SUPPRESSION OF GEOSMIN AND 2-MIB PRODUCTION IN RAS BY … · 2018-11-23 · v Acknowledgements...

SUPPRESSION OF GEOSMIN AND 2-MIB

PRODUCTION IN RAS BY BIOFLOCS

Number of words: 36776

Dissanayake Mudiyanselage Anusha Edirisinghe

Student Number: 01600768

Promotors: Dr. ir. Nancy Nevejan, Prof. Dr. ir. Sven Mangelinckx

Tutors: Brecht Stechele, Elias Bonneure

Master’s Dissertation submitted to Ghent University in partial fulfilment of the requirements for

the degree of Master of Science in Aquaculture

Academic year: 2017 – 2018

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"The author and the promoters give permission to make this master dissertation available for

consultation and to copy parts of this master dissertation for personal use. In the case of any other

use, the copyright terms have to be respected, in particular with regard to the obligation to state

expressly the source when quoting results from this master dissertation."

Gent University, 24th August 2018

Promoter: …………………… Promoter: ………………………

Dr. ir. Nancy Nevejan Prof. Dr. ir. Sven Mangelinckx

Author: ……………………....

Dissanayake Mudiyanselage Anusha Edirisinghe

v

Acknowledgements

Foremost, I would like to express my sincere gratitude to my promoter Dr. Ir. Nancy Nevejan for

her continuous support for my research, for her patience, motivation, enthusiasm, and immense

knowledge. Her guidance helped me in all the time of research and writing of this thesis. Also, I

would like to thank Prof. Dr. Ir. Sven Mangelinckx, for his encouragement and insightful comments.

My sincere thanks also goes to Brecht Stechele, Yoshi Mertens and Eli Bonneure for their valuable

guidance and endless support. You always helped me to choose the right direction and successfully

complete my dissertation. My thanks goes to Project AquaVlan 2, because this work would not

have been possible without the support of the Project AquaVlan 2 which is financed by the Interreg

V programme Flanders-The Netherlands, the cross-border collaboration programme with financial

support from the European Fund for Regional Development (www.grensregio.eu). Results

presented in this thesis are obtained with infrastructure funded by EMBRC Belgium - FWO

agreement 20151029-03 and Hercules agreement 20140910-03.

I wish to convey my sincere gratitude to Prof. Dr. ir. Peter Bossier, for accepting me as MSc student at

the Laboratory of Aquaculture and Artemia Reference Centre (ARC) allowing me to gain fruitful

experience and knowledge during the period of life in Ghent University. Very special thanks are going

to the administrative and technical staff of ARC and SynBioC laboratories for their friendly behaviour,

valuable assistance and tireless effort to provide the best study conditions. I would like to thank my

colleagues from Masters of Science in Aquaculture 2016 - 2018 for their wonderful collaboration.

They supported me greatly and were always willing to help me.

Nobody has been more important to me in the pursuit of this project than the members of my

family. I would like to thank my parents and sister; whose love and guidance are with me in

whatever I pursue. Most importantly, I wish to thank my loving and supportive husband,

Anuruddha, who provides unending inspiration and dedication.

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Table of contents

Copy right .......................................................................................................................................... iii

Acknowledgements............................................................................................................................ v

Table of contents .............................................................................................................................. vi

List of figures ...................................................................................................................................... x

List of tables .................................................................................................................................... xiv

List of abbreviations ......................................................................................................................... xv

Abstract .......................................................................................................................................... xvii

Chapter 1: Introduction ................................................................................................................... 19

1.1. Background information ................................................................................................... 19

1.2. Research problem identification and justification ............................................................ 20

1.3. Objectives .......................................................................................................................... 21

Chapter 2: Literature review ............................................................................................................ 22

2.1. Introduction to geosmin and 2-methylisoborneol (2-mib) ................................................... 22

2.2. Presence of geosmin and 2-mib in aquaculture systems ..................................................... 26

2.2.1 Uptake of geosmin and 2-mib by fish .............................................................................. 29

2.2.2. Disadvantages due to the taste and odour compounds in aquaculture ........................ 30

2.2.3. Removal of geosmin and 2-mib from water................................................................... 31

2.2.4. Potential of bioflocs to reduce the production of geosmin and 2-mib .......................... 34

2.3. Analysis of geosmin and 2-mib by solid-phase microextraction .......................................... 35

2.3.1. Principle of gas chromatography-mass spectrometry (GC-MS) ..................................... 35

2.3.2. Solid-phase microextraction (SPME) in GC-MS analysis ................................................ 36

Chapter 3: Materials and Methods .................................................................................................. 38

3.1. Section A – Biofloc stock preparation and management ..................................................... 38

3.1.1. Biofloc reactor preparation ............................................................................................ 38

3.1.2. Balancing input Carbon/Nitrogen (C/N) ratio ................................................................ 39

3.1.3. Biofloc characteristics ..................................................................................................... 41

3.1.4. Determination of floc morpho-structure ....................................................................... 43

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3.1.5. Main water quality parameters ...................................................................................... 43

3.2. Section B – Optimization of GC-MS for geosmin and 2-mib analysis ................................... 44

3.2.1. Gas chromatography-mass spectrometry (GC-MS) optimization .................................. 44

3.2.2. Chemicals for GC-MS optimization ................................................................................. 44

3.2.3. Solid-phase microextraction standard protocol ............................................................. 44

3.2.4. Gas chromatography-mass spectrometry (GC-MS) conditions in the laboratory ......... 45

3.2.4.1. Optimum incubation temperature ......................................................................... 48

3.2.4.2. Optimum extraction time ....................................................................................... 49

3.2.4.3. Salting out effect ..................................................................................................... 50

3.2.4.4. Sample storage time and stability .......................................................................... 50

3.2.4.5. Repeatability and reproducibility of GC-MS for developed method ...................... 51

3.2.4.6. Limit of detection (LOD).......................................................................................... 51

3.3. Section C - Experiments done to determine the suppression of geosmin and 2-mib by

bioflocs ......................................................................................................................................... 52

3.3.1. Chemicals used for the experiment ............................................................................... 52

3.3.2. Experimental setups to determine the effect of bioflocs on geosmin and 2-mib ......... 52

3.4. Section D - Statistical analysis ............................................................................................... 58

Chapter 4: Results ............................................................................................................................ 59

4.1. Section A: Biofloc stock management................................................................................... 59


4.1.2. Floc morpho structure .................................................................................................... 60

4.1.3. Water quality parameters .............................................................................................. 61

4.2. Section B: Experiments for optimized geosmin and 2-mib analysis using GC-MS................ 63

4.2.1. Optimum incubation temperature ................................................................................. 63

4.2.2. Optimum extraction time ............................................................................................... 63

4.2.3. Salting out effect ............................................................................................................ 64

4.2.4. Sample storage time and stability .................................................................................. 65

4.2.5. Repeatability and reproducibility of GC-MS analysis for geosmin and 2-mib ............... 66

4.2.6. Limit of detection (LOD) ................................................................................................. 68

4.2.7. Optimized SPME GC-MS analysis method ...................................................................... 69

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4.3. Section C: Analysis of bioflocs for the suppression of geosmin and 2-mib using optimized GC-

MS method ................................................................................................................................... 71

4.3.1. Experiment 1: Identification of the naturally occurring geosmin and 2-mib in

experimental water and carryover effect of GC-MS ................................................................ 71

4.3.2. Experiment 2: Bioflocs spiked with geosmin and 2-mib with aeration ......................... 71

4.3.3. Experiment 3: Effect of aeration on geosmin and 2-mib using different aeration

techniques ................................................................................................................................ 72

4.3.4. Experiment 4: Effect of salinity and aeration on losses of geosmin and 2-mib in

experimental bottles ................................................................................................................ 73

4.3.5. Experiment 5: Effect of bioflocs on geosmin and 2-mib without aeration .................... 75

4.3.6. Experiment 6: Changes of water quality parameters of bioflocs stored in closed vials

placed on a rotator versus shaker ............................................................................................ 77

4.3.7. Experiment 7: Effect of bioflocs on suppression of geosmin and 2-mib without aeration

on a shaker ............................................................................................................................... 79

4.3.8. Experiment 8: Effect of bioflocs on geosmin and 2-mib without aeration on a rotator 81

Chapter 5: Discussion ....................................................................................................................... 83

5.1. Section A: Biofloc stock management................................................................................... 83


5.1.2. Water quality management ........................................................................................... 85

5.2. Section B: Experiments for optimized geosmin and 2-mib analysis using GC-MS................ 86

5.2.1. Optimum incubation temperatures for geosmin and 2-mib.......................................... 86

5.2.2. Optimum extraction time ............................................................................................... 87

5.2.3. The salting out effect for optimum extraction ............................................................... 90

5.2.4. Sample storage time and stability .................................................................................. 91

5.2.5. Assessment of the repeatability and reproducibility of SPME GC-MS ........................... 91

5.2.6. Limit of detection (LOD) ................................................................................................. 95

5.3. Section C: Experiments done to determine the suppression of geosmin and 2-mib by bioflocs

...................................................................................................................................................... 95

5.3.2. Experiment 2: Bioflocs spiked with geosmin and 2-mib with aeration ......................... 96

5.3.3. Experiment 3: Effect of aeration for geosmin and 2-mib using different aeration

techniques ................................................................................................................................ 97

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experimental bottles ................................................................................................................ 99

5.3.5. Experiment 5: Effect of bioflocs on geosmin and 2-mib without aeration .................. 100


placed on a rotator versus shaker .......................................................................................... 101

5.3.7. Experiment 7: Effect of bioflocs for suppression of geosmin and 2-mib without aeration

on a shaker ............................................................................................................................. 102

5.3.8. Experiment 8: Effect of bioflocs on geosmin and 2-mib without aeration on a rotator

................................................................................................................................................ 103

Chapter 6: General conclusions ..................................................................................................... 106

Chapter 7: Recommendations for further research ...................................................................... 108

8: List of references........................................................................................................................ 109

9: Appendices ................................................................................................................................. 135

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List of figures

Figure 1: Different organisms responsible for geosmin and 2-mib production .............................. 24

Figure 2: The biochemical pathways for formation of geosmin and 2-mib in Streptomycetes and

myxobacteria (Juttner & Watson, 2007) ......................................................................................... 25

Figure 3: Sketch of a RAS system in aquaculture (adapted from Yoshino, et al., 1997) ................. 27

Figure 4: Schematic description of the geosmin occurrences and transport of geosmin (Chung, et

al., 2016) .......................................................................................................................................... 29

Figure 5: Purging of fish in RAS (Llyn Aquaculture Ltd. (2009)) ...................................................... 31

Figure 6: Morphology of floc under microscope ............................................................................. 34

Figure 7: Schematic of a GC-MS system (Wu, et al., 2012) ............................................................. 36

Figure 8: Diagram of SPME analysis with GC-MS (Schmidt & Podmore, 2015)............................... 37

Figure 9: Biofloc stock tank .............................................................................................................. 39

Figure 10: Commercial feed (grinded) and glucose for feeding of biofloc ...................................... 40

Figure 11: Imhoff cone test .............................................................................................................. 42

Figure 12: Tetra water quality test kit ............................................................................................. 43

Figure 13: CERTAN capillary vial ...................................................................................................... 44

Figure 14: GC-MS equipment used for the analysis ........................................................................ 45

Figure 15: The adsorption and desorption process with SPME GC-MS (adapted from Yang, et al.,

2018) ................................................................................................................................................ 47

Figure 16: GC-MS component (Goodman, 2015). ........................................................................... 48

Figure 17: Incubation of the sample and head space SPME (Schmidt & Podmore, 2015) ............. 49

Figure 18: Principles of extraction by headspace SPME (Schmidt & Podmore, 2015) .................... 50

Figure 19: Experimental setup with four treatments (right to left: SW+, BF+, SBF+, BF) Note that

SBF+ was covered with tissue due to foaming effect before starting the experiment ................... 54

Figure 20: Experimental setup to evaluate the effect of biofloc on geosmin and 2-mib without

aeration ............................................................................................................................................ 56

Figure 21: Experimental design to analyse the effect of bioflocs for suppression geosmin and 2-mib

without aeration on a shaker .......................................................................................................... 57

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Figure 22: Experimental setup to analyse the effect of bioflocs on geosmin and 2-mib without

aeration on a rotator ....................................................................................................................... 58

Figure 23: Biofloc morpho structure ................................................................................................ 60

Figure 24: Water quality parameters of the biofloc stock during the study period ....................... 62

Figure 25: The area of geosmin with different extraction times ..................................................... 64

Figure 26: The area of 2-mib with different extraction times ......................................................... 64

Figure 27: The area of geosmin with different storage periods ...................................................... 65

Figure 28: The area of 2-mib with different storage periods .......................................................... 66

Figure 29: Limit of detection of geosmin ......................................................................................... 69

Figure 30: Limit of detection of 2-mib ............................................................................................. 69

Figure 31: Oven temperature program of GC-MS ........................................................................... 70

Figure 32: The GSM/TCA with different salinity and aeration (FW a-, FW a+, SW a-, SW a+) during

0 h, 2 h, 6 h and 24 h periods spiked with 50 µg L-1 geosmin at 0 h. Significant differences between

treatments at 0 h and 24 h were represented as a, b and c, (n=3) ................................................. 74

Figure 33: The 2-mib/TCA with different salinity and aeration (FW a-, FW a+, SW a-, SW a+) during

0 h, 2 h, 6 h and 24 h periods spiked with 50 µg L-1 2-mib at 0 h. Significant differences between

treatments at 0 h and 24 h were represented as a, b and c, (n=3) ................................................. 75

Figure 34: The GSM/TCA with four treatments (BF, BF+, SBF+, SW+) during 24 h without aeration

spiked with 50 µg L- 1 geosmin at 0 h. Significant differences between treatments at 0 h and 24 h

were represented as a, b and c, (n=3) ............................................................................................. 76

Figure 35: The 2-mib/TCA with four treatments (BF, BF+, SBF+, SW+) during 24 h without aeration

spiked with 50 µg L- 1 2-mib at 0 h. Significant differences between treatments at 0 h and 24 h were

represented as a, b and c, (n=3) ...................................................................................................... 76

Figure 36: The temperature of the bioflocs stored on rotator and shaker during five days (n=1). 77

Figure 37: The DO% of the bioflocs stored on rotator and shaker during five days (n=1). ............. 78

Figure 38: The pH of the bioflocs stored on rotator and shaker during five days (n=1). ................ 78

Figure 39: The ammonium concentration (mg L-1) of the bioflocs stored on rotator and shaker

during five days (n=1). ..................................................................................................................... 79

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Figure 40: The GSM/TCA with three treatments (BF, BF+, SW+) during 72 h without aeration spiked

with 50 µg L- 1 geosmin at 0 h. Significant differences between treatments at 0 h and 72 h were

represented as a and b (n=3) ........................................................................................................... 80

Figure 41: The 2-mib/TCA with three treatments (BF, BF+, SW+) during 72 h without aeration

spiked with 50 µg L- 1 2-mib at 0 h. Significant differences between treatments at 0 h and 72 h were

represented as a and b (n=3) ........................................................................................................... 80

Figure 42: The GSM/TCA for four treatments (BF, BF+, SBF+, SW+) during 240 h period on a rotator

spiked with 50 µg L-1 of geosmin at 0 h. Significant differences between treatments at 0 h and 240

h were represented as a, b and c (n=3) ........................................................................................... 82

Figure 43: The 2-mib/TCA for four treatments (BF, BF+, SBF+, SW+) during 240 h period on a rotator

spiked with 50 µg L-1 of 2-mib at 0 h. Significant differences between treatments at 0 h and 240 h

were represented as a, b and c (n=3) .............................................................................................. 82

Figure 44: The effect of iIncubation temperature on the HS-SPME of geosmin and 2-mib (Saito, et

al., 2008). ......................................................................................................................................... 87

Figure 45: Effect of incubation temperature on the extraction efficiencies of geosmin (red) and 2-

mib (blue) (Arachchige, & Indrajith, 2016) ...................................................................................... 87

Figure 46: Time effect for SPME extraction (Supelco, 2001). .......................................................... 88

Figure 47: The effect of extraction time on area of geosmin and 2-mib with 1 ng mL-1 of geosmin

and 2-mib in water saturated with sodium chloride (Saito, et al., 2008). ...................................... 89

Figure 48: Effect of extraction time of the sample on extraction efficiencies for geosmin (red) and

2-mib (blue) using 0.01 µg L-1 geosmin and 2-mib (Arachchige, & Indrajith, 2016). ....................... 89

Figure 49: Addition of more salt reduces the volume of head space which allows the efficient

extraction ......................................................................................................................................... 91

