘Cr(VI) concentration, mg/L’) ;
87
APPENDIX
C
Flow direction
Proposed barrier
Figure C: Target Site
88
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