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OPTIMISATION OF THE BIOCATALYTIC COMPONENT IN A FERRICYANIDE

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					     OPTIMISATION OF THE BIOCATALYTIC

  COMPONENT IN A FERRICYANIDE MEDIATED

  APPROACH TO RAPID BIOCHEMICAL OXYGEN

                DEMAND ANALYSIS



A thesis submitted in fulfilment of the requirements for

          the Degree of Doctor of Philosophy



                           By

                   KRISTY MORRIS




       School of Environmental and Applied Sciences
            Faculty of Environmental Sciences
                    Griffith University
                        Australia

                       March, 2005
                              CERTIFICATE




This work has not previously been submitted for a degree or diploma in any

university. To the best of my knowledge and belief, the thesis contains no

material previously published or written by another person except where

due reference is made in the thesis itself.




                                                   Signature of Candidate.




                                                 …………………………….




                                                                        II
                       ACKNOWLEDGEMENT

I would like to take this special opportunity to thank those people that have
contributed to the production of this thesis. Without their support, this time may
not have been such a rewarding experience.


Firstly, I would like to thank my supervisors Associate Professor Richard John and
Associate Professor Huijun Zhao. In particular, I would like to thank Richard John
for the time, friendship, encouragement and guidance he has provided to me over
my time at Griffith University. I am truly appreciative.


I would like to thank Huijun Zhao for his time, encouragement and support
throughout my undergraduate and graduate studies at Griffith University.
Thankyou to Dr Peter Teasdale and Dr Neal Pasco (Lincoln Ventures, New Zealand)
who also provided friendship and technical support.




I would like to extend my appreciation to my colleagues and close friends Calvin
Gladman, Kylie Catterall and Kelly O’Halloran as well as fellow students Colin
Hutchins, Lisa Hamilton, Eddie Zhang, Dianlu and Weija, Simon Hodgkinson,
Michelle Stock, Michaela Guest and Nicola Sander.


Thankyou to Coombabah WWTP and local industry including; Golden Circle,
Paul’s Milk, Carlton United Breweries and many more who provided me with
samples.


I am truly thankful to my mother Dianne for her continuing support and friendship.


I would finally like to thank my family, Rodney Ellis and Belah Ellis for their
undoubting support and patience as well as their love and friendship.




                                                                                III
                              ABSTRACT

A novel rapid method for the determination of biochemical oxygen demand
(BOD) has been developed. By replacing oxygen, the terminal electron
acceptor in the microbial oxidation of organic substrate, with the ferricyanide
ion, a significant increase in the rate of the biochemical reaction could be
achieved. This arises from the high solubility of the ferricyanide ion
(compared to oxygen); therefore allowing for elevated microbial populations
without rapid depletion of the electron acceptor. Therefore, the BOD of a
sample can be determined within 1-3 hours compared to 5-days with the
standard BOD5 assay.



A range of microorganisms were shown to be able to use the ferricyanide ion
as an alternative electron acceptor for the biodegradation of a range of
organic compounds in the ferricyanide mediated BOD (FM-BOD) assay. The
most suitable biocatalyst in the FM-BOD method, however, was shown to be
a mixture of microorganisms that was capable of degrading large amounts
and types of compounds. These mixed consortia of microorganisms included
a synthetic mixture prepared in our laboratory and two commercially
available consortia, BODseedTM and Bi-ChemTM. When these seed materials
were employed in the FM-BOD assay, the method was shown to closely
estimate the BOD5 values of real wastewater samples. The linear dynamic
working range of the FM-BOD method was also greatly extended compared
to the standard BOD5 assay (nearly 50 times greater) and other oxygen based
BOD biosensors.


The immobilisation of the microbial consortia by both gel entrapment and
freeze-drying methods was shown to greatly reduce the preparation and
handling time of the mixed consortia for use in the FM-BOD method.
Immobilisation of the mixed microbial consortium in LentiKats®, a PVA
hydrogel, resulted in a marked increase in the stability of the biocatalyst.