Figure 50: The area of geosmin, 2-mib and TCA of three successive injections during 0 h, 24 h and

96 h (spiked with1 µg L-1 geosmin, 2-mib and TCA) ......................................................................... 94

Figure 51: Influence of aeration rate of 150 mL min-1, 120 mL min-1, 60 mL min-1, 30 mL min-1 on

geosmin in dark condition (Bellu, et al., 2008) ................................................................................ 98

Figure 52: Volatilization rates of three different concentrations of geosmin (Rescorla, 2012) ...... 98

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Figure 53: Mean reduction [%] ± confidence interval (p < 0.05) of 2-MIB and GSM in tap water, RAS

fresh water and RAS sea water. ..................................................................................................... 100

Figure 54: Geosmin (left) and 2-mib (right) concentrations in water spiked with these two

compounds and incubated for 2 weeks in beakers containing sterilized bioflocs, non-sterilized

bioflocs, no bioflocs, no geosmin or 2-mib. Data are given as mean ± SD (Ma, et al., 2016) ....... 104

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List of tables

Table 1: Chemical and physical parameters of geosmin and 2-mib ................................................ 23

Table 2: Microorganisms associated with geosmin and 2-mib production in RAS ......................... 28

Table 3: Microorganisms implicated in the biodegradation of 2-mib and geosmin ....................... 33

Table 4: Proximate composition of commercial feed ...................................................................... 39

Table 5: Supelco method (Supelco, 2001). ...................................................................................... 45

Table 6: GC-MS conditions at the beginning of the experiment ..................................................... 46

Table 7: Temperature profile ........................................................................................................... 47

Table 8: The results of Imhoff cone test, TSS, SVI and ash content of the biofloc reactor tank during

each week of the experimental period. ........................................................................................... 59

Table 9: Calculation of RSD values for repeatability of GC-MS (n=3), spiked with 1 µg L-1 geosmin

and 1 µg L-1 2-mib ............................................................................................................................ 66

Table 10: Calculation of %RSD values for (GSM/TCA) during repeated injections for three time

periods (0 h, 24 h and 96 h), (n=5), spiked with 1 µg L-1 geosmin. .................................................. 67

Table 11: Calculation of %RSD values for 2-mib/TCA during repeated injections for three time

periods (0 h, 24 h and 96 h) (n=5) spiked with 1 µg L-1 2-mib ......................................................... 68

Table 12 : Analysis of sea water samples (SW_1 and SW_2) to check the presence of geosmin and

2-mib, SW+ sample spiked with 1 µg L-1 geosmin and 1 µg L-1 2-mib, (n=3) ................................... 71

Table 13: The calculation of GSM/TCA and 2-mib/TCA for the experiment with bioflocs spiked with

geosmin (1 µg L-1) and 2-mib (1 µg L-1) with aeration (n=3) ............................................................ 72

Table 14: GSM/TCA ± SD for three treatments (NA, AP and AS) with different time periods (0 h, 2

h, 6 h and 24 h) spiked with 50 µg L-1 geosmin at 0 h. Significant differences between treatments

at each time period was represented as a and b, (n=3) .................................................................. 73

Table 15: 2-mib/TCA ± SD for three treatments (NA, AP and AS) with different time periods (0 h, 2

h, 6 h and 24 h) spiked with 50 µg L-1 2-mib at 0 h. Significant differences between treatments at

each time period was represented as a and b, (n=3) ...................................................................... 73

Table 16: The area of geosmin, 2-mib, TCA and their ratio reference to the first injection ........... 92

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List of abbreviations

2-mib 2-methylisoborneol

2-mib/TCA Area of 2-mib/Area of TCA

ANOVA Analysis of variance

AOP Advanced oxidation processes

AP Air pipette

APHA American public health association

ARC Aquaculture and Artemia Reference Centre

AS Air stone

BC Biological filter

BF Pure biofloc

BF+ Bioflocs spiked with geosmin and 2-mib

CFU Colony forming unit

CORDIS Community research and development information service

CPEO Centre for public environmental oversight

CSID Chemspider identification

DO Dissolved oxygen

DVB/CAR/PDMS Divinylbenzene/Carboxen/Polydimethylsiloxane fibres

EC Electrical conductivity

EI Electron ionization

EMBRC European marine biological resource centre

EPS Extracellular polymeric substances

FBF Floating bead filters

FW a- Fresh water without aeration

FW a+ Fresh water with aeration

FWO Research foundation-Flanders

GC-MS Gas chromatography – Mass spectrometry

GF/F Glass fibre F grade

GSM/TCA Area of geosmin/Area of TCA

HPLC High-performance liquid chromatography

ILVO Institute for Agricultural and Fisheries Research (Instituut voor Landbouw-

en Visserijonderzoek)

LEU leucine pathway

M Mean

MEP 2-methylerythritol-4-phosphate pathway

MEV mevalonate pathway

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MSD Mass selective detector

NA No aeration

NIS Nikon instruments software

PTFE Polytetrafluoroethylene

RAS Recirculated aquaculture systems

rpm Revolutions per minute

RSD Relative standard deviation

S/N Signal to noise ratio

SBF+ Sterile biofloc spiked with geosmin and 2-mib

SD Standard deviation

SIM Selected ion monitoring mode

SPME Solid-Phase Micro Extraction

SPSS Statistical package for the social sciences

SVI Sludge volume index

SW a- Sea water without aeration

SW a+ Sea water with aeration

SW+ Sea water spiked with geosmin and 2-mib

SynBioC Synthesis, Bio resources and Bioorganic Chemistry

TAN Total ammonium nitrogen

TCA 2,4,6-trichloroanisole

TSS Total suspended solids

UV Ultra Violet

w/v Weight/volume

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Abstract

Geosmin and 2-methylisoborneol (2-mib) are two of the most common odorous compounds that

critically affect the quality of fish in recirculating aquaculture systems (RAS). At present, depuration

procedure is the only trustworthy way to eliminate these odour compounds in aquaculture, which

is expensive and time-intensive. Hence as an alternative approach, the potential of bioflocs to

decrease the concentration of geosmin and 2-mib in sea water was investigated during this study.

In order to accurately quantify geosmin and 2-mib in the laboratory, Supelco, (2001) protocol was

optimized using solid phase micro extraction (SPME) with gas chromatography-mass spectrometry

(GC-MS) up to a detection limit of 0.01 µg L-1 of geosmin and 0.05 µg L-1 of 2-mib. Then the

suppression of geosmin and 2-mib using the bioflocs was investigated using the upgraded SPME

GC-MS analysis method. During the optimization of SPME GC-MS, incubation temperature (60 °C),

extraction time (20 min) and salting out effect (6.25 g) were improved. The repeatability and

reproducibility of the developed process was exceeding the 15% relative standard deviation (RSD)

indicating fine adjustments are necessary further to achieve the most appropriate method for

geosmin and 2-mib analysis.

According to the results of the experiments with bioflocs, the geosmin and 2-mib was shown to be

involved in rapid volatilization with the supply of aeration regardless the salinity of water. When

the aeration was not supplied to the experimental setup, bioflocs show the production of geosmin and

2-mib after 24 h period. This may be due to the death of the bacterial cells in bioflocs and release of

these odour compounds. The bioflocs spiked with geosmin and 2-mib (50 µg L-1) in sealed containers

kept in suspension using a rotator demonstrated for a decrease of geosmin and 2-mib 15.1% and

56% respectively after 10 days. In addition, sterile bioflocs showed a depletion of geosmin of 9.5%

and 2-mib of 36.4%, in the same experimental setup. This points out the degradation of geosmin

and 2-mib using bioflocs, can be less significant than chemical/physical sorption and longer

experimental period is required to identify the effect of bioflocs on the geosmin and 2-mib. In

conclusion, although biodegradation of geosmin and 2-mib using bioflocs was not assured, this

experiment proved that live bioflocs did not produce geosmin and 2-mib.

Keywords: geosmin, 2-methylisoborneol, bioflocs, solid phase micro extraction, gas

chromatography-mass spectrometry, recirculating aquaculture system

19

Chapter 1: Introduction

1.1. Background information

Geosmin and 2-methylisoborneol (2-mib) are two of the most common compounds that impart an

earthy–musty taste and odour to water (Guttman & van Rijn, 2008). Furthermore, they are

responsible for unwanted aromas in fish, dry beans, canned mushrooms, red beets and even wine

(Murray, et al., 1975; Buttery, et al., 1976; Darriet, et al., 2000; Lloyd and Grimm, 1999; Fontana,

2012). An increasing number of complaints about off-flavour water are recorded every year by

drinking water utility operators (Turgeon, et al., 2004) caused by the changes in organoleptic

quality (Parinet, et al., 2013).

In fact, geosmin and 2-mib are secondary metabolites produced by members of cyanobacteria and

actinomycetes (McGuire, 1999). Those compounds are detectable by the human nose at

concentrations of as low as 10 ng L-1 in water (Lloyd, et al., 1998). Due to these extremely low

odour thresholds, conventional processes such as coagulation, sedimentation, sand filtration etc.

are not suitable for the removal of these compounds (Srinivasan & Sorial, 2011). Since these

compounds have a clear impact on the quality and consumer acceptability of products,

identification, quantification and removal of these compounds from water are essential (Sung, et

al., 2005).

The presence of geosmin and 2-mib in recirculated aquaculture systems (RAS) has a significant

negative influence on the fish production (Persson, 1980). Due to poor flavour quality of produced

fish, there is an increased risk of rejection by fish processors (Klausen & Grønborg, 2010). The

process of purging fish is required to ensure that the fish reaches the marketplace without off-

flavours (Lim & Webster, 2006). This process commonly involves transferring fish to a separate

clean water system and, at the same time, depriving them of food for a period from a few days to

many weeks (Masser, et al., 2000). However, a significant drawback of prolonged purging

procedures is that, due to starvation, there is an unavoidable weight loss, consequently an obvious

economic loss and high treatment costs for water supplies (Persson, 1980; Lim & Webster, 2006).

As an example, Tucker, (2000) reported that the annual cost to catfish producers of off‐flavour is

estimated at 10–60 million US dollars annually.

20

Despite this contemporary problem, only a few studies have addressed the possible causes and

prevention of off-flavour compounds' accumulation in RAS systems so far (Schrader, et al.,

2005, Guttman & van Rijn, 2008).

1.2. Research problem identification and justification

The production of fish in RAS continues to be hampered by problems with microbial derived

geosmin and 2-mib. Their effect is best described as an earthy and musty taste of the fish fillet

(Schrader, et al., 2010). A wide variety of microorganisms have been shown to produce these

secondary metabolites (Dickschat, et al., 2005; Izaguirre & Taylor, 1998; Ludwig, et al., 2007).

These taste and odour compounds are typically removed by the addition of powdered activated

carbon or strong oxidants (Bruce, et al., 2002). However, oxidants such as chlorine and ozone are

not entirely effective for their removal (Glaze, et al., 1990). Activated carbon adsorption is

adversely affected by the presence of natural organic material (Newcombe, et al., 1997, Cook, et

al., 2001). Therefore as an alternative, the biodegradability of geosmin and 2-mib in water suggests

the possibility to use biological processes as a worthwhile treatment option for removing these

compounds (Ho, et al., 2007). Recently, biological degradation of geosmin and 2-mib was observed

in anaerobic sludge derived from the digestion treatment of a RAS (Guttman and van Rijn, 2009).

McDowall, et al., (2009) showed that the presence of a biofilm has an effect on the removal of

geosmin, because the biofilm enhances the attachment of the geosmin-degrading bacterial

conglomerate and boosts the removal of geosmin.

Based on the biodegradability of geosmin and 2-mib, bioflocs offer an attractive solution to deal

with musty and earthy compounds. The use of biofloc technology production systems continues

to increase in the aquaculture industry worldwide (Schrader, et al., 2011). Bioflocs consist of

phytoplankton, bacteria, masses of living and dead particulate organic matter and grazers of

bacteria (Hargreaves, 2006). They are irregular by shape, have a broad distribution in particle size,

easily compressible and permeable to fluids (Chu and Lee, 2004). The predominant microbes in

bioflocs belong to Bacillus sp., which have been reported to potentially degrade geosmin and 2-

mib (Schrader, et al., 2011). Meaning that, the adsorption and degradation of geosmin and 2-mib

21

(Tucker, 2000) by microbial flocs should be expected. Possible implications of the use of bioflocs

for the removal of geosmin and 2-mib in aquaculture water are investigated in this research.

Various extraction and quantification techniques for geosmin and 2-mib, such as solvent extraction

and closed-loop stripping analysis, exist (Bao, et al., 1997; Kim, et al., 2015). Furthermore, these

methods are time-consuming and labour-intensive (Lopez, et al., 2002). Solid-Phase Micro

Extraction (SPME) is a relatively simple, fast, inexpensive, portable and solvent-free technique.

Various reports (Saito, et al., 2008; Fujise, et al., 2010) show its excellent analytical utility and

applicability to other common taste-odorants as well. SPME has been widely used for the

extraction of volatile and semi-volatile organic compounds from environmental, biological and

food samples (Natangelo, et al., 1999). In this study, tests were designed to optimize the analytical

conditions of GC-MS for geosmin and 2-mib and the impact of different factors such as incubation

temperature, incubation time, storage etc. were examined.

1.3. Objectives

This study was designed to achieve two major objectives. The first aim was to develop and optimize

a method to quantify geosmin and 2-mib using gas chromatography-mass spectrometry (GC-MS)

analysis. Secondly, by using the optimized analysis method, to determine the impact of bioflocs on

the biodegradation of taste and odour compounds more particularly, geosmin and 2-mib by using

SPME.

22

Chapter 2: Literature review

2.1. Introduction to geosmin and 2-methylisoborneol (2-mib)

The presence of certain metabolites, including aliphatic hydrocarbons, sulfur-containing

compounds, aldehydes, ketones and in particular alicyclic alcohols such as geosmin and 2-

methylisoborneol (2-mib), are one of the major causes of taste and odour problems in aquaculture

(Juttner, 1983; Mallevialle and Suffet, 1987; Kenefick, et al., 1992). Major contributors to this

include geosmin, 2-mib, 2-isobutyl-3-methoxypyrazine, and 2-isopropyl-3-methoxypyrazine

(Izaguirre and Taylor, 2007; Ma, et al., 2012; Lu, et al., 2016). Among these compounds, geosmin

and 2-mib have been known to be the main compounds contributing to a typical earthy-musty

smell of water (Suffet, et al., 1999). The undesirable effect of these compounds is not restricted to

water only. Many reports describe their presence in other liquid and solid samples, such as

wine (Lizarraga, et al., 2004; Aung and Jenner, 2004), cork stoppers (Ezquerro and Tena, 2005;

Soleas, et al., 2002), fruit juice (Siegmund and Pöllinger-Zierler, 2006), catfish (Grimm, et al., 2004;

Conte, et al., 1996) and beet sugar (Marsili, et al., 1994).

Juttner and Watson (2007) stated that geosmin and 2-mib are tertiary alcohols, each of which

existing as (+) and (-) enantiomers and odour outbreaks are caused by naturally occurring

(-) enantiomers. Similarly, Polak & Provasi (1992) proposed that, (-) geosmin has, a lower threshold

(×11) than (+) isomer. In addition, these compounds have a slow rate of biodegradation and high

lipophilic affinity (Ho, et al., 2007). Table 1 describes the chemical and physical properties related

to geosmin and 2-mib.

According to a flavour profile analysis panel at the University of California, geosmin has an earthy,

wet muddy, beet, river-bed odour and 2-mib has a musty, camphor, mouldy, basement odour

(Suffet, et al., 1999). Humans can detect these compounds in concentrations of 10 to 30 ng L-1

(Srinivasan & Sorial, 2011). Moreover, Petersen, et al., (2011) confirmed that the threshold

concentrations of geosmin and 2-mib in water, which may affect fish taste, was as low as 20 ng L-1.

23

Table 1: Chemical and physical parameters of geosmin and 2-mib

Parameter geosmin 2-mib reference

Molecular formula C12H22O C11H20O CSID:27642

CSID:16024

Molecular weight (g Mol-1) 182.33 168.28 CSID:27642

CSID:16024

Boiling point

(°C at 760 mmHg) 270 207 to 209

CSID:27642

CSID:16024

Aqueous solubility (mg L-1) 150 195 Pirbazari, et

al., 1992

Enthalpy of vaporization

(kJ Mol-1) 59.0 52.69 Li, 2015

Log Kow

Octanol/water partition

coefficient

3.57 3.31 Howgate,

2004

Chemical structure

Li, 2015

Geosmin and 2-mib were first identified in the late 1960s, from Streptomycetes (aerobic

filamentous Actinomycete bacteria) and blue green algae (cyanobacteria) (Gerber and Lechevalier,

1965; Bentley and Meganathan, 1981; Gerber, 1979). In addition, myxobacteria (Myxococcales)

(Breheret, et al., 1999) and a number of eukaryotes such as fungi (Schulz, et al., 2004; Smith, et al.,

2008), amoeba Vannella (Hayes, et al., 1991) and a liverwort (Sporle, et al., 1991) have also been

identified as potential producers of geosmin and 2-mib (Figure 1).

24

Figure 1: Different organisms responsible for geosmin and 2-mib production

A: Colonies of Streptomyces ambofaciens. The fuzzy surface of these geosmin-producing colonies is made up of

millions of hydrophobic spores. (Keith, 2015); B: Blue-green algae producers of geosmin and 2-mib belonging to the

taxonomic family Oscillatoriaceae (Westerhoff, et al., 2002); C: Vannella amoeba which can produce geosmin is moving

towards a piece of algae. The fan-like "hyaline veil" is the anterior edge (Issaquah, 2004); D: The strong and distinct

mossy odour of Lophocolea bidentata (liverwort) is due to a mixture of geosmin and 2-mib (Asakawa, et al., 2013)

The seasonal variation of cyanobacteria is more frequently monitored than Actinomycetes because

the seasonal variation links with poor water quality. Therefore, cyanobacteria were more easily

identified as odour compound producers, than Actinomycetes (Watson, et al., 2007). Several

studies show that cyanobacteria produce geosmin and 2-mib during the growth. These off-flavours

are secondary products, related to photosynthesis and pigment synthesis. Depending on the

growth phase and environmental factors of cyanobacteria, the cells store or release these

compounds (Naes, et al., 1988; Rashash, et al., 1996; Srinivasan and Sorial, 2011).

A B

C D

25

In contrast to cyanobacteria, Actinomycetes abundance has poor correlation with geosmin and 2-

mib concentrations. This is due to several reasons. To identify and enumerate Actinomycetes,

highly selective media are necessary. When different media are used, they generate different

biomass (Dionigi, et al., 1992). Further, not all Streptomycetes are producing geosmin or 2-mib

(Cross, 1981), and it is essential to note that active individual isolates have considerable variation

in the cell-specific capacity to produce geosmin or 2-mib (Kenefick, et al., 1992).

According to Juttner & Watson, (2007), these two compounds are believed to be produced by three

different pathways in Streptomycetes and myxobacteria; 2-methylerythritol-4-phosphate (MEP)

pathway, mevalonate pathway (MEV) and/or the leucine pathway (LEU) (Figure 2).

Figure 2: The biochemical pathways for formation of geosmin and 2-mib in Streptomycetes and myxobacteria

(Juttner & Watson, 2007)

Juttner & Watson, (2007) stated that the MEP pathway is the major biosynthetic isoprenoid route

in many bacterial groups. The MEV pathway functions exclusively in the synthesis of geosmin and

other isoprenoids in some groups such as myxobacteria (Dickschat, et al., 2005) and contributes to

26

geosmin production in the stationary growth phase of Streptomycetes (Seto, et al., 1998).

Myxobacteria also use the MEV pathway as a major route to synthesize a range of isoprenoid

compounds, including geosmin. Nevertheless, there is some evidence that some Streptomycetes

may use both pathways during different growth stages; with the MEP as a major pathway during

active growth and the MEV pathway in the stationary phase (Seto, et al, 1996).

2.2. Presence of geosmin and 2-mib in aquaculture systems

In the fish culture industry, studies on off-flavour have mostly been conducted in conventional,

earthen pond systems (Tucker, 2000). It is now firmly established that cyanobacteria are

responsible for producing geosmin and 2-mib, in nutrient enriched aquaculture ponds (Juttner,

1995). However, geosmin and 2-mib have been detected in fish ponds during winter periods with

low algal biomass (Durrer, et al., 1999; Lanciotti, et al., 2003); this contradicts that cyanobacteria

are the only source of odours in freshwaters. The observations by Lanciotti, et al., (2003) indicated

that Actinomycetes, possibly in association with microalgae, were the major odour producers in

the winter.

Lately, evidence is accumulating that taste and odour compound accumulation is a common

problem in RAS (Masser et al., 2000, Schrader et al., 2005). RAS are developed as a technology for

onshore-intensive fish farming, that is based on the recirculation and filtration of fish culture water

with a efficiency of up to 99% (Badiola, et al., 2012). A simplified sketch of RAS used in aquaculture

is illustrated in Figure 3. RAS can be indoor or outdoor tank-based systems which can be freshwater

or marine water systems, although farming freshwater fish at present, the most common choice

(Burnell and Allan, 2009; Tal et al., 2009).

Lukassen, et al., (2017) indicated that Myxococcales, Actinomycetales, and genus Sorangium were

the main geosmin producing bacteria in European RAS. Out of all bacteria in RAS, 0.001-1% were

quantified as geosmin producers. Specifically, four species of Actinomycetes (Nocardia

cummidelens, Nocardia fluminea, Streptomyces albidoflavus, and Streptomyces luridiscabiei) were

isolated from biosolids from a RAS used for rainbow trout production (Schrader & Summerfelt,

2010). Relatively high geosmin and 2-mib concentrations and higher in vitro production was

reported in the aerobic components (drum filter and a trickling filter) of the RAS (Guttman & van

27

Rijn, 2008). This is a strong evidence for the important role of oxygen in geosmin and 2-mib

production. Similar observations from the literature were summarized as indicated in Table 2.

Figure 3: Sketch of a RAS system in aquaculture (adapted from Yoshino, et al., 1997)

FBF- Floating Bead Filters; BC- Biological Filter; UV- Ultra Violet

In aquaculture systems, a variety of organisms produce geosmin and 2-mib as both cellular (cell-

bound) and dissolved fractions (Figure 4). When their cell damages due to death, senescence or

biodegradation these organisms release geosmin and 2-mib into the water (Srinivasan and Sorial,

2011). As described by Watson, et al., (2016) the severity and timing of these events are ultimately

governed by a multidimensional scale of interacting processes, ranging from intracellular coding,

signalling, temperature, light, nutrients and food web interactions.

The cell-bound volatile fraction can be transferred rapidly into the dissolved form (Juttner &

Watson, 2007; Sugiura & Nakano, 2000) via cell degradation (Sugiura, et al., 1994). The cell

degradation process liberates geosmin from the cell protein matrix, facilitating the transformation

of cell-bound material into the dissolved form. During this transformation, geosmin is degraded

more slowly than other cell components by most bacteria (Juttner and Watson, 2007). This process

affects the efficiency of treatment and removal of these odour compounds, which is far more

challenging for dissolved fractions (Watson, et al., 2016). The cell-bound geosmin and 2-mib

concentrations were lower when the organisms are growing faster (Wu, et al., 1991; Juttner, 1995;

28

Van der Ploeg, et al., 1995). Dissolved concentrations of off-flavours tend to be higher in old

cultures and nutrient-limited conditions (Miwa and Morizane, 1988; Naes et al., 1988).

Table 2: Microorganisms associated with geosmin and 2-mib production in RAS

Group Microorganisms Source Reference

Actinomycetes Streptomyces roseoflavus,

S. thermocarboxydus

Organic rich conditions,

more in aerobic (drum and

trickling filter) and

anaerobic treatment loops

(sedimentation/digestion

basin), freshwater RAS

(tilapia)

Guttman & van

Rijn, (2008)

Actinomycetes Streptomyces

cyaneofuscatus, Nocardia

cf. fluminea, Nocardia

salmonicida

Drum filter effluent and

inside drum filter of

Atlantic salmon RAS

Burr, et al.,

(2012)

Actinomycetes Nocardia cummidelens, N.

fluminea, Streptomyces

luridiscabiei, and

Streptomyces cf.

Albidoflavus

Water, biosolids within the

RAS drum filters and heat

exchangers, fillet in

Rainbow trout

Schrader &

Summerfelt,

(2010)

Cyanobacteria Pseudanabaena sp. Biofilm of Arctic charr RAS Houle, et al.,

(2011)

Cyanobacteria Microcoleus sp.,

Phormidium tenue

Water and flesh in

recirculating trout farm

Robin, et al.,

(2006)

Myxobacteria Sorangium nannocystis Water and flesh in RAS

trout farm

Auffret, et al.,

(2013)

29

Figure 4: Schematic description of the geosmin occurrences and transport of geosmin (Chung, et al., 2016)

2.2.1 Uptake of geosmin and 2-mib by fish

According to the study of Pimolrat, et al., (2015), when geosmin and 2-mib are present in water,

they enter into the bloodstream of fish via the gills and accumulate in the fatty tissues. Gills are

the primary sites of uptake because their structure and function enhance diffusion of substances

between water and blood (Tucker, 2000). Additionally, geosmin-producing bacteria were found in

the stomach, skin and the intestinal mucus layer of fish, suggesting that fish may also feed on

potential geosmin-producing microorganisms (Lukassen, 2017; Gutierrez, et al., 2006; Watson, et

al., 2016).

Rurangwa and Verdegem, (2015) emphasized that geosmin absorption is relatively fast, but its

excretion is much slower. When Johnsen and Lloyd (1992) exposed 0.5 kg channel cat fish to 2-mib

dissolved in water at 0.5 µg L-1, fish became off-flavoured within the first 2 h of exposure but

continued to accumulate 2-methylisoborneol throughout the first 24 h of exposure, after which

equilibrium was achieved. However, when uptake trials were conducted at different water

temperatures (ranging from 6.5 °C to 34 °C), the effect of water temperature was found to be more

important than lipid content in controlling accumulation of 2-methylisoborneol (Johnsen et al.,

1996).

30

2.2.2. Disadvantages due to the taste and odour compounds in aquaculture

Due to its high economic impact, off-flavour in fish is still one of the most severe difficulties in the

aquaculture industry worldwide (Jonns, et al., 2017). In the United States catfish industry,

off-flavour problems were calculated to have increased production costs by U.S. $47 million in

1999 (Hanson, 2001). At the production level, economic losses due to off-flavour range from U.S.

$0.04 to U.S. $0.26 per kg of catfish (farm gate price per kg of catfish was U.S. $2) (Keenum

&Waldrop, 1988; Engle, et al., 1995; Hanson, 2003). Social costs of off-flavour problems in catfish

production have been estimated to be equivalent to 12% of the annual revenue received by catfish

farmers (Kinnucan, et al., 1988).

Moreover, the European aquaculture industry with the worth of 4 billion euro, estimated the

annual loss of fish biomass due to off-flavour depuration has a value of more than 8 million euro.

In addition, it is clear that off-flavour seriously impinges upon profit margin and economic

feasibility of individual fish producers in RAS. Three main causes for economic damage to the

European aquaculture industry related to off-flavour are consumer rejection of off-flavoured fish,

reduction of market volumes and prices and costs of depurating off-flavours from fish crops

(CORDIS, European Commission, 2018).

It has been estimated that 30% of potential revenue is lost annually due to off-flavour problems.

Because of delays in harvest that result in additional feed costs, forfeiture of income from foregone

sales because producers are forced to delay restocking ponds, and loss of fish during the holding

period from disease, water quality deterioration, and bird depredation (Tucker, 2000; Smith, et al.,

2008). Similarly, adverse impacts of these earthy off-flavours include loss of market demand due

to inconsistent fish quality, inhibition of growth into new markets, and economic losses associated

with delays in stocking a new stock while holding the off-flavour fish until flavour quality improves

(Schrader, et al., 2015). In addition, another drawback is that the purging process can lead to loss

of body mass of fish and the time taken for leaching may be several days depending on the intensity

of taint (Hathurusingha & Davey, 2016; Tucker & Van der Ploeg, 1999).

Until now, the only reliable way to reduce off-flavours in aquaculture is moving the fish to clean

and odour-free water for a certain time prior to harvest which is referred to as depuration (Burr,

et al., 2012) (Figure 5). However, this is a high cost, time intensive, as well as a capacity demanding

31

process (Nam-Koong, et al., 2016). In addition, Burr, et al., (2012) indicate that the purging rate

depends on the initial level of 2-mib and geosmin in the fish, water temperature, and size and fat

content of the fish. Schram, et al., (2016) reported a controversial argument stating that no

significant interaction between the temperature and purging rate could be detected in their

studies.

Figure 5: Purging of fish in RAS (Llyn Aquaculture Ltd. (2009))

A: Crystal clear purging system, 1st week prior to slaughter with empty guts; B: Sea bass RAS in UK, 20 tonnes per

annum, purging tank with clean water, Water exchange 10% per day

According to Schram, et al., (2016), exercise also can be used to reduce the time required to

depurate off-flavours from fish, because physiological responses aimed at increasing oxygen

uptake also affect the branchial exchange of lipophilic xenobiotic chemicals between the fish and

its surroundings.

Unfortunately, alternative strategies and processes for efficient prevention of taste and odour

compounds are still lacking in aquaculture (Nam-Koong, et al., 2016). Thus, the economic impacts

associated with off-flavours have encouraged research into the bioregulation of geosmin synthesis

and elimination (Dionigi, 1994). Practices that can reduce geosmin production have been

extensively explored, but cost-effective methods remain elusive (Schrader, et al., 2013).

2.2.3. Removal of geosmin and 2-mib from water

Because consumers can detect these compounds as musty-earthy odours at very low levels, the

treatment methods must be very effective (Agus, et al., 2011). Studies conducted to investigate

A B

32

removal of these compounds using alum coagulation and oxidants including Cl2, ClO2, and KMnO4

demonstrated that they are not effective (Bruce, et al., 2002; Glaze, et al., 1990).

Currently, the main available technologies for geosmin and 2-mib removal include oxidation

processes which include ozone, UV, and H2O2, granular activated carbon and powdered activated

carbon adsorption, biological treatment (Srinivasan and Sorial, 2011). Experiments reported that

90% geosmin and 60% 2-mib were removed (40–43 ng L-1 initial concentration) at the UV dose of

1200 mJ cm-2 with 6 mg L-1 H2O2 (Jo, et al., 2011; Collivignarelli & Sorlini, 2004). Though oxidation

processes have been proven effective, there are significantly high energy and capital costs

associated with these technologies (Srinivasan and Sorial, 2011) and limited by dissolved/non-

dissolved substances of aquaculture water (Klausen & Grønborg, 2010) and the production of fish

toxic disinfection byproducts (Tango & Gagnon, 2003). A major influence on the application of

activated carbon is the competitive effect of natural organic material, which significantly reduces

the adsorption (Chen, et al., 1997). Biological degradation of geosmin and 2-mib achieved by

biological filters has been proven a positive alternative (Egashira, et al., 1992, Ho, et al., 2012b). As

an example McDowall, et al., (2009) reported geosmin removals of up to 75% through sand

columns which had been inoculated with the geosmin-degrading bacteria.

Until now, the available literature on full-scale treatment processes for geosmin and 2-mib removal

is rare (Zamyadi, et al., 2015). In addition, due to the nature of RAS and the microbial sources of

earthy off-flavour, the use of biocides to control the Actinomycetes responsible for earthy and

musty off-flavour problems would be difficult (Schrader and Summerfelt, 2010). Therefore,

establishment of novel purification methods to manage off-flavour problems in RAS is necessary.

Silvey and Roach, (1964), first reported the biological degradation of taste and odour compounds.

They subsequently demonstrated that strains of Bacillus cereus were responsible for the

degradation (Silvey, et al., 1970). Narayan and Nunez (1974) who identified B. subtilis to be

efficient confirmed these results. However, MacDonald et al. (1987) and Danglot et al. (1983) could

not reproduce these results although they used the same strains. Several studies have been done

to identify these types of bacteria and Table 3 summarizes the literature of geosmin and 2-mib

degrading bacteria.

33

Several authors have proven that Bacillariaceae are a primary member of the microbial

community, which are capable to degrade geosmin and 2-mib efficiently (De Schryver, et al., 2012,

Guttman & van Rijn, 2011, Lauderdale, et al., 2004). It is considered that bacteria can use geosmin

and 2-mib as a primary carbon source (Guttman & van Rijn, 2011). However, Saito, et al., (1999)

found that geosmin is extremely difficult to degrade microbially when it was used as the sole

carbon source. In his experiments, an acceleration of the reaction was realized by adding ethanol.

Experiments performed by Luo, et al., (2016) showed that the amount of 2-mib removal in the

inoculated reactors was significantly greater than that of geosmin, suggesting that the removal of

2-mib is more efficient than that of geosmin. These findings differ from earlier results of Ho, et al.,

(2012a) which demonstrate that geosmin appears to be degraded more easily than 2-mib by the

bacteria within the sand filters and bioreactors.