                                                                            IV
Diffusion limitations resulting from the gel matrix, however, reduced the rate
and extent of the bioreaction as well as the linear dynamic working range of
the method.


Freeze-drying techniques were shown to circumvent some of the limitations
identified with gel entrapment for the immobilisation of the mixed consortia.
The freeze-dried consortia could be used off-the-shelf and demonstrated
reduced diffusional restrictions. A marked decrease in the viability of the
microorganisms was observed directly following the freeze-drying process
and in subsequent storage. Carrageenan, however, was shown to afford a
significant degree a protection to the cells during the freeze-drying process.




                                                                                 V
                       PUBLICATIONS


1. A review of ferricyanide-mediated microbial reactions for environmental
   monitoring
   K. Morris, H. Zhao, R. John,
   Australian Journal of Chemistry, 58(4) 237–245


2. The use of a mixed microbial consortium in a rapid ferricyanide mediated
   Biochemical Oxygen Demand assay
   K. Morris, H. Zhao, R. John,
   Water Pollution VII: Modelling, Measuring and Prediction, 2003
   Editors, C.A. Brebbia, D. Almorza and D. Sales, Progress in Water
   Resources Vol 9

3. Ferricyanide mediated Biochemical Oxygen Demand– development of a rapid
   biochemical oxygen demand assay
   K. Morris, K. Catterall, H. Zhao, N. Pasco, R. John, 2001,
   Analytica Chimica Acta, 442, 129-139

4. The use of microorganisms with broad substrate specificity for the
   Ferricyanide rapid BOD determination
   Catterall K, Morris K, Gladman C, Zhao H, John R, 2001,
   Talanta, 55(6):1187-1194




                                                                        VI
                    LIST OF ABBREVIATIONS
A         Ampere
Ag/AgCl   Silver/silver chloride reference electrode
(aq)      Aqueous
ATP       Adenosine triphosphate
APHA      American Public Health Association
BOD       Biochemical oxygen demand
oC        Degree Celsius
COD       Chemical Oxygen Demand
DO        Dissolved oxygen
Eapp      Applied potential
EPA       Environmental Protection Authority
ETS       Electron transport system
FM-BOD    Ferricyanide mediated biochemical oxygen demand
g         Gram
(g)       Gas
GGA       Glucose/ Glutamic acid
ilim      Limiting current
(l)       Liquid
L         Litre
M         Molar
m         Slope
m         Milli-(prefix)
n         Nano- (prefix)
nA        Nano-ampere
NADH      Nicotinamide adenine dinucleotide
OECD      Organisation for Economic Co-operation and Development
p         p-value
R         Pearson correlation co-efficient
rsd       Relative standard deviation
TCA       Tricarboxylic Acid Cycle


                                                                   VII
TOC    Total organic carbon
TSB    Trypticase soy broth
µ      Micro- (prefix)
V      Volt(s)
WWTP   Waste water treatment plant




                                     VIII
                            LIST OF FIGURES
FIGURE 1.1 SCHEMATIC PRESENTATION OF A BOD BIOSENSOR.                           13