Table 3: Microorganisms implicated in the biodegradation of 2-mib and geosmin

geosmin 2-mib

Microorganisms Literature

sources Microorganisms Literature sources

Bacillus cereus Silvey, et al.,

(1970) Candida spp. Sumitomo (1988)

Bacillus subtilis

Narayan &

Nunez, (1974) Bacillus subtilis

Yagi, et al., (1988);

Lauderdale, et al.,

(2004)

Arthrobacter atrocyaneus Saadoun & El-

Migdadi, (1998)

Pseudomonas

aeruginosa

Egashira, et al.,

(1992)

Arthrobacter globiformis Saadoun & El-

Migdadi, (1998) Pseudomonas spp.

Egashira, et al.,

(1992)

Rhodococcus moris Saadoun & El-

Migdadi, (1998)

Flavobacterium

multivorum

Egashira, et al.,

(1992)

Chlorophenolicus strain N-

1053

Saadoun & El-

Migdadi, (1998) Flavobacterium spp.

Egashira, et al.,

(1992)

Pseudomonas putida Oikawa, et al., (1995)

Enterobacter spp. Tanaka, et al., (1996)

34

2.2.4. Potential of bioflocs to reduce the production of geosmin and 2-mib

Biofloc technology was first developed at the beginning of 1990's to solve water quality problems

by developing and controlling dense heterotrophic bacteria within the culture (Avnimelech, 2006;

Avnimelech, 2007). The growth of these bacterial communities can be stimulated in culture

systems by manipulating the carbon/nitrogen ratio (Avnimelech, 1999; Ebeling, et al., 2006) and

when they reache a density of 107 CFU ml-1, they tends to form bioflocs that contain bacteria,

protozoa, zooplankton and other micro-organisms (Burford, et al., 2003) (Figure 6).

Figure 6: Morphology of floc under microscope

A: Biofloc particle flocculation with filamentous algae and bacteria, B: Biofloc flocculates with nematodes (Rajkumar,

et al., 2016)

Large bioflocs can be seen with the naked eye, but most are microscopic (Hargreaves, 2013). The

biofloc consists of different types of microorganisms which can be divided into five groups; floc

forming organisms, saprophytes, nitrifying bacteria, algae grazers, and pathogenic bacteria

(Manan, et al., 2017). These organisms and materials are conglomerated by sticky extracellular

polymeric substances (EPS) secreted by bacteria, bounded by filamentous microorganisms, and

attached by electrostatic attraction (Hargreaves, 2013; Medina & Neis, 2007). These EPS were

known to have a significant effect on the physico-chemical properties of the microbial aggregates

including structure, surface charge, flocculation, settling properties, dewatering and absorptive

capacity (Sheng, et al., 2010).

A B

35

The in vitro experiments demonstrated the ability of bioflocs to remove geosmin and 2-mib via

biological degradation. However, they found biodegradation was less important than

chemical/physical sorption in the overall removal of geosmin and 2-mib (Ma et al., 2016).

2.3. Analysis of geosmin and 2-mib by solid-phase microextraction

Since the threshold odour concentrations of geosmin and 2-mib are very low, a highly sensitive

method with a detection limit of 1 ng L-1 is needed. Lu, et al., (2003) explained that several

techniques have been developed to concentrate geosmin and 2-mib including liquid-liquid

extraction, closed-loop stripping, purge and trap technique, solid-phase extraction, headspace

microextraction, etc. However, these methods have some shortcomings, which hinder their wide

application.

For example, closed-loop stripping requires relatively complex instrumentation (Sun, et al., 2012).

Liquid-liquid extraction needs a toxic solvent, and the up-concentration is a problem because of

the volatile feature of the target compound (Rezaee, et al., 2006). Solid-phase extraction was quick

and simple, but appropriate solid-phase extraction columns are needed. Purge and trap technique

is time-consuming and has inferior stability while needing some special tools (Ho, et al., 2012c).

Solid-phase microextraction (SPME) has been widely used for the extraction of volatile and semi-

volatile organic compounds from environmental, biological and food samples (Nagasawa, et al.,

1996; Ng, et al., 1996; Matich, et al., 1998; Beltran, et al., 2000; Potter and Pawliszyn, 1994). Arthur,

et al., (1992) has applied SPME to the analysis of geosmin and 2-mib (Lloyd, et al., 1999) because

SPME is a simple and effective technique.

2.3.1. Principle of gas chromatography-mass spectrometry (GC-MS)

Gas chromatography-mass spectrometry (GC-MS) is a common combined technique, comprising a

gas chromatograph (GC) coupled to a mass spectrometer (MS), by which complex mixtures of

chemicals may be separated, identified and quantified (Nebrodensis, 2010). A schematic diagram

of a GC-MS is shown in Figure 7.

The GC-MS instrument separates chemical mixtures by the GC component and identifies at a

molecular level by the MS component. The principle of GC is that a mixture will separate into

36

individual substances when heated (Cpeo.org., 2018). The heated gases are carried through a

column with an inert gas (He). When separated substances emerge from the column opening, they

flow into the MS component, which identifies compounds by its mass. Mass spectrometry is

considered the only definitive analytical detection method; therefore, many GC instruments are

coupled with a mass spectrometer (Nebrodensis, 2010).

Figure 7: Schematic of a GC-MS system (Wu, et al., 2012)

2.3.2. Solid-phase microextraction (SPME) in GC-MS analysis

SPME is a very simple and efficient, solventless sample preparation method. Solutes from a sample

are directly extracted into an absorptive polymeric layer coated onto a solid fused-silica fibre

(Arthur & Pawliszyn, 1990). After the sample is exposed to the fibre for some time, equilibrium is

reached and the extracted mass is proportional to the concentration in the sample. Then the SPME

fibre and captured solutes are transferred into an injection system that desorbs the solutes into

the gas mobile phase (He) of the gas chromatograph, and at the same time the analysis run is

started (Figure 8) (Gorecki & Pawliszyn, 1995; Pawliszyn, 1999; Kataoka, 2000).

37

SPME is an ideal extraction method characterised by low detection limits, rapidity, solvent

elimination, high sensitivity, low costs, compatibility with a wide variety of detection methods,

automation, simplicity in use, suitability for on-site analysis and process monitoring (Prosen &

Zupancic-Kralj., 1999; Kataoka et al., 2000; Mills and Walker, 2000).

Figure 8: Diagram of SPME analysis with GC-MS (Schmidt & Podmore, 2015)

SPME has been widely used in the analysis of many organic compounds in water, including odour

compounds such as geosmin and 2-mib (Lloyd, et al., 1998; Watson, et al., 2000). SPME is already

a part of the standardized protocol for the analysis of geosmin and 2-mib in drinking water, as

standard method 6040D (APHA, 2000).

38

Chapter 3: Materials and Methods

In this chapter, methodology is divided in to four major sections. Section A describes about the

biofloc stock preparation and management done in the Laboratory of Aquaculture and Artemia

Reference Centre (ARC). Optimization of GC-MS for analysis of geosmin and 2-mib in the Synthesis,

Bioresources and Bioorganic Chemistry laboratory (SynBioC) is discussed in section B. Section C

explains the experiments done to determine the suppression of geosmin and 2-mib by bioflocs

while section D explains the statistical analysis used for the experiments.

3.1. Section A – Biofloc stock preparation and management

3.1.1. Biofloc reactor preparation

The biofloc stock was maintained in a conical shaped indoor tank (50 L) in the Laboratory of

Aquaculture and Artemia Reference Centre (ARC), Faculty of Bioscience Engineering, Ghent

University, Belgium. Biofloc inoculum was collected from the Institute for Agricultural and Fisheries

Research (Instituut voor Landbouw en Visserijonderzoek /ILVO) Oostende, Belgium. A total of 5 L

of inoculum biofloc was added to the reactor and 20 L of sea water was added equivalent to a

working volume of 25 L. The stock tank was not covered and the photoperiod of the experimental

room was 12 h day /12 h night, provided through an artificial lighting (Figure 9). The room

temperature was maintained at 28 °C. Dechlorinated fresh water was added daily to restore the

volume lost through evaporation. Salinity was maintained at 35 g L-1. Vigorous aeration was used

(approximately 10 L min-1) to prevent the settlement of the flocs and to provide sufficient oxygen.

Biofilm growing on the walls of the biofloc stock tank was manually removed using aquarium

cleaning magnets. Excess solids were removed by periodic flushing of the biofloc reactor.

39

Figure 9: Biofloc stock tank

3.1.2. Balancing input Carbon/Nitrogen (C/N) ratio

Commercial pellet feed (SUPREME-22) produced by Alltech Coppens, Netherlands and glucose D

(glucose anhydrous), Merck, Germany were used for the feeding of the biofloc. Commercial feed

has gross energy of 22.3 MJ kg-1 and digestible energy of 20.5 MJ kg-1. The proximate composition

of feed was detailed in Table 4.

Table 4: Proximate composition of commercial feed

Proximate composition %

Protein 44

Fat 22

Crude fibre 1.2

Ash 7.2

Total phosphorus 1.16

Commercial feed was grinded into fine powders using a grinder (Braun KMM30 Coffee/Espresso

Mill). Glucose and grinded feed were stored in sealed containers at 4 °C for daily feeding

(Figure 10).

40

Figure 10: Commercial feed (grinded) and glucose for feeding of biofloc

To obtain the C/N ratio of 20/1, calculations for the daily feeding of biofloc was done as follows.

0.5 g – 1.0 g of feed (commercial feed + glucose) was used for 5 L day-1 in ILVO

Total volume of biofloc reactor was 25 L

2.5 g – 5.0 g of feed (commercial feed + glucose) was used for biofloc reactor tank

Commercial pellet feed

1 g of feed 44% protein

(1 g of feed 0.44 g of protein)

16% protein is N

1 g of feed 0.0704 g of N

1 g of feed 51% of C

(1 g of feed 0.51 g of C)

Glucose C6H12O6

180.156 g/mol

1 g of glucose 0.42 g of C

41

Nitrogen from feed

Carbon from feed + Carbon from glucose =

1

20

0.0704 g

0.51 g + X 0.42 g =

1

20

Carbon can be supplied by increasing glucose content, X = weight of glucose needed.

X = 2.183 g

Commercial feed (1 g) + Glucose (2.138 g) = 3.138 g/day

3.1.3. Biofloc characteristics

Two types of tests, namely Imhoff cone test and gravimetric determination using vacuum filtration,

were used to estimate TSS. The Imhoff cone was used to measure the volume of settleable solids

in a specific volume of water, while gravimetric determination using vacuum filtration was used to

determine total solids (settleable + fixed + volatile). SVI test was volume in mL occupied by 1 g of

a suspension after settling. This test typically is used to monitor settling characteristics of biological

suspensions. Test for ash content was used to estimate fixed solids.

A) Imhoff cone test

Imhoff or settling cones are a simple way to index the concentration of suspended solids. The cones

have marked graduations on the outside that can be used to measure the volume of solids that

settle from 1 L of biofloc water (Bakar, et al., 2015).

In brief, every day, 1 L of the mixed biofloc was removed from the reactors and allowed to settle

for 60 min of quiescent settling in an Imhoff cone (Figure 11). The volume of the floc plug

accumulating on the bottom of the cone was determined (APHA, 1995). According to

Avnimelech (2009), floc plug can be 2-200 mL L-1 in fish ponds. Therefore, Imhoff cone value was

maintained 2 – 70 mL L-1 by regulating the solids in biofloc stock tank (by daily dilution and

removing the excess solid). During the regulation of biofloc in stock tank, aeration was stopped

and flocs were allowed to settle for 10 – 15 min. Then turn-knob at the bottom tip of the biofloc

stock tank was opened and excess flocs were allowed to drain. Then biofloc tank was readjusted

to 25 L by filling with sea water and the aeration was started.

42

Figure 11: Imhoff cone test

B) Gravimetric determination using vacuum filtration

Total solid (mg L-1) was determined once a week using a gravimetric method as described by

Strickland and Parsons (1972). 20 mL of biofloc water was filtered under vacuum pressure through

pre-dried and pre-weighed Whatman GF/F 50-A glass microfiber filters (0.7 µm). Ammonium

formate 0.5 M was used to remove salt from sea water. After filtering, filter paper containing

suspended materials was dried in an oven for 4 h at 103 °C. After cooling in a desiccator, dried

samples were weighed to 0.0001 g using an analytical balance (Sartorius Micro, MC 210P). The

total solid was calculated from the weight differences. The concentration of total solid in the

reactor was set to 2000 mg L-1 at the beginning of the experiment.

C) Sludge volume index (SVI)

SVI was calculated weekly using Imhoff cone test and total solid measurements using following

equation.

SVI (mL g-1) = Settled sludge volume

Suspended solids concentration × 1000

D) Ash content

Ash was determined using a pre-weighed crucible with lid. The sample was placed in the muffle

furnace (Carbolite ashing furnace) at 550 °C for 4 h to complete combustion of the sample. The

crucible, lid and ash were then cooled in a desiccator and re-weighed.

43

3.1.4. Determination of floc morpho-structure

The floc morpho-structure was observed by biological microscope (Nikon, Eclipse E200) and

photographs were taken with a zoom stereomicroscope (9SMZ1270, Nikon) connected to a lens

(Plan Apo, 1×, WFWD: 70) and using imaging software, NIS elements (version 4.40, Nikon) once a

week.

3.1.5. Main water quality parameters

Different water quality parameters such as pH, temperature, oxygen and salinity were measured

on a daily basis with a portable pH/EC/DO multi-parameter (HANNA, HI 98194). The concentrations

of total ammonium nitrogen (TAN) (NH3 + NH4+) and nitrite (NO2

-) were measured daily using Tetra

(Figure 12) and JBL test kit. Tetra test kit provides readings between 0.0 - 5.0 mg L-1 while JBL test

kit affords the range of 0.05 - 5 mg L-1 for TAN. Tetra NO2- test kit offers readings between 0.3 mg L-1

and 3.0 mg L-1 and JBL test kit provides the range of 0.01-1.0 mg L-1 by using colour plates. The

nitrate (NO3-) was determined once a week with JBL Test set within the range 0.5 - 240 mg L-1.

Figure 12: Tetra water quality test kit

44

3.2. Section B – Optimization of GC-MS for geosmin and 2-mib analysis

3.2.1. Gas chromatography-mass spectrometry (GC-MS) optimization

Optimization of GC-MS for the analysis of geosmin and 2-mib was done in the Synthesis,

Bioresources and Bioorganic Chemistry laboratory (SynBioC), Faculty of Bioscience Engineering,

Gent University, Belgium.

3.2.2. Chemicals for GC-MS optimization

During the optimization of the GC-MS analysis, ampule of 1 mL (Supelco CRM47525) was used

containing geosmin and 2-mib solution, 100 μg mL-1 of each component, in methanol. The stock

solution was diluted in methanol (HPLC grade, Sigma Aldrich) and 0.1 µg L-1 solutions were

prepared for the experiments. The prepared diluted solutions were stored in capillary vials (10 mL,

CERTAN, Sigma Aldrich) at 4 °C during the analysis (Figure 13). 2,4,6-Trichloroanisole (TCA)

(235393 Aldrich) was used as an internal standard to compensate for the variability in the SPME

process which is not carried out to equilibrium.

Figure 13: CERTAN capillary vial

3.2.3. Solid-phase microextraction standard protocol

To obtain optimal conditions for the experiment, a new protocol involving the use of SPME has

been developed for the analysis of geosmin and 2-mib in sea water. The basic method was derived

from Supelco Standard Methods (6040D: Solid phase microextraction of odours in drinking water

for analysis with GC/MS) as described in Table 5.

45

Table 5: Supelco method (Supelco, 2001).

desorption process 260 °C for 3 min

extraction headspace, 65 °C (30 min)

SPME fibre 2 cm Stable Flex coated with 50/30 µm DVB/CAR/PDMS

column Equity-5, 30 m x 0.25 mm, 0.25 µm film

oven 60 °C (2 min), 8 °C.min-1 to 200 °C

carrier gas Helium, 37 cm sec-1 at 60 °C (1 mL min-1 constant flow)

injection SPME fibre, splitless opened at after 1 min at 50 mL min-1

sample 25 mL of water containing 25% NaCl and drinking water odours kit

GC liner 0.75 mm interior diameter, SPME liner

detector 5973 MSD, interface at 280 °C

scan range Selected Ion Monitoring mode (SIM), mass (m)/charge number of

ions (z) = 95, 112, 124, 137, 197

3.2.4. Gas chromatography-mass spectrometry (GC-MS) conditions in the laboratory

A GC-MS - Hewlett Packard 6890 GC system coupled to a 5973N mass selective detector, equipped

with an Equity-5, (30 m x 0.25 mm x 0.25 μm) column and a quadrupole mass analyser (EI, 70 eV)

were used for the analysis (Figure 14). The protocol for geosmin and 2-mib was upgraded up to

the conditions mentioned in Table 6 in the SynBioC laboratory before starting the experiments.

Figure 14: GC-MS equipment used for the analysis

46

Table 6: GC-MS conditions at the beginning of the experiment

desorption process 250 °C for 1 min

extraction headspace, 40 °C (20 min)

SPME fibre Supelco SPME Fibre assembly: 50/30 μm DVB/CAR/PDMS, 24 Ga

Column Equity-5, 30 m x 0.25 mm, 0.25 µm film

Oven 40 °C to 250 °C (Table 7)

carrier gas Helium, 37 cm sec-1 at 60 °C (1.2 mL min-1 constant flow)

injection SPME fibre, splitless opened at after 1 min at 50 mL min-1

Sample 12 mL of water containing 6.25 g NaCl

GC liner 0.75 mm I.D., SPME liner

Detector 5973 MSD, interface at 280 °C

scan range SIM, m/z = 95, 107, 112, 149

During the sample preparation, a 20 mL SPME vial (75.5 mm x 22.6 mm, clear glass, 1st hydrolytic

class, rounded bottom, special crimp neck, Gerstel) was used. For the optimization experiments,

these SPME vials were filled with 12 mL of HPLC grade water. Then, the sample was spiked with

geosmin, 2-mib and TCA (as internal standard).

During the experiments with bioflocs, 12 mL of biofloc water (sea water) was used to fill the SPME

vial. During these experiments, only TCA was spiked because bioflocs were already spiked at the

beginning of the experiment with geosmin and 2-mib.

Next step was adding 6.25 g of table salt to the SPME vial in order to increase the ionic strength.

This step is used to enhance the escaping tendency of the volatiles (geosmin, 2-mib and TCA) from

the matrix (salting out effect) and hence to enhance extraction. Then the vial was capped

immediately using a Crimp cap (silicon creme / PTFE red, 55° Shore A, 1.5 mm, magnetic, golden,

Gerstel).

The SPME vial containing the sample was incubated at 40 °C for 5 minutes, while shaken vigorously

at 500 rpm in GC-MS agitator. The odour compounds were extracted, under slow agitation, during

20 min, after which it was desorbed during 1 min at 250 °C (Figure 15).

47

Figure 15: The adsorption and desorption process with SPME GC-MS (adapted from Yang, et al., 2018)

The oven (Figure 16) temperature of 40 °C was increased to 250 °C, using the profile in Table 7.

The column flow was maintained at 1.2 mL min-1. Retention times of geosmin and 2-mib were 9.27

and 15.6 min, respectively.

The MS (Figure 16) was operated in SIM mode, measuring m/z = 95, 107, 112 and 149 (dwell = 50

μs). The Supelco SPME fibre assembly was 50/30 μm DVB/CAR/PDMS, 24 Ga. The GC-MS - Hewlett

Packard 6890 GC system was coupled to a 5973N mass selective detector, equipped with an Equity-

5, (30 m x 0.25 mm x 0.25 μm) column and a quadrupole mass analyser (EI, 70 eV)

Table 7: Temperature profile

Temperature Duration Speed

40 °C 1 min

Increase to 80 °C 2 min 20 °C min-1

Increase to 115 °C 7 min 5 °C min-1

Increase to 250 °C 3.4 min 40 °C min-1

250 °C 3 min

Adsorption

48

Figure 16: GC-MS component (Goodman, 2015).

To optimize the analysis for geosmin and 2-mib, several experiments were performed (optimum

incubation temperature, optimum extraction time, amount of salt for optimum extraction, sample

storage time and stability). Moreover, repeatability, reproducibility and the limit of detection was

identified in the optimized method for geosmin and 2-mib analysis.

3.2.4.1. Optimum incubation temperature

Incubation temperature of the vial is an important consideration in headspace GC-MS

development. If the sample is incubated at a low temperature, less of the analyte will be in the

headspace, which can affect overall area counts. After a certain point, however, the analyte and

the solution will settle into equilibrium; a longer incubation will not result in any more sample

entering the vapor phase and may result in sample degradation or cause secondary reactions.

Figure 17 illustrates the simple diagram of incubation process.

49

Figure 17: Incubation of the sample and head space SPME (Schmidt & Podmore, 2015)

In order to determine the effect of different incubation temperatures on geosmin and 2-mib

extraction, different temperatures (40 °C, 60 °C, and 80 °C) were tested. SPME vials were filled with

HPLC grade water (12 mL) and spiked with 1 µg L-1 geosmin, 2-mib and TCA (internal standard).

6.25 g of table salt was added to each sample and were capped immediately. The samples were

analysed in triplicate per each treatment as described under 3.2.4. (GC-MS conditions in the

laboratory). They were randomized during the analysis.

3.2.4.2. Optimum extraction time

During the extraction step, the SPME needle is exposed to the sample head phase. Over time, the

amounts of the compounds absorbed to the SPME needle reach an equilibrium level with their

surroundings. When sampling from the headspace, odour compounds must first cross the liquid–

gas interface before encountering the SPME layer. The time to reach equilibrium may be influenced

strongly because nonpolar and volatile solutes that strongly favour the headspace phase will come

to equilibrium more rapidly. Figure 18 illustrates the extraction process during SPME.

In order to determine the effect of different extraction times on geosmin and 2-mib, different

extraction times of 5 min, 10 min, 15 min, 20 min, and 40 min were tested. SPME vials were filled

with HPLC grade water (12 mL) and spiked with 1 µg L-1 geosmin, 1 µg L-1 2-mib and 1 µg L-1 TCA

(internal standard). 6.25 g of table salt was added to each sample. Then samples were immediately

capped and analysed in triplicate per each treatment. Analysis conditions were as described under

3.2.4. (GC-MS conditions in the laboratory) and samples were randomized during the analysis.

50

Figure 18: Principles of extraction by headspace SPME (Schmidt & Podmore, 2015)

3.2.4.3. Salting out effect

In SPME analysis, the addition of salt to an aqueous solution minimizes the variability in ionic

strength in the sample, helping to normalize the results obtained. Increasing the ionic strength of

a sample induces an effect referred to as the salting out effect. This improves sensitivity in most

applications by driving compounds toward the fibre, promoting the mass transfer of analytes to

the headspace and improving reproducibility for samples.

To identify the saturation with salt to ensure maximum ionic strength different amounts of salt

were added. SPME vials were filled with HPLC grade water (12 mL) and spiked with 1 µg L-1

geosmin, 1 µg L-1 2-mib and 1 µg L-1 TCA (internal standard). SPME vials were prepared with four

replicates per treatment and different salt amounts (0.00 g, 3.125 g and 6.25 g) were added, and

the vial was capped immediately. The analysis conditions were as described under 3.2.4. (GC-MS

conditions in the laboratory) and samples were randomized during the analysis.

3.2.4.4. Sample storage time and stability

This experiment was designed to identify the stability of geosmin, 2-mib and TCA after preparation

in SPME vials at room temperature (25 °C). The different storage times of 0 h, 24 h, 72 h, 96 h were

tested. Five replicate samples were prepared for each treatment in SPME vials filled with HPLC

grade water (12 mL) and spiked with 1 µg L-1 geosmin, 1 µg L-1 2-mib and 1 µg L-1 TCA (internal

51

standard). 6.25 g of table salt was added to each sample. Then SPME vials were immediately

capped. Analysis conditions were as described under 3.2.4. (GC-MS conditions in the laboratory)

and samples were randomized during the analysis.

3.2.4.5. Repeatability and reproducibility of GC-MS for developed method

The major objective of this experiment was to identify the repeatability and reproducibility of the

newly developed analysis method of geosmin and 2-mib using GC-MS. This experiment was

performed in two sub experiments. In each experiment, triplicate samples were prepared using

SPME vials filled with HPLC grade water (12 mL) and spiked with 1 µg L-1 geosmin, 1 µg L-1 2-mib

and 1 µg L-1 TCA (internal standard). After, 6.25 g of table salt was added to each sample. The SPME

vials were immediately capped. During successive injections, SPME vials were stored in room

temperature (25 °C). Analysis conditions were as described under 3.2.4. (GC-MS conditions in the

laboratory) and samples were randomized during the analysis.

Experiment 1: Same sample was injected consecutively three times to GC-MS to check the

repeatability of the method in triplicate. RSD (Relative Standard Deviation) was calculated as

follows;

RSD = SD/ X mean

SD – standard deviation

X mean - mean value of the measurement results

Experiment 2: Same sample was injected consecutively three times to GC-MS stored for 0 h, 24 h

and 96 h to test the reproducibility of the geosmin and 2-mib analytical method with five replicates.

RSD was calculated for each time period to compare the reproducibility.

3.2.4.6. Limit of detection (LOD)

The lowest concentration of geosmin and 2-mib that can be reliably detected with GC-MS was

determined using this experiment.

52

SPME vials were filled with HPLC grade water (12 mL). Different concentrations of geosmin and 2-

mib namely, 0.100 µg L-1, 0.050 µg L-1, 0.010 µg L-1 and 0.001 µg L-1 were spiked to each SPME vial

in triplicate. 1 µg L-1 TCA was spiked to each sample as the internal standard. After, 6.25 g of table

salt was added. The samples were immediately capped and analysed as described under 3.2.4. (GC-

MS conditions in the laboratory). The samples were randomized during the analysis.

3.3. Section C - Experiments done to determine the suppression of geosmin and 2-

mib by bioflocs

The objective of these experiments was to determine the suppression of geosmin and 2-mib by

using bioflocs grown in sea water. These experiments were done using the optimized GC-MS

protocol.

3.3.1. Chemicals used for the experiment

For these experiments, geosmin was obtained from SIGMA (± Geosmin, UC18-5MG, analytical

grade, Switzerland), and 2-MIB was purchased from Wako (2-Methylisoborneol Standard, 132-

07071, analytical grade, Japan). Dilutions of 50 mg L-1 were prepared with distilled water. The

prepared diluted solutions were stored in falcon tubes (Corning™ Falcon™ 15 mL Conical

Centrifuge Tubes) at -20 °C during the analysis.

3.3.2. Experimental setups to determine the effect of bioflocs on geosmin and 2-mib

These experiments were designed to identify the effect of bioflocs for suppression of geosmin and

2-mib. They were designed in a chronological order to achieve the objectives of the research.

Experiment 1 was aimed to identify the presence of geosmin and 2-mib in natural sea water used

for the experiment and sample carryover effect of GC-MS. Subsequently experiment 2 was used

to recognise the effect of bioflocs for geosmin and 2-mib (1 µg L-1). According to the results, the

geosmin and 2-mib disappeared from the experimental setup less than 48 h. Therefore,

Experiment 3 was planned to identify the effect of aeration on geosmin and 2-mib in sea water.

During next experiments the concentration of geosmin and 2-mib was increased up to 50 µg L-1.

53

Not only the effect of aeration but also the effect of salinity (sea water and fresh water) was tested

during experiment 4. According to the results achieved, aeration was the major factor for the

volatilization of the geosmin and 2-mib from the experimental setup. Consequently, experiment 5

was designed to identify the influence of bioflocs on geosmin and 2-mib without supplying

aeration. During this experiment bioflocs were not performing well due to lack of aeration.

Considering that, experiment 6 was designed to identify the survival of biofloc (using water quality

parameters) in a closed vial, allowing it to keep in suspension using shaker and rotator. Results of

the test indicate that both shaker and rotator can be used to keep biofloc alive for few days while

mixing. Therefore experiment 7 was designed on a shaker to evaluate the effect of bioflocs on

geosmin and 2-mib. As this experiment also revealed volatilization of geosmin and 2-mib from the

experimental setup, experiment 8 was designed using closed vials attached to the rotator.

Experiment 1: Identification of the naturally occurring geosmin and 2-mib in experimental water

and carryover effect of GC-MS

Purpose: To confirm that the sea water used for experiments does not contain natural geosmin

and 2-mib. Also to ensure that the GC-MS does not retain the geosmin and 2-mib in the SPME

needle from the previously analysed sample (carryover effect of GC-MS)

Set-up: Mainly, sea water (SW) and sea water spiked with geosmin (1 µg L-1) and 2-mib (1 µg L-1)

(SW+) was used for the experiment. As internal standard, 1 µg L-1 concentration of TCA was spiked

to each treatment. Each treatment was run in triplicate using the optimized GC-MS method.

Experiment 2: Bioflocs spiked with geosmin and 2-mib with aeration

Purpose: This experiment was designed to evaluate the suppression of geosmin and 2-mib by

bioflocs grown in sea water.

Set-up: The experiment counted four treatments (Figure 19): The first treatment was sea water

spiked with geosmin and 2-mib (SW+) to estimate the natural loss of the compounds due to their

volatility. 2000 mL of sea water was added into 2 L glass bottles (SIMAX) during this treatment. The

second treatment was glass bottles (2 L SIMAX) with bioflocs spiked with geosmin and 2-mib (BF+)

54

to determine the possible suppression of the compounds by bioflocs. For this treatment 1800 mL

of sea water and 200 mL of biofloc sample from stock tank was used.

The third treatment was glass bottles (2 L SIMAX) with sterile bioflocs spiked with geosmin and 2-

mib (SBF+) to make a distinction between possible physical absorption of the compounds to the

bioflocs and microbial interference. 200 mL of bioflocs were autoclaved (20 min at a pressure of

1.2 psi and 121 °C) and mixed with 1800 mL of autoclaved sea water.

The fourth treatment was glass bottles (2 L SIMAX) with pure bioflocs (BF) to check the production

of the two compounds by the bioflocs themselves. 1800 mL of sea water and 200 mL of biofloc

sample from stock tank was used for each bottle.

Each treatment was prepared in triplicates and they were spiked with 1 µg L-1 geosmin and 1 µg L-1

2-mib at 0 h. Aeration was supplied to the experimental setup using aquarium air stones in order

to keep bioflocs in suspension. The water samples from each treatment were analysed at 0 h, 48

h, and 72 h using GC-MS. In order to avoid interference of bioflocs with SPME analysis, water

samples taken from each bottle were centrifuged (Harrier, MSE, 18/80) at 4000 rpm for 10 min

and 12 mL of supernatant was used for the GC-MS analysis with 1 µg L-1 TCA as internal standard.

Figure 19: Experimental setup with four treatments (right to left: SW+, BF+, SBF+, BF) Note that SBF+ was covered

with tissue due to foaming effect before starting the experiment

Experiment 3: Effect of aeration on geosmin and 2-mib using different aeration techniques

Purpose: This experiment was designed to evaluate the effect of aeration rate on the

concentration of geosmin and 2-mib in sea water.

55

Set-up: Three types of treatments were used, air stone (AS), air pipette (AP) and no aeration (NA).

No aeration treatment facilitates air supply only through the water-air interface. The experiment

was performed in 2 L glass bottles (SIMAX) filled with 2000 mL of sea water in triplicate. A higher

concentration of 50 µg L-1 of geosmin and 50 µg L-1 of 2-mib were spiked at 0 h. The samples were

prepared and analysed with internal standard (TCA, 1 µg L-1) after 0 h, 2 h, 6 h and 24 h using the

optimized GC-MS analysis.

Experiment 4: Effect of salinity and aeration on losses of geosmin and 2-mib in experimental

bottles

Purpose: This experiment was designed to evaluate the influence of salinity and aeration on the

depletion of geosmin and 2-mib in the experimental setup.

Set-up: For this experiment four types of treatments were used, sea water with aeration using air

pipette (SW a+), sea water without aeration (SW a-), fresh water with aeration using air pipette

(FW a+) and fresh water without aeration (FW a-). The experiment was carried out in 2 L glass

bottles (SIMAX) in triplicate per each treatment. Aeration was supplied with an air pipette for SW

a+ and FW a+ treatments. Each bottle was filled with 2000 mL of sea water/fresh water. 50 µg L-1

of geosmin and 50 µg L-1 of 2-mib were spiked at 0 h. The samples were prepared and analysed

with TCA 1 µg L-1 as internal standard after 0 h, 2 h, 6 h and 24 h using the optimized GC-MS analysis.

Experiment 5: Effect of bioflocs on geosmin and 2-mib without aeration

Purpose: This experiment was designed to evaluate the ability of bioflocs to suppress geosmin and

2-mib. This experiment was performed without aeration in order to minimize the volatilization of

geosmin and 2-mib.

Set-up: For this experiment four types of treatments were used as described in Experiment 2.