FIGURE 1.2 PRINCIPLE OF THE AMPEROMETRIC FERRICYANIDE-MEDIATED BOD

             BIOSENSOR.                                                         18

FIGURE 2.1   INCUBATION APPARATUS INCLUDING A SHAKING WATER BATH AND

             CONTROLLED NITROGEN SPARGING UNIT                                  47

FIGURE 2.2 POTENTIAL WAVEFORM AND CURRENT RESPONSE FOR A

             CHRONOAMPEROMETRIC EXPERIMENT.                                     48

FIGURE 2.3   CHRISTTM ALPHA 1-4 FREEZE DRYING UNIT WITH A

             JAVACTM DD-75 DOUBLE STAGE HIGH VACUUM PUMP                        60

FIGURE 3.1   LIMITING CURRENT VALUES FOR A 3 HOUR INCUBATION

             OF E. COLI AND THE SYNTHETIC ORGANIC SOLUTIONS IN THE

             PRESENCE OF FERRICYANIDE.                                          70

FIGURE 3.2   HISTOGRAMS AND DENSITY FUNCTIONS FOR THE 10 ORGANIC SOLUTIONS.     71

FIGURE 3.3   A BIPLOT OF THE FIRST TWO PRINCIPLE COMPONENTS FOR THE SYNTHETIC

             ORGANIC SOLUTIONS AND THE DIFFERENT MICROBIAL SPECIES.             73

FIGURE 3.4   A BIPLOT OF THE FIRST PRINCIPLE COMPONENT AGAINST THE THIRD

             PRINCIPLE COMPONENT FOR THE SYNTHETIC ORGANIC SOLUTIONS AND

             DIFFERENT SPECIES OF MICROORGANISMS.                               74

FIGURE 3.5   HIERARCHICAL CLUSTER ANALYSIS FOR THE 23 MICROBIAL SPECIES         75

FIGURE 3.6   SCATTERPLOT OF BOD5 VALUES VERSUS FM-BOD5 VALUES FOR

             THE 10 SYNTHETIC ORGANIC SOLUTIONS AND THE MIXED

             CONSORTIUM AS THE MICROBIAL SEED.                                  79

FIGURE 4.1   LIMITING CURRENT VALUES OBTAINED AT VARIOUS

             INCUBATION TIMES FOR INDIVIDUAL MICROBIAL SEEDS AND THE

             MIXED MICROBIAL CONSORTIUM.                                        90

FIGURE 4.2   LIMITING CURRENT VALUES OBTAINED FOR INCREASING

             CONCENTRATIONS OF THE STANDARD GGA AND OECD

             CALIBRATION SOLUTIONS                                              93

FIGURE 4.3   EFFECT OF MICROBIAL CONSORTIUM CONCENTRATION ON THE LIMITING

             CURRENT RESPONSE FOR THE GGA SOLUTION                              95

FIGURE 4.4   EFFECT OF FERRICYANIDE CONCENTRATION ON THE LIMITING CURRENT

             RESPONSE FOR THE MIXED MICROBIAL CONSORTIUM                        96

FIGURE 4.5   LIMITING CURRENT VALUES OBTAINED AT VARIOUS DILUTIONS FOR

             CONFECTIONARY AND DAIRY SAMPLES                                    98

FIGURE 4.6   SCATTERPLOT OF BOD5 VALUES VERSUS FM-BOD5 EQUIVALENT

             VALUES FOR INDUSTRIAL WASTEWATER SAMPLES                           101




                                                                                IX
FIGURE 4.7    SCATTERPLOT OF UNDILUTED BOD5 VALUES VERSUS FM-BOD5

              EQUIVALENT VALUES FOR INDUSTRIAL WASTEWATER SAMPLES           102

FIGURE 5.1    COMMERCIALLY AVAILABLE BODSEED AND BI-CHEM CONSORTIA          112

FIGURE 5.2    SCATTERPLOT OF BOD5 VALUES OBTAINED WITH ACTIVATED SLUDGE

              AND BOD5 VALUES OBTAINED WITH BODSEEDTM REAL SAMPLES          116

FIGURE 5.3    SCATTERPLOT OF BOD5 VALUES OBTAINED WITHACTIVATED SLUDGE

              AND BOD5 VALUES OBTAINED WITH BI-CHEMTM                       117

FIGURE 5.4    BODSEED AFTER 16 HOURS INCUBATION IN TRYPTICASE SOY BROTH

              AT 27OC IN A HORIZONTAL SHAKER BATH                           119