Namely, pure bioflocs (BF), bioflocs spiked with geosmin and 2-mib (BF+), sterile bioflocs spiked

with geosmin and 2-mib (SBF+) and sea water spiked with geosmin and 2-mib (SW+). The

experiment was done in 2 L glass bottles (SIMAX) in triplicate per each treatment. The volume of

water in each bottle was 1000 mL, 1 L of headspace was allowed since this experiment was not

performed with aeration. Each treatment was prepared with 500 mL of sea water and 500 mL

56

bioflocs/sterile bioflocs, except SW+ treatment which used 1000 mL of sea water. Bottles were not

aerated and kept closed throughout the experimental period (Figure 20). When spiked, a total of

50 µg L-1 of geosmin and 50 µg L-1 of 2-mib was used. Samples were taken at 0 h, 2 h, 6 h, 10 h and

24 h, they were centrifuged, prepared and analysed with 1 µg L-1 TCA as internal standard using

optimized GC-MS method.

Figure 20: Experimental setup to evaluate the effect of biofloc on geosmin and 2-mib without aeration

Experiment 6: Changes of water quality parameters of bioflocs stored in closed vials placed on

a rotator versus shaker

Purpose: This experiment was designed to evaluate the survival of bioflocs without supply of

aeration but with continuous mixing. For the mixing purpose, a shaker and rotator were evaluated

measuring the water quality parameters.

Set-up: For this experiment two sets identical setups were operated on a rotator and shaker.

Tightly capped 40 mL transparent glass vials (Schott Duran, 40 mL) filled with 5 mL of bioflocs and

15 mL of sea water were used. The water quality was checked for five days continuously. The levels

of pH, temperature, dissolved oxygen, total ammonia (TAN: NH3 + NH4 +) and nitrite (NO2-) were

measured daily in each setup.

57

Experiment 7: Effect of bioflocs for suppression of geosmin and 2-mib without aeration on a

shaker

Purpose: This was designed to reduce the settlement of bioflocs using a shaker and to test the

effect of bioflocs on odour compounds (geosmin and 2-mib).

Set-up: During this experiment three types of treatments were used specifically, bioflocs (BF),

bioflocs spiked with geosmin and 2-mib (BF+), and sea water spiked with geosmin and 2-mib (SW+).

Sterile bioflocs treatment was not used in this experiment due to the capacity limitation of the

shaker. The experiment was performed in Erlenmeyer flasks (Schott Duran, 500 mL) in triplicate.

They were filled up to 300 mL of sea water for SW+ treatment and 200 mL of sea water + 100 mL

bioflocs was used for the BF and BF+ treatments. 50 µg L-1 of geosmin and 50 µg L-1 of 2-mib were

spiked at 0 h. Erlenmeyer flasks were kept covered with parafilm throughout the experimental

period on a multifunctional orbital shaker (Biosan) with 120 rpm (Figure 21). Samples were taken

at 0 h, 24 h, 48 h and 72 h, they were centrifuged, prepared and analysed with 1 µg L-1 TCA as

internal standard using optimized GC-MS method.

Figure 21: Experimental design to analyse the effect of bioflocs for suppression geosmin and 2-mib without

aeration on a shaker

Experiment 8: Effect of bioflocs on geosmin and 2-mib without aeration on a rotator

Purpose: This experiment was designed in sealed containers to completely cease the depletion of

geosmin and 2-mib in samples while providing better mixing.

Set-up: For this experiment four types of treatments were used as described in experiment 2,

specifically, bioflocs (BF), bioflocs spiked with geosmin and 2-mib (BF+), sterile bioflocs spiked with

58

geosmin and 2-mib (SBF+) and sea water spiked with geosmin and 2-mib (SW+). The experiment

was performed in transparent glass vials (Schott Duran, 40 mL). Four stock solutions were made

for each treatment as BF (200 mL sea water + 50 mL bioflocs), BF+ (200 mL sea water + 50 mL

bioflocs), SBF+ (200 mL sterile sea water + 50 mL sterile bioflocs) and SW+ (250 mL sea water). 50

µg L-1 of geosmin and 50 µg L-1 of 2-mib were spiked at 0 h to the stock solutions of BF+, SBF+ and

SW+ treatments. They were distributed into glass vials (15 mL per each vial) and capped tightly and

attached to the rotator with 6 rpm speed (Figure 22). Each vial represents a replicate, therefore 12

vials were used for analysis per each time period (4 treatments * 3 replicates). The analysis was

done at 0 h, 24 h, 48 h and 240 h. Samples were centrifuged, prepared and analysed with 1 µg L-1

TCA as internal standard using optimized GC-MS method.

Figure 22: Experimental setup to analyse the effect of bioflocs on geosmin and 2-mib without aeration on a

rotator

3.4. Section D - Statistical analysis

Two sample t test and one way ANOVA statistical tests were used to compare the difference

between the treatments of the experiments. During the data analysis normality and the

homogeneity of variances were checked using Q-Q plots and Levene test, respectively. Significant

differences were assumed at p < 0.05 level and post-hoc analysis was performed using Tukey HSD

test. All statistical analysis was conducted using SPSS Statistics 17.

59

Chapter 4: Results During this study different types of experiments were conducted. In this chapter, the results of

each experiment are divided into three major sections as described in Chapter 3. Section A

illustrates the results related to bioflocs stock management done in the Laboratory of Aquaculture

and Artemia Reference Centre (ARC). Results of the experiments to optimize GC-MS for analysis of

geosmin and 2-mib in the Synthesis, Bioresources and Bioorganic Chemistry laboratory (SynBioC)

was discussed in section B. Section C explains the results of the experiments done to determine

the suppression of geosmin and 2-mib by bioflocs.

4.1. Section A: Biofloc stock management


The results of Imhoff cone tests, TSS value, SVI value and the ash content are expressed in Table 8

during the experimental period.

Table 8: The results of Imhoff cone test, TSS, SVI and ash content of the biofloc reactor tank during each week of the experimental period.

Week Imhoff cone

(mL L-1)

TSS

(mg L-1)

SVI

(mL g-1)

Ash

%DW

1 22.12 1252.21 17.66 36.52

2 58.23 2286.62 25.47 35.53

3 45.25 2754.21 16.43 34.57

4 57.31 2415.52 23.73 35.68

5 64.52 1854.21 34.80 39.06

6 65.64 1956.64 33.55 37.21

7 60.93 2427.41 25.10 36.29

Average± SD 53.43±14.21 2135.26±456.79 25.25±6.52 36.41±1.44

60

4.1.2. Floc morpho structure

Figure 23: Biofloc morpho structure

A; Biofloc particle flocculation with filamentous algae, B; Copepod in biofloc, C; flocculation with nematode,

copepod, filamentous algae, D; copepod, E; Ciliate, F; Nematode

A B

C D

E F

61

Several changes in characteristics of the bioflocs stock culture could be witnessed during the

course of the experiments. Although these characteristics were not measured, they can hold

valuable indications.

Throughout the seven weeks of the experiments, biofloc stock has changed substantially in

colour. Based on visual observation, first they had a milky brown appearance, which continuously

changed into brownish gray in colour. During the experimental period, the biofloc characteristics

changed considerably. Sometimes flocs settled to the bottom and sometimes floated on the

surface, suggesting qualitative changes in flocs. Furthermore, the size of the flocs was changed

drastically within few days from smaller flocs to larger flocs and vice versa. Foaming was very

frequent in the biofloc reactor tank and after adding the feed, it disappeared suddenly. Sometimes

excessive foaming was evident before feeding the flocs. As illustrated in Figure 23 diverse species

of microalgae and different species of ciliates and flagellate protozoans, rotifers and crustaceans

and a species of nematode were observed in the biofloc reactor tank.

4.1.3. Water quality parameters

The different water quality parameters during the experimental period were measured daily

(Appendix I). According to the measurements (Figure 24), the average water temperature of the

biofloc stock tank was 25.10 ± 1.34 °C. A sudden decrease of temperature was reported on day 19

due to a temperature regulation failure of the experimental room. The average dissolved oxygen

of the biofloc stock tank was 6.38 mg L-1 ± 0.54 ranging between 5.38 - 7.33 mg L-1. Average pH of

the biofloc stock tank was 8.04 ± 0.19 ranging between 7.50 and 8.39. Average salinity of the

biofloc stock tank was 33.16 PSU ± 1.83 ranging between 29.45 - 37.24 PSU. The average

ammonium concentration was 0.29 mg L-1 ± 1.21 ranged in between 0.0 mg L-1 and 1.5 mg L-1.

Average nitrite concentration of the biofloc stock tank was 0.19 mg L-1 ± 0.13 ranged in between

0.01 mg L-1 and 0.30 mg L-1.

62

Figure 24: Water quality parameters of the biofloc stock during the study period A; temperature (°C), B; dissolved oxygen (mg L-1), C; pH, D; salinity (PSU), E; ammonium and nitrite concentrations

(mg L-1)

18

21

24

27

0 10 20 30 40 50

Tem

per

atu

re (

°C)

Number of Days

A

4

5

6

7

8

0 10 20 30 40 50

Dis

solv

ed o

xyge

n (

mg

L-1)

Number of Days

B

6

7

8

9

0 10 20 30 40 50

pH

Number of Days

C

28

30

32

34

36

38

0 10 20 30 40 50

Salin

ity

(PSU

)

Number of Days

D

0.0

0.5

1.0

1.5

2.0

0 10 20 30 40 50

Co

nce

ntr

atio

n (

mg

L-1)

Number of Days

Ammonium concentration Nitrite concentration

E

63

4.2. Section B: Experiments for optimized geosmin and 2-mib analysis using GC-MS

Different types of experiments were designed to determine the optimum GC-MS conditions for

geosmin and 2-mib analysis. The results of these experiments are statistically analysed and

graphically illustrated in this section.

4.2.1. Optimum incubation temperature

During this experiment, samples were spiked with 1 µg L-1 geosmin, 1 µg L-1 2-mib and 1 µg L-1 TCA

and tested for three different incubation temperatures (40 °C, 60 °C, and 80 °C) (Appendix II).

Geosmin

The area of geosmin value at 80 °C (M = 162259 ± 9763, p = 0.002) and 60 °C (M = 145030 ± 12065,

p = 0.000) were significantly higher than incubation at 40 °C (M = 93221 ± 8755). There was no

statistically significant difference between incubation at 80 °C and incubation at 60 °C (p = 0.181).

2-mib

When considering the impact of different incubation temperatures on the area of 2-mib in SPME

GC-MS, at 60 °C (M = 66663 ± 6910) the area of 2-mib was significantly higher than at 80 °C (M =

46873 ± 5097, p = 0.017) and at 40 °C (M = 47989 ± 6105, p = 0.022). There was no statistically

significant difference between incubation at 40 °C and 80 °C (p = 0.973).

Therefore, 60 °C was selected as optimal incubation temperature for the experiments.

4.2.2. Optimum extraction time

Different extraction times of 5 min, 10 min, 15 min, 20 min, and 40 min were verified to determine

the best condition (Appendix III).

Geosmin

Figure 25 illustrated the results of statistical analysis of area of geosmin for tested extraction times.

2-mib

Figure 26 illustrated the results of statistical analysis of area of 2-mib for tested extraction times.

Agreeing to the results, 20 min was selected as the best extraction time for geosmin and 2-mib.

64

Figure 25: The area of geosmin with different extraction times (n=3), spiked with 1 µg L-1 geosmin, error bars represent SD and a, b, c represent significant differences

Figure 26: The area of 2-mib with different extraction times (n=3), spiked with 1 µg L-1 2-mib, error bars represent SD and a, b, c represent significant differences

4.2.3. Salting out effect

Different amounts of table salt were added to the SPME vial, being 0.00 g, 3.13 g and 6.25 g

(Appendix IV).

Geosmin

According to the outcomes, the area of geosmin obtained using 6.25 g salt (M = 138553 ± 30945)

was not significantly different from using 3.125 g salt (M = 102832 ± 23561) (p = 0.116). The

c

bcb

a

a

8

13

18

23

28

33

0 5 10 15 20 25 30 35 40 45

Are

a o

f ge

osm

inx

10

00

0

Extraction time (min)

c

bcabc

a ab

3

5

7

9

11

13

15

0 5 10 15 20 25 30 35 40 45

Are

a o

f 2

-mib

x 1

00

00


65

significantly lowest area of geosmin was obtained without adding salt to the sample (0 g) (M =

9233 ± 1317).

2-mib

The significantly highest area of 2-mib was obtained using 6.25 g salt (M = 70241 ± 20059, p =

0.002) and 3.125 g salt (M = 56743 ± 17552, p = 0.001) than without salt (0 g) (M = 4439 ± 424).

Area of 2-mib obtained using 6.25 g salt and 3.125 g salt was not statistically different from each

other at p = 0.461.

4.2.4. Sample storage time and stability

Different periods of 0 h, 48 h, 72 h and 96 h were used for the storage of prepared samples

(Appendix V).

According to the results (Figure 27), there were no significant differences for the storage periods

for area of geosmin (p = 0.487) and for area of 2-mib (p = 0.405) (Figure 28).

The results elucidated that although there are no significant differences among storage periods;

standard deviation is higher for longer storages. For that reason, immediate analysis was done

throughout the experiments.

Figure 27: The area of geosmin with different storage periods (n=3), spiked with 1 µg L-1 geosmin, error bars represent SD

30

40

50

60

70

80

90

100

110

120

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Are

a ge

osm

inTh

ou

san

ds

Storage time (h)

66

Figure 28: The area of 2-mib with different storage periods (n=3), spiked with 1 µg L-1 2-mib, error bars represent SD)

4.2.5. Repeatability and reproducibility of GC-MS analysis for geosmin and 2-mib

Experiment 1: Repeatability of GC-MS (Appendix VI)

Table 9 represents the calculated RSD values for each sample analysed in SPME GC-MS. The

average %RSD value for ratio (area of geosmin/area of TCA) (GSM/TCA) was 45% and %RSD value

for ratio (area of 2-mib/area of TCA) (2-mib/TCA) was 49%.

Table 9: Calculation of RSD values for repeatability of GC-MS (n=3), spiked with 1 µg L-1 geosmin and 1 µg L-1 2-mib

Sample (GSM/TCA) and (2-mib/TCA)

Average SD %RSD First injection Second Injection Third injection

geosmin_1 1.34 1.88 3.09 2.10 0.90 43

geosmin_2 1.45 1.68 3.47 2.20 1.10 50

geosmin_3 1.28 1.94 3.01 2.08 0.87 42

2-mib_1 0.79 1.11 2.09 1.33 0.68 51

2-mib_2 0.75 1.00 1.93 1.23 0.62 51

2-mib_3 0.71 1.00 1.67 1.13 0.49 44

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Are

a 2

-mib

Tho

usa

nd

s

Storage time (h)

67

Experiment 2: Reproducibility of GC-MS (Appendix VII)

Geosmin

Reproducibility of GC-MS was estimated by calculating %RSD value (Table 10) for consecutive

injections at each time (0 h, 24 h and 96 h) for geosmin and 2-mib analysis method. Average %RSD

value at 0 h was 66%, at 24 h was 32% and at 96 h was 29%.

Table 10: Calculation of %RSD values for (GSM/TCA) during repeated injections for three time periods (0 h, 24 h and 96 h), (n=5), spiked with 1 µg L-1 geosmin.

Time

(h)

(GSM/TCA)

Average SD RSD %RSD First

injection

Second

injection

Third

Injection

0 0.76 1.28 3.26 1.77 1.32 0.75 66

0 0.89 1.67 3.12 1.89 1.13 0.60

0 0.70 1.47 2.51 1.56 0.91 0.58

0 0.77 1.38 3.08 1.74 1.20 0.69

0 0.76 1.65 3.46 1.96 1.38 0.70

24 1.73 2.53 4.29 2.85 1.31 0.46 32

24 1.81 2.12 2.83 2.25 0.52 0.23

24 1.77 2.46 3.26 2.50 0.75 0.30

24 1.74 2.46 3.01 2.40 0.63 0.26

24 1.70 2.42 3.32 2.48 0.82 0.33

96 1.27 1.46 1.80 1.51 0.27 0.18 29

96 1.05 1.80 2.32 1.73 0.64 0.37

96 1.18 1.83 1.87 1.63 0.39 0.24

96 1.16 1.72 2.02 1.63 0.44 0.27

96 0.88 1.93 1.88 1.57 0.59 0.38

2-mib

Table 11 depicts the calculated RSD values of 2-mib analysis by SPME GC-MS for consecutive

injections during three time periods (0 h, 24 h and 96 h). Average %RSD value at 0 h was 66%, at

24 h was 53% and at 96 h was 52%.