FIGURE 5.5    BI-CHEM AFTER 16 HOURS INCUBATION IN TRYPTICASE SOY BROTH

                  AT 27OC IN A HORIZONTAL SHAKER BATH                       120

FIGURE 5.6    EFFECT OF COMMERCIAL CONSORTIUM CONCENTRATION ON THE

              LIMITING CURRENT RESPONSE FOR THE GGA SOLUTION                122

FIGURE 5.7    EFFECT OF FERRICYANIDE CONCENTRATION ON THE LIMITING

              CURRENT RESPONSE FOR THE COMMERCIAL MICROBIAL CONSORTIA       123

FIGURE 5.8    EFFECT OF GGA CONCENTRATION ON THE LIMITING CURRENT

              RESPONSE FOR THE COMMERCIAL CONSORTIA                         124

FIGURE 5.9    EFFECT OF OECD CONCENTRATION ON THE LIMITING CURRENT

              RESPONSE FOR THE COMMERCIAL CONSORTIA                         126

FIGURE 5.10   SCATTERPLOT OF BOD5 VALUES WITH ACTIVATED SLUDGE VERSUS

              FMBOD5 EQUIVALENT VALUES WITH BODSEED FOR REAL SAMPLES        128

FIGURE 5.11   SCATTERPLOT OF BOD5 VALUES WITH BODSEED VERSUS

              FMBOD5 EQUIVALENT VALUES WITH BODSEED FOR REAL SAMPLES        129

FIGURE 5.12   SCATTERPLOT OF BOD5 VALUES WITH ACTIVATED SLUDGE VERSUS

              FMBOD5 EQUIVALENT VALUES WITH BI-CHEM FOR REAL SAMPLES        130

FIGURE 5.13   SCATTERPLOT OF BOD5 VALUES VERSUS FMBOD5 EQUIVALENT

              VALUES WITH BI-CHEM FOR REAL SAMPLES                          131

FIGURE 6.1    IMMOBILISATION METHODS AND THEIR CLASSIFICATION IN TERMS OF

              THE PHYSICAL MECHANISM OF LOCALISATION (MODIFIED FROM [2]).   137

FIGURE 6.2    SCHEMATIC OF LENTIKAT® PRODUCTION                             142

FIGURE 6.3    SCHEMATIC VIEW OF A LENTIKAT®.                                143

FIGURE 6.4    TOP VIEW OF A LENTIKAT ®                                      143

FIGURE 6.5    CONFOCAL LASER SCANNING MICROSCOPY OF THE DISTRIBUTION

              AND DENSITY OF PROPIDIUM IODIDE STAINED MICROORGANISMS        144

FIGURE 6.6    CONFOCAL LASER SCANNING MICROSCOPY OF THE DISTRIBUTION AND

              DENSITY OF PROPIDIUM IODIDE STAINED MICROORGANISMS            144

FIGURE 6.7    LIMITING CURRENT VALUES AT VARIOUS INCUBATION TIMES FOR

              DIFFERENT INITIAL BIOMASS CONCENTRATIONS OF THE LENTIKAT ®

              IMMOBILIZED MICROBIAL CONSORTIUM.                             146


                                                                             X
FIGURE 6.8    LIMITING CURRENT VALUES AT DIFFERENT INITIAL BIOMASS

              CONCENTRATIONS OF THE LENTIKAT ® IMMOBILIZED MICROBIAL

              CONSORTIUM.                                                    147

FIGURE 6.9    LIMITING CURRENT RESPONSES FOR INCREASING MASS OF LENTIKATS®   148

FIGURE 6.10   LIMITING CURRENT VALUES FOR INCREASING CONCENTRATIONS

              OF THE STANDARD GGA SOLUTION FOR THE IMMOBILISED MICROBIAL

              CONSORTIUM                                                     150

FIGURE 6.11   EFFECT OF FERRICYANIDE CONCENTRATION ON THE LIMITING CURRENT

              RESPONSE FOR LENTIKATS ®                                       152

FIGURE 6.12   LIMITING CURRENT RESPONSES FOR LENTIKATS® AFTER INCUBATION

              IN THE STANDARD GGA SOLUTION                                   153

FIGURE 6.13   LIMITING CURRENT VALUES OBTAINED AT VARIOUS DILUTIONS FOR

              THE CEREAL AND CANNERY SAMPLES                                 155

FIGURE 6.14   SCATTERPLOT OF BOD5 VALUES VERSUS FM-BOD5 EQUIVALENT

              VALUES FOR REAL SAMPLES                                        156

FIGURE 7.1    IDEALISED STRUCTURES OF KAPPA AND IOTA TYPE CARRAGEENANS       173

FIGURE 7.2    LIMITING CURRENT RESPONSES FREEZE-DRIED E. COLI                175

FIGURE 7.3    SCATTERPLOT OF BOD5 VALUES VERSUS FM-BOD5 EQUIVALENT VALUES FOR

              REAL SAMPLES AND THE FREEZEDRIED CONSORTIUM                    177

FIGURE 7.4    SCATTERPLOT OF BOD5 VALUES VERSUS FM-BOD5 EQUIVALENT VALUES FOR

              REAL SAMPLES AND FREEZEDRIED BODSEED                           178

FIGURE 7.5    SCATTERPLOT OF BOD5 VALUES VERSUS FM-BOD5 EQUIVALENT VALUES

              FOR REAL SAMPLES AND FREEZEDRIED BODSEED USING OECD AS THE

              CALIBRATION STANDARD                                           179




                                                                                XI
                             LIST OF TABLES

TABLE   1.1   COMMERCIALLY AVAILABLE BOD INSTRUMENTS                          16

TABLE   1.2   MICROORGANISM- MEDIATOR COMBINATIONS AND

              THEIR APPLICATIONS REPORTED IN LITERATURE                      22

TABLE   2.1   SOURCE OF MICROORGANISMS EMPLOYED AS BIOCATALYSTS

              IN THE FM-BOD METHOD.                                          44

TABLE   3.1 MICROORGANISMS REPORTED IN LITERATURE TO USE

              FERRICYANIDE AS AN ALTERNATIVE ELECTRON ACCEPTOR               63

TABLE   3.2   COMPOSITION OF SYNTHETIC ORGANIC SOLUTIONS                     65

TABLE   3.3   DESCRIPTION OF MICROORGANISMS INVESTIGATED AS

              POTENTIAL BIOCATALYST IN THE FM-BOD METHOD                     66

TABLE   3.4 LOADINGS OF EACH ORGANIC SOLUTION GIVEN

              BY EACH PRINCIPLE COMPONENT.                                   70

TABLE   3.5   BOD5 AND COD VALUES FOR THE TEN STANDARD

              ORGANIC SOLUTIONS                                              77

TABLE   3.6   CORRELATION INFORMATION FOR THE BOD5 AND FM-BOD5 EQUIVALENT

              VALUES OF THE TEN STANDARD ORGANIC SOLUTIONS                   79

TABLE   4.1   BOD5 VALUES, COD VALUES AND THE BIODEGRADABILITY

              (BOD5/ COD) OF VARIOUS INDUSTRIAL EFFLUENTS                    98

TABLE   4.2   COMPARISON OF CALIBRATION SOLUTIONS FOR FOUR SAMPLES

              WITH THE MIXED CONSORTIUM                                     102

              THE BIOCOMPONENT AND MEASURING IN THE STEADY STATE MODE        112

TABLE   7.1   CRYOPROTECTIVE AGENTS IN 100ML DEIONISED WATER                 166

TABLE   7.2   LIMITING CURRENT RESPONSES OBTAINED FOR A ONE-HOUR INCUBATION OF

              FREEZE-DRIED E.COLI                                            170




                                                                             XII
                       TABLE OF CONTENTS

CERTIFICATE                                                                II
ACKNOWLEDGEMENTS                                                          III
ABSTRACT                                                                  IV
PUBLICATIONS                                                              VI
LIST OF ABBREVIATIONS                                                    VII
LIST OF FIGURES                                                           IX
LIST OF TABLES                                                            XII
TABLE OF CONTENTS                                                        XIII