68

Table 11: Calculation of %RSD values for 2-mib/TCA during repeated injections for three time periods (0 h, 24 h and 96 h) (n=5) spiked with 1 µg L-1 2-mib

Time (h)

2-mib/TCA

Average SD RSD %RSD First

injection

Second

injection

Third

Injection

0 0.76 1.28 3.26 1.77 1.32 0.75 66

0 0.89 1.67 3.12 1.89 1.13 0.60

0 0.70 1.47 2.51 1.56 0.91 0.58

0 0.77 1.38 3.08 1.74 1.20 0.69

0 0.76 1.65 3.46 1.96 1.38 0.70

24 0.88 1.48 3.66 2.01 1.46 0.73 53

24 1.26 1.16 2.17 1.53 0.56 0.37

24 0.75 1.49 2.80 1.68 1.04 0.62

24 0.83 1.76 2.25 1.61 0.72 0.45

24 0.76 1.57 2.31 1.55 0.78 0.50

96 0.49 0.92 1.51 0.97 0.51 0.53 52

96 0.45 1.06 1.79 1.10 0.67 0.61

96 0.45 1.12 1.36 0.98 0.47 0.48

96 0.54 1.09 1.56 1.06 0.51 0.48

96 0.43 1.05 1.29 0.92 0.45 0.48

4.2.6. Limit of detection (LOD)

Samples with different concentrations of geosmin and 2-mib were prepared, namely 0.100 µg L-1,

0.050 µg L-1, 0.010 µg L-1 and 0.001 µg L-1. Then 1 µg L-1 TCA was spiked to each sample as internal

standard and analysed (Appendix VIII). and 10, respectively,

Geosmin

According to the results of SPME GC-MS, (GSM/TCA) can be detected at 0.01 µg L-1 (Figure 29) level

with signal to noise ratio (S/N) of 170

2-mib

Detection of 2-mib was only down to 0.05 µg L-1 (Figure 30) with S/N of 61.

69

Figure 29: Limit of detection of geosmin (n=3, error bars represent SD)

Figure 30: Limit of detection of 2-mib (n=3, error bars represent SD)

4.2.7. Optimized SPME GC-MS analysis method

Based on the above explained experiments, a final method of SPME GC-MS was designed to

analyse geosmin and 2-mib. Only the incubation temperature and extraction time were adjusted

in this new method. Sample preparation was done by filling a 20 mL SPME vial with 12 mL of HPLC

grade water and spiking with geosmin and 2-mib with TCA as internal standard. After adding 6.25 g

of table salt, the vial was capped immediately.

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.10 0.05 0.01 0.001

Are

a ge

osm

in/A

rea

TCA

Concentration of geosmin (µg L-1)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.10 0.05 0.01 0.001

Are

a 2

-mib

/Are

a TC

A

Concentration of 2-mib (µg L-1)

70

The vial containing the sample was incubated at 60 °C for 5 minutes, while shaken vigorously at

500 rpm. The odour compounds were extracted, under slow agitation, during 20 min, after which

desorption was done during 3 min at 250 °C. The GC oven temperature of 40 °C was increased to

250 °C, using the profile in Figure 31.

The column flow was maintained at 1.2 mL min-1. Retention times of geosmin and 2-mib were

15.58 min and 9.25 min, respectively. TCA, which was used as an internal standard, had a retention

time of 13.58 min. The MS was operated in SIM mode, measuring m/z = 95, 112 and 149 (dwell =

50 μs).

Figure 31: Oven temperature program of GC-MS

0

50

100

150

200

250

300

0 1 3 10 13.4 16.4

Tem

per

atu

re (

°C)

Time (min)

71

4.3. Section C: Analysis of bioflocs for the suppression of geosmin and 2-mib using

optimized GC-MS method

In this section, results of the experiments of biofloc samples spiked with geosmin and 2-mib were

described. All the samples were analysed using the optimized SPME GC-MS method for geosmin

and 2-mib, using TCA as an internal standard.

4.3.1. Experiment 1: Identification of the naturally occurring geosmin and 2-mib in

experimental water and carryover effect of GC-MS

In this experiment, raw sea water (SW) samples and sea water spiked with geosmin (1 µg L-1) and

2-mib (1 µg L-1) (SW+) were used as two treatments. According to the results (Table 12), sea water

samples (SW_1) did not contain geosmin and 2-mib naturally. The SW+ samples had 1.85 ± 0.04

GSM/TCA and 0.69 ± 0.02 area of 2-mib/TCA. Subsequently, another raw SW sample (SW_2) was

analysed to ensure the complete desorption of odour compounds from the SPME needle in order

to identify the carryover effect. According to the results, the SPME needle did not retain geosmin

and 2-mib.

Table 12 : Analysis of sea water samples (SW_1 and SW_2) to check the presence of geosmin and 2-mib, SW+ sample spiked with 1 µg L-1 geosmin and 1 µg L-1 2-mib, (n=3)

Sample GSM/TCA 2-mib/TCA

SW_1 0.00 0.00

SW+ 1.85 (± 0.04) 0.69 (± 0.02)

SW_2 0.00 0.00

4.3.2. Experiment 2: Bioflocs spiked with geosmin and 2-mib with aeration

This experiment counted four treatments, sea water spiked with geosmin and 2-mib (SW+),

bioflocs spiked with geosmin and 2-mib (BF+), sterile bioflocs spiked with geosmin and 2-mib

(SBF+) and pure bioflocs (BF). The samples were spiked with 1 µg L-1 geosmin and 1 µg L-1 2-mib at

0 h. The water samples from each treatment were analysed at 0 h, 48 h and 72 h using SPME GC-

MS.

72

The GC-MS results obtained from the experiment were illustrated in Table 13.

Table 13: The calculation of GSM/TCA and 2-mib/TCA for the experiment with bioflocs spiked with geosmin (1 µg L-1) and 2-mib (1 µg L-1) with aeration (n=3)

Time (h) Treatment Area of TCA Area of GSM Area of 2-mib GSM/TCA 2-mib/TCA

0 SW+ 77089 4459 17672 0.06 0.23

0 BF+ 56286 6682 15709 0.12 0.28

0 SBF+ 62287 10560 14787 0.17 0.24

0 BF 63066 0 5886 0.00 0.09

48 SW+ 62023 0 0 0.00 0.00

48 BF+ 18481 0 0 0.00 0.00

48 SBF+ 79180 0 0 0.00 0.00

48 BF 66675 0 0 0.00 0.00

72 SW+ 19657 0 0 0.00 0.00

72 BF+ 68720 0 0 0.00 0.00

72 SBF+ 30710 0 0 0.00 0.00

72 BF 26804 0 0 0.00 0.00

According to the analysis by SPME GC-MS, geosmin and 2-mib became undetectable in this

experimental setup in less than 48 h. Therefore, the effect of aeration in the removal of these

compounds was investigated further in the next experiments.

4.3.3. Experiment 3: Effect of aeration on geosmin and 2-mib using different aeration

techniques

Three types of treatments were used, air stone (AS), air pipette (AP) and no aeration (NA). A

concentration of 50 µg L-1 of geosmin and 50 µg L-1 of 2-mib were spiked at 0 h. The samples were

analysed with internal standard (TCA, 1 µg L-1) after 0 h, 2 h, 6 h and 24 h using optimized SPME

GC-MS (Appendix IX).

73

Geosmin

For 0 h, statistical analysis was not performed because only one sample was analysed per

treatment. At 2 h, 6 h and 24 h statistical analysis was illustrated in Table 14.

Table 14: GSM/TCA ± SD for three treatments (NA, AP and AS) with different time periods (0 h, 2 h, 6 h and 24 h) spiked with 50 µg L-1 geosmin at 0 h. Significant differences between treatments at each time period was represented as a and b, (n=3)

Treatment 0 h 2 h 6 h 24 h

NA 34.69 5.38 ± 5.26b 18.50 ± 10.99a 18.15 ± 5.17a

AP 5.64 28.69 ± 6.2a 18.28 ± 1.02a 2.10 ± 0.41b

AS 30.09 0.87 ± 0.5b 0.01 ± 0.02b 0.00 ± 0.00b

2-mib

At 0 h statistical analysis was not performed because only one sample was analysed per treatment.

At the 2 h, 6 h and 24 h statistical analysis was illustrated in Table 15.

Table 15: 2-mib/TCA ± SD for three treatments (NA, AP and AS) with different time periods (0 h, 2 h, 6 h and 24 h) spiked with 50 µg L-1 2-mib at 0 h. Significant differences between treatments at each time period was represented as a and b, (n=3)

Treatment 0 h 2 h 6 h 24 h

NA 33.24 5.85 ± 4.38b 14.09 ± 7.22a 14.12 ± 2.85a

AP 7.39 20.21 ± 7.42a 12.84 ± 1.97b 1.35 ± 0.86b

AS 28.67 1.47 ± 0.76b 0.00 ± 0.00b 0.00 ± 0.00b


experimental bottles

For the experiment four types of treatments were used, sea water with aeration using air pipette

(SW a+), sea water without aeration (SW a-), fresh water with aeration using air pipette (FW a+)

and fresh water without aeration (FW a-). 50 µg L-1 of geosmin and 50 µg L-1 of 2-mib were spiked

at 0 h. The samples were prepared and analysed with TCA 1 µg L-1 after 0 h, 2 h, 6 h and 24 h using

optimized SPME GC-MS (Appendix X).

74

Geosmin

The results at the beginning of the experiment (0 h) indicated a significant difference between FW

a- with other three treatments (p = 0.002) (Figure 32).

The results after 24 h of the experiment indicated a significantly higher GSM/TCA value for SW a-

than for FW a- (p = 0.006), FW a+ (p = 0.000) and SW a+ (p = 0.000).

Figure 32: The GSM/TCA with different salinity and aeration (FW a-, FW a+, SW a-, SW a+) during 0 h, 2 h, 6 h and 24 h periods spiked with 50 µg L-1 geosmin at 0 h. Significant differences between treatments at 0 h and 24 h were represented as a, b and c, (n=3)

2-mib

The results at the beginning of the experiment (0 h) indicated that none of the treatments were

significantly different (Figure 33).

The results after 24 h of the experiment indicated a significantly higher 2-mib/TCA value for SW a-

than for FW a- (p = 0.026), FW a+ (p = 0.000) and SW a+ (p = 0.000).

b

b

a a

c0

5

10

15

20

25

30

35

0 2 4 6 8 10 12 14 16 18 20 22 24

Are

a o

f ge

osm

in/A

rea

of

TCA

Time (h)

FW No aeration

FW Aeration

SW No Aeration

SW Aeration

75

Figure 33: The 2-mib/TCA with different salinity and aeration (FW a-, FW a+, SW a-, SW a+) during 0 h, 2 h, 6 h and 24 h periods spiked with 50 µg L-1 2-mib at 0 h. Significant differences between treatments at 0 h and 24 h were represented as a, b and c, (n=3)

4.3.5. Experiment 5: Effect of bioflocs on geosmin and 2-mib without aeration

Four types of treatments were used, pure bioflocs (BF), bioflocs spiked with geosmin and 2-mib

(BF+), sterile bioflocs spiked with geosmin and 2-mib (SBF+) and sea water spiked with geosmin

and 2-mib (SW+). A total of 50 µg L-1 of geosmin and 50 µg L-1 of 2-mib was used and samples were

taken at 0 h, 2 h, 6 h, 10 h and 24 h (Appendix XI).

Geosmin

At the beginning of the experiment (0 h) (Figure 34), the significantly highest GSM/TCA was

obtained for BF+ (M = 31.64 ± 2.23, p = 0.000), SBF+ (M = 27.83 ± 2.12, p = 0.000) and SW+ (M =

24.11 ± 5.16, p = 0.000) treatments. BF treatment (M = 0.02 ± 0.03) had significantly lowest value.

After 24 h of the experiment, the highest GSM/TCA was obtained for BF+ (M = 47.50 ± 9.74). SBF+

treatment (M = 28.08 ± 0.07) and SW+ treatment (M = 24.07 ± 2.45) were not significantly different

from each other. BF (M = 0.14 ± 0.01) treatment was significantly lower than SBF+ (p = 0001) and

BF+ (p = 0.000) treatments.

b

a

c0

5

10

15

20

25

30

35

0 2 4 6 8 10 12 14 16 18 20 22 24

Are

a o

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-mib

/A

rea

of

TCA

Time (h)

FW No aeration

FW Aeration

SW No Aeration

SW Aeration

76

Figure 34: The GSM/TCA with four treatments (BF, BF+, SBF+, SW+) during 24 h without aeration spiked with 50 µg L- 1 geosmin at 0 h. Significant differences between treatments at 0 h and 24 h were represented as a, b and c, (n=3)

Figure 35: The 2-mib/TCA with four treatments (BF, BF+, SBF+, SW+) during 24 h without aeration spiked with 50 µg L- 1 2-mib at 0 h. Significant differences between treatments at 0 h and 24 h were represented as a, b and c, (n=3)

bc

a

a

b

5

15

25

35

45

55

65

0 2 4 6 8 10 12 14 16 18 20 22 24

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BF+

SBF+

SW+

c c

a

a b

b bc

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A

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BF+

SBF+

SW+

77

2-mib

At the beginning of the experiment (0 h) (Figure 35), higher 2-mib/TCA was obtained for BF+ (M =

28.64 ± 5.87) and SBF+ (M = 27.83 ± 8.20) in comparison to SW+ (M = 12.85 ± 2.16) and BF

treatment (M = 0.00 ± 0.00). SW+ was significantly higher than BF treatments (p = 0.042).

After 24 h of the experiment, the significantly lowest 2-mib/TCA was obtained from BF (M = 0.02

± 0.04) in comparison to SBF+ (M = 27.30 ± 4.83, p = 0.005) and BF+ (M = 48.33 ± 12.44, p = 0.000).

SBF+ and SW+ treatments were not significantly different (p = 0.144). BF+ treatment was

significantly higher than SBF+ (p = 0.021) and SW+ (p = 0.001).


placed on a rotator versus shaker

For this experiment two sets identical setups were operated on rotator versus shaker. Tightly

capped 40 mL transparent glass vials (Schott Duran, 40 mL) filled with 5 mL of bioflocs and 15 mL

of sea water were used. The water quality was checked for five days continuously. The levels of

pH, temperature, dissolved oxygen, total ammonia (TAN: NH3 + NH4 +) and nitrite (NO2-) were

measured daily in each setup (Appendix XII).

Two sample independent t test for temperature of bioflocs on rotator (26.24 ± 1.50 °C) and shaker

(27.02 ± 0.88 °C) indicated no significant difference, (t (8) = -1.004, p = 0.345) (Figure 36).

Figure 36: The temperature of the bioflocs stored on rotator and shaker during five days (n=1).

22

23

24

25

26

27

28

29

1 2 3 4 5

Tem

per

atu

re (

0 C)

Numer of days

On rotator

On shaker

78

Two sample independent t test for DO% of bioflocs on rotator (56.64 ± 6.93%) and shaker (57.12 ±

5.72%) indicated no significant difference, (t(8) = -0.119, p = 0.908) (Figure 37).

Figure 37: The DO% of the bioflocs stored on rotator and shaker during five days (n=1).

Two sample independent t test for pH of bioflocs on rotator (6.75 ± 0.31%) and shaker (6.74 ± 0.30

%) indicated no significant difference, (t(8) = 0.031, p = 0.976) (Figure 38).

Figure 38: The pH of the bioflocs stored on rotator and shaker during five days (n=1).

Two sample independent t test for ammonium of biofloc on rotator (3.30 ± 2.39 mg L-1) and shaker

(3.05 ± 2.67 mg L-1) indicated no significant difference, (t(8) = 0.156, p = 0.880) (Figure 39).

40

45

50

55

60

65

70

1 2 3 4 5

DO

%

Number of days

On rotator

On shaker

6.4

6.6

6.8

7.0

7.2

7.4

1 2 3 4 5

pH

Number of days

On rotator

On shaker

79

Figure 39: The ammonium concentration (mg L-1) of the bioflocs stored on rotator and shaker during five days

(n=1).

Nitrite concentration of the bioflocs on rotator and shaker was 0.3 mg L-1 from day 1 to day 5.

4.3.7. Experiment 7: Effect of bioflocs on suppression of geosmin and 2-mib without aeration

on a shaker

During the experiment three types of treatments were used specifically, bioflocs (BF), bioflocs

spiked with geosmin and 2-mib (BF+), and sea water spiked with geosmin and 2-mib (SW+).

50 µg L-1 of geosmin and 50 µg L-1 of 2-mib were spiked at 0 h. Samples were taken at 0 h, 24 h,

48 h and 72 h, they were centrifuged, prepared and analysed with 1 µg L-1 TCA via optimized SPME

GC-MS (Appendix XIII).