CHAPTER 1
INTRODUCTION
1.1           BIOCHEMICAL OXYGEN DEMAND                                   1
       1.1.1 BOD5 standard method                                         4
       1.1.2 Limitations of the BOD5 standard method                      6
       1.1.3 Alternative wet chemical techniques for BOD determination    8
       1.1.4 Significance of the BOD5 assay                               10
1.2.          ALTERNATIVE METHODS FOR THE RAPID
              DETERMINATION OF BOD                                        11
       1.2.1 BOD Biosensors                                               12
       1.2.2 Limitations of BOD Biosensors                                13
            1.2.2.1 Commercial BOD Analysers                              15
       1.2.3 Ferricyanide-mediated microbial BOD sensors                  17
1.3           BIOCHEMICAL MEDIATOR DEMAND- FERRICYANIDE
              MEDIATED RAPID BOD (FM-BOD)                                 18
       1.3.1 Redox mediators in microbial reactions                       21
       1.3.2 Mechanisms of mediator reduction by microbial whole cells    23
       1.3.3 Selection of Microorganisms for a rapid ferricyanide
              mediated BOD method                                         25
       1.3.4 Limitations of the current Ferricyanide mediated rapid
              BOD method (FM-BOD)                                         29
1.4           AIMS AND SCOPE OF THIS STUDY                                31


                                                                          XIII
1.5          REFERENCES                                                   35


CHAPTER 2
GENERAL EXPERIMENTAL METHODS
2.1          GENERAL EXPERIMENTAL METHODS FOR
             CHAPTER 3                                                    43
      2.1.1 Reagents                                                      43
      2.1.2 Microorganism preparation                                     44
      2.1.3 Sample Preparation                                            45
      2.1.4 Incubation and Detection                                      45
             2.1.4.1 Chronoamperometric detection                         47
      2.1.5 BOD5 and COD analysis                                         49
      2.1.6 FM-BOD5 Equivalent Values                                     50
      2.1.7 Correlation analysis                                          50
2.2          GENERAL EXPERIMENTAL METHODS FOR
             CHAPTER 4                                                    51
      2.2.1 Microorganism preparation                                     51
      2.2.2 Sample preparation                                            52
      2.2.3 Preparation of real samples                                   53
      2.2.4 Preparation of the OECD solution                              53
      2.2.5 FM-BOD5 Equivalent Values                                     53
2.3          GENERAL EXPERIMENTAL METHODS FOR
             CHAPTER 5                                                    54
      2.3.1 BODseed and Bi-Chem culture conditions                        54
2.4          GENERAL EXPERIMENTAL METHODS FOR
             CHAPTER 6                                                    55
      2.4.1 Preparation of Microorganisms                                 55
           2.4.1.2 Principle of entrapment in poly(vinyl alcohol) (PVA)   56
      2.4.2 Sample Preparation                                            57
2.5          GENERAL EXPERIMENTAL METHODS FOR
             CHAPTER 7                                                    58
      2.5.1 Preparation of freeze-dried Microorganisms                    58
      2.5.2 Preparation of samples                                        59



                                                                          XIV
2.6           REFERENCES                                                      61


CHAPTER 3
EVALUATION OF A RANGE OF BIOCATALYSTS IN THE
FERRICYANIDE MEDIATED RAPID BOD ASSAY

3.1          INTRODUCTION                                                     62
3.2          METHODS                                                          64
      3.2.1 Multivariate statistical analysis                                 67
3.3          RESULTS AND DISCUSSION                                           68
      3.3.1 Evaluation of microorganisms to use ferricyanide as an
             alternative electron acceptor for the degradation of synthetic
             organic solutions                                                68
      3.3.2 Evaluation of Microorganisms to estimate BOD5 values
             of synthetic organic solutions                                   76
3.4          CONCLUSIONS                                                      80
3.5          REFERENCES                                                       82


CHAPTER 4
EVALUATION OF A MICROBIAL CONSORTIUM IN THE
FERRICYANIDE MEDIATED RAPID BOD ASSAY

4.1          INTRODUCTION                                                     86
4.2          METHODS                                                          88
4.3          RESULTS AND DISCUSSION                                           88
      4.3.1 Comparison of single species seeds and the mixed microbial
           consortium- rate and extent of the biochemical reaction            88
      4.3.2 Linear dynamic range for standard calibration solutions           90
      4.3.3 Effect of concentration of the mixed microbial consortium         93
      4.3.4 Effect of mediator concentration                                  95
      4.3.5 Effect of varying real wastewater sample concentration            96
      4.3.6 Comparison of BOD5 values and equivalent FM-BOD5 values for
             industrial wastewater samples                                    97



                                                                              XV
      4.3.7 Comparison of calibration solutions                             101
4.4          CONCLUSIONS                                                    105
4.5          REFERENCES                                                     107


CHAPTER 5
EVALUATION EVALUATION OF COMMERCIAL CONSORTIA
IN THE FERRICYANIDE MEDIATED RAPID BOD ASSAY

5.1           INTRODUCTION                                                  110
5.2          METHODS                                                        113
5.3          RESULTS AND DISCUSSION                                         114
      5.3.1 Comparison of Activated sludge and the commercially available
             consortia as seed material in the standard BOD5 assay          114
      5.3.2 Microbial consortia composition after harvesting                117
      5.3.3 Effect of microbial concentration in the FM-BOD assay           120
      5.3.4 Effect of ferricyanide concentration in the FM-BOD assay        121
      5.3.5 Effect of GGA Concentration in the FM-BOD assay                 122
      5.3.6 Effect of OECD concentration in the FM-BOD assay                124
      5.3.7 Comparison of BOD5 values and FM-BOD5 equivalent values
             for industrial samples                                         125
5.4          CONCLUSION                                                     131
5.5          REFERENCES                                                     133


CHAPTER 6
IMMOBILISATION OF THE MICROBIAL CONSORTIA IN
LENTIKATS® FOR USE IN THE FERRICYANIDE MEDIATED
RAPID BOD ASSAY

6.1          INTRODUCTION                                                   135
6.2          METHODS                                                        139
      6.2.1 Preparation of Lentikats                                        140
6.3          RESULTS AND DISCUSSSION                                        141




                                                                            XVI
      6.3.1    Investigation of the distribution of the microbial consortium
               within LentiKats                                                142
      6.3.2 Effect of initial biomass loading in LentiKats ®                   144
      6.3.3 Effect of LentiKat ® bead mass147
      6.3.4 Linear dynamic range for standard GGA solution                     148
      6.3.5 Effect of ferricyanide concentration                               150
      6.3.6 Stability of LentiKats ®                                           151
      6.3.7 Effect of Industrial sample concentration                          153
      6.3.8 Analysis of real industrial wastewater samples using LentiKats®    154
6.4             CONCLUSIONS                                                    156
6.5             REFERENCES                                                     159
CHAPTER 7
EVALUATION OF FREEZE-DRIED MICROBIAL CONSORTIA
IN THE FERRICYANIDE MEDIATED RAPID BOD ASSAY
7.1             INTRODUCTION                                                   162
7.2             METHODS                                                        165
      7.2.1 Preparation of E.coli                                              166
      7.2.2 Solution Preparation                                               166
7.3            RESULTS AND DISCUSSION                                          167
      7.3.1 Evaluation of cryoprotective additives                             167
      7.3.2 Evaluation of the viability of E. coli during storage
              following freeze-drying in carrageenan                           171
      7.3.3 Analysis of real wastewater samples using a mixed
              freeze-dried microbial consortia                                 174
      7.3.4 Analysis of real industrial samples using the
              freeze-dried commercially available BODseed                      176
CHAPTER 8
GENERAL CONCLUSIONS                                                            185

APPENDIX A                                                                     190

APPENDIX B                                                                     192

APPENDIX C                                                                     195




                                                                               XVII

				
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