Geosmin

At the beginning of the experiment (0 h) (Figure 40), the significantly highest GSM/TCA was

obtained for SW+ (M = 21.21 ± 0.41) and BF+ (M = 19.37 ± 3.52) in comparison to BF treatment

(M = 0.06 ± 0.01, p = 0.000 and p = 0.000, respectively). There was no significant difference

between SW+ and BF+ treatments (p = 0.547). After 72 h of the experiment, there was no

statistically significant difference between the treatments for the GSM/TCA.

2-mib

At the beginning of the experiment (0 h) (Figure 41), the significantly highest 2-mib/TCA was

obtained for BF+ (M = 28.32 ± 3.86) and SW+ (M = 19.90 ± 4.81) in comparison to BF treatment (M

= 0.00 ± 0.00, p = 0.000 and p = 0.001, respectively). There was no significant difference between

0

1

2

3

4

5

6

1 2 3 4 5

Am

mo

niu

m (

mg

L-1)

Number of days

On rotator

On shaker

80

BF+ and SW+ treatments (p = 0.062). After 72 h of the experiment, the analysed samples via SPME

GC-MS did not indicate the presence of 2-mib.

Figure 40: The GSM/TCA with three treatments (BF, BF+, SW+) during 72 h without aeration spiked with 50 µg L- 1

geosmin at 0 h. Significant differences between treatments at 0 h and 72 h were represented as a and b (n=3)

Figure 41: The 2-mib/TCA with three treatments (BF, BF+, SW+) during 72 h without aeration spiked with 50 µg L- 1

2-mib at 0 h. Significant differences between treatments at 0 h and 72 h were represented as a and b (n=3)

a

b0

5

10

15

20

25

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

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BF+

BF

a

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25

30

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Time (h)

SW+

BF+

BF

81


For this experiment four types of treatments were used, biofloc (BF), biofloc spiked with geosmin

and 2-mib (BF+), sterile bioflocs spiked with geosmin and 2-mib (SBF+) and sea water spiked with

geosmin and 2-mib (SW+). 50 µg L-1 of geosmin and 50 µg L-1 of 2-mib were spiked at 0 h to the

stock solutions of BF+, SBF+ and SW+ treatments. The analysis was done at 0 h, 24 h and 240 h.

Samples were centrifuged, prepared and analysed with 1 µg L-1 TCA using the optimized SPME GC-

MS method (Appendix XIV).

Geosmin

At the beginning of the experiment (0 h) (Figure 42), the highest GSM/TCA was obtained for SW+

(M = 47.18 ± 7.35, p = 0.000), BF+ (M = 31.25 ± 6.22, p = 0.004) and SBF+ (M = 29.99 ± 7.80, p =

0.002) in comparison to BF treatment (M = 0.00 ± 0.00). There was no significant difference

between SW+, BF+ and SBF+ treatments (p = 0.950, p = 0.996, respectively).

At the end of the experiment (240 h), the highest GSM/TCA was obtained for SW+ (M = 46.00 ±

3.69) compared to other treatments. SBF+ (M = 27.15 ± 5.50) and BF+ (M = 26.53 ± 5.70) were

significantly lower than SW+ treatment with p = 0.004 and p = 0.003, respectively. BF treatment

(M = 0.00 ± 0.00) had the lowest GSM/TCA and was significantly different from all other

treatments. There was no significant difference between BF+ and SBF+ treatments (p = 0.388).

2-mib

At the beginning of the experiment (0 h) (Figure 43), the highest 2-mib/TCA was obtained for SW+

(M = 36.59 ± 8.54, p = 0.005), SBF+ (M = 34.75 ± 12.25, p = 0.007) and BF+ (M = 26.82 ± 8.72, p =

0.044) compared to BF treatment (M = 0.00 ± 0.00). There was no significant difference of SW+

with SBF+ and BF+ treatments (p = 0.993, p = 0.751, respectively).

At the end of the experiment (240 h), the highest 2-mib/TCA was obtained for SW+ (M = 23.05 ±

0.41) and SBF+ (M = 17.60 ± 3.65) compared to other treatments. BF+ (M = 11.80 ± 1.52) was

significantly lower than SW+ and SBF+ treatment with p = 0.000. BF treatment (M = 0.00 ± 0.00)

had the lowest 2-mib/TCA and significantly differed from all other treatments. There was no

significant difference between SW+ and SBF+ treatments (p = 0.349).

82

Figure 42: The GSM/TCA for four treatments (BF, BF+, SBF+, SW+) during 240 h period on a rotator spiked with

50 µg L-1 of geosmin at 0 h. Significant differences between treatments at 0 h and 240 h were represented as a, b

and c (n=3)

.

Figure 43: The 2-mib/TCA for four treatments (BF, BF+, SBF+, SW+) during 240 h period on a rotator spiked with

50 µg L-1 of 2-mib at 0 h. Significant differences between treatments at 0 h and 240 h were represented as a, b and

c (n=3)

bc

b

a

a

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10 30 50 70 90 110 130 150 170 190 210 230 250

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BF+

SBF+

SW+

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SW+

83

Chapter 5: Discussion

In this chapter, results of experiments were discussed in three major sections as described in the

methodology and results chapters. Section A discusses the results related to biofloc stock

management done in the Laboratory of ARC. Results of the experiments for optimization of GC-MS

for analysis of geosmin and 2-mib in the SynBioC laboratory were discussed in Section B. Section

C explained the results of experiments performed to determine the suppression of geosmin and 2-

mib by bioflocs.

5.1. Section A: Biofloc stock management

The discussion in this section is concerning the results related to biofloc stock management done

in the Laboratory of ARC.


During the present study, the biofloc management was done to achieve optimum growth

conditions.

The result of the Imhoff cone test (Table 8) was 53.43 mL L-1 (± 14.21) on average during the

experimental period. This value was low at the beginning of the experimental period and then

gradually increased until the end of the experiment. This parameter was managed by daily flushing

of the excess solid from biofloc reactor tank. Particularly, after the 4th week the Imhoff cone value

increased indicated the formation of more settling bioflocs. The recommendation of settling solids

for marine shrimp is up to 15 mL L-1 (Taw, 2010) and for Nile tilapia between 25 mL L-1 and 50 mL

L-1 (Hargreaves, 2006). Therefore, during this experiment, the Imhoff cone value was maintained

at recommended levels for fish but not suitable for shrimp.

The average TSS value of the bioflocs was 2135.26 mg L-1 (± 456.79) during the experiment. As

indicated by Hargreaves (2006), biofloc systems are typically operated at TSS concentration less

than 1000 mg L-1 and most often less than 500 mg L-1. According to that, the TSS value of this

experiment was exceeding the optimum level for RAS. Even though the aquaculture conditions in

RAS use the bioflocs with a low TSS value, when experiments were done to determine the effect

84

of bioflocs on odour compounds, usually the average amount of TSS was 1920 ± 147 mg L-1 (Ma, et

al., 2016). Hargreaves (2006) also described that, over time and with adequate mixing, solids can

accumulate to undesirably high levels such as 2000 to 3000 mg L-1. However, during the current

research TSS level of bioflocs was intentionally maintained at higher concentration in order to

achieve a visible effect of the bioflocs on the suppression of odour compounds. According to the

literature, the TSS value in this experiment is exceeding the concentration sufficient for good

system functionality in RAS as reported of 200 to 500 mg L-1 (Hargreaves, 2006) and 100 to 300 mg

L-1 for shrimp (Krummenauer, et al., 2014).

The average SVI value of the experiment was 25.25 mL g-1. The SVI values are normally linked to

shifts in the characteristics of the biofloc microbial community (Graddy, et al., 1999). Higher SVI

values, above 60 mL g-1, are usually found in bioflocs with a high abundance of filamentous forms

of bacteria and cyanobacteria, where the amount of free space within the flocs increases (Graddy,

et al., 1999; De Schryver, et al., 2008). Bioflocs with high SVI levels have the capacity to remain

suspended in the water column without excessive settling (De Schryver, et al., 2008). Hence, they

can be easily harvested by culture species, as they are accessible in the water column and do not

tend to sink and build up at the bottom of the tanks (Liu, et al., 2014). Therefore, the bioflocs of

this experiment with 25.25 mL g-1 on average SVI value is indicated not to consist with filamentous

forms.

The average ash content was 36.41 (± 1.44) expressed in %dry weight. According to the literature,

the ash content has an average value ranging from 17% to 27% dry weight (Crab, et al., 2010) and

27% to 39% (Xu, et al., 2012). When comparing with these values, our ash content was relatively

high which may be due to the high TSS values.

Azim & Little, (2008) reported that TSS levels became uncontrollable at the last two sampling dates

of their experiment reaching up to 1000 mg L-1. The same strong growth conditions were observed

in the current research. The variation of floc settling characteristics indicated qualitative changes

in flocs during the experimental period. This characteristic was also observed by Little, et al., (2008)

in biofloc systems. Therefore, characterization of the flocs is a pre-requisite for effective

management of the system to ensure homogeneous samples throughout the different

experiments.

85

5.1.2. Water quality management

The major water quality parameters were monitored daily in order to maintain the bioflocs at

optimum conditions throughout the experimental period (Figure 24). The average water

temperature was 25.10 °C ± 1.34 during the experimental period. At the beginning the

temperature of the biofloc stock was lower because the biofloc stock (in ILVO) was adapted to the

environmental temperature (18 °C) and it was gradually increased to the room temperature

(25 °C). Except for the sudden temperature regulation failure in the middle of the experiment (19th

day), the biofloc temperature was regulated without major variations throughout the study period.

Deviller, et al., (2004) reported that the temperature in RAS increased with indoor air temperature

during the summer to a maximal temperature of 29 °C. The average dissolved oxygen was

6.38 mg L-1 ± 0.54, which is at its optimum level as discussed by Masser, et al., (2000). Dissolved

oxygen concentrations should be maintained above 60% of saturation or above 5 ppm for optimum

fish growth in RAS. The fluctuation of the dissolved oxygen concentrations is due to the frequent

clogging of the air stones due to the high concentration of bioflocs.

The average pH was 8.04 ± 0.19, during the experimental period. Fish generally can tolerate a pH

range from 6 to 9.5 in RAS although a rapid pH change of 2 units or more is harmful, especially to

fry. In addition, biofilter bacteria in RAS are efficient in the optimum pH range from 7 to 8 (Masser,

et al., 2000). During this experiment, the pH tends to decline as organisms in bioflocs produce acids

and carbon dioxide. Carbon dioxide reacts with water to form carbonic acid, which drives the pH

downward. Below a pH of 6.8, the nitrifying bacteria are inhibited and do not remove toxic nitrogen

wastes (Masser, et al., 2000). Therefore, maintaining the optimum pH range is very important in

biofloc management. To regulate the pH, frequent water exchange and flushing were done without

the addition of alkaline buffers during the experiment. The average salinity level was 33.16 PSU ±

1.83 and this shows a higher variation throughout the experimental period. The major reason for

this was the evaporation and the dilution of the biofloc reactor. The average ammonium

concentration was 0.29 mg L-1 ± 0.46 and average nitrite concentration was 0.19 mg L-1 ± 0.13 in

the biofloc stock during the experimental period. The fluctuations of the ammonium and nitrite

concentrations are mainly due to the handling of the biofloc stock tank. Because, when the water

quality tests indicate excessive concentrations of ammonium and nitrite, immediate dilution and

86

removal of excess solids were done in the biofloc reactor tank. Guttman & van Rijn (2008)

explained that in the RAS, ammonium levels were generally lower than 1 mg L-1 and nitrite levels

were lower than 0.5 mg L-1. Therefore, during the experiment all of the water quality parameters

were maintained to similar conditions as in RAS.

5.2. Section B: Experiments for optimized geosmin and 2-mib analysis using GC-MS

Results of the experiments for optimization of GC-MS analysis of geosmin and 2-mib in the

SynBioC laboratory were discussed in this section.

5.2.1. Optimum incubation temperatures for geosmin and 2-mib

The distribution constant of geosmin and 2-mib between the fibre and the sample depends on the

incubation temperature (Fettig, et al., 2014). An increase in temperature facilitates the migration

of geosmin and 2-mib from solution to the head space, accelerating sorption of odour substances

on the fibre (Reade, et al., 2014).

According to the results of the experiment for geosmin, the incubations at 80 °C and 60 °C were

producing higher areas of geosmin than at 40 °C. For 2-mib, the incubation at 60 °C was the

optimum condition. Therefore, 60 °C was selected as the best incubation temperature for both

compounds during the experiment. However, an excessive increase in temperature can cause

premature desorption of the odour compounds (Ezquerro, et al., 2002). According to the results,

geosmin can be well extracted up to 80 °C but 2-mib gives maximum extraction at 60 °C. This may

be due the differences of molecular weight, boiling point and enthalpy of vaporization of both

compounds as explained in Table 1.

The SPME GC-MS analysis of geosmin and 2-mib, several authors used different incubation

temperatures. Ng, et al., (2002) and Guttman & van Rijn (2009) used 65 °C, while Ikai, et al., (2003)

and Saito, et al., (2008) set incubation temperature of 70 °C. As shown in Figure 44, Saito, et al.,

(2008) obtained maximum extraction efficiency at 70 °C during their studies. During the study by

Arachchige, & Indrajith, (2016) from 30 °C to 70 °C of incubation temperatures, they concluded

that 60 °C produced the best extraction for geosmin and 2-mib (Figure 45).

87

Figure 44: The effect of iIncubation temperature on the HS-SPME of geosmin and 2-mib (Saito, et al., 2008).

Figure 45: Effect of incubation temperature on the extraction efficiencies of geosmin (red) and 2-mib (blue)

(Arachchige, & Indrajith, 2016)

5.2.2. Optimum extraction time

The extraction time is a critical parameter in the SPME process. Figure 46 shows the typical

relationship between extraction time and analyte absorbed on the fibre. According to that, before

reaching the equilibrium between the fibre and the sample (pre-equilibrium), the time factor is

very critical (Supelco, 2001). On the steep part of the graph, even small variations in the extraction

88

time can result in significant variations in the amount extracted (Mester & Sturgeon, 2005). At

equilibrium, small variations in the extraction time do not affect the amount of analyte extracted

by the fibre, therefore no critical influence on the quantitative results (Supelco, 2001). An optimal

approach to SPME analysis is to allow the analyte to reach equilibrium between the sample and

the fibre coating (Mester & Sturgeon, 2005).

Figure 46: Time effect for SPME extraction (Supelco, 2001).

According to results for the current experiment (Figure 25), extraction for 40 min and 20 min

produced a higher area of geosmin than extraction for 5 min, 10 min and 15 min. When considering

2-mib (Figure 26), the extraction for 40 min, 20 min and 15 min produced higher areas. These

values tend to follow the same pattern as in Figure 46. Up to 20 min, the area values of both

compounds are increasing suggesting the pre-equilibrium phase. Afterward for 40 min, the area

value of geosmin showed a slight increase while area of 2-mib was decreased. Therefore, 20 min

was selected as the best extraction time for both compounds.

Different authors used different extraction times during their studies which produce different

optimum results such as 20 min at 65 °C (Guttman & van Rijn, 2008; Guttman & van Rijn, 2011)

40 min at 60 °C (Ma, et al., 2016). As shown in Figure 47, Saito, et al., (2008) also obtained an

optimum extraction time at 20 min during their studies. Arachchige, & Indrajith, (2016) used 30

min of extraction time for their studies at 50 °C (Figure 48).

89

Figure 47: The effect of extraction time on area of geosmin and 2-mib with 1 ng mL-1 of geosmin and 2-mib in water saturated with sodium chloride (Saito, et al., 2008).

Figure 48: Effect of extraction time of the sample on extraction efficiencies for geosmin (red) and 2-mib (blue) using 0.01 µg L-1 geosmin and 2-mib (Arachchige, & Indrajith, 2016).

The extraction time is very important. A longer period favours the occupation of more sites on the

fibre by analyte molecules, but prolonged time when all sites are occupied does not affect the

preconcentration efficiency and sometimes can cause desorption (Zhang, et al., 1994; Namies´nik

& Jamro´giewicz, 1998). Extraction time and extraction temperature are parameters closely related

to each other (Mestres, et al., 2000). As an example, an increase in extraction temperature enables

shorter extraction time. Therefore, considering the results, 20 min of extraction time and 60 °C of

incubation temperature was selected during this study.

Are

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in/2

-mib

90

5.2.3. The salting out effect for optimum extraction

The suitability of the headspace SPME technique for the extraction of geosmin and 2-mib in water

depends on the transfer of these compounds from the aqueous phase to the gaseous phase

(Buchholz & Pawliszyn, 1994). Salt addition could significantly decrease their solubility in water,

resulting in a higher concentration of these compounds in the headspace (Guichard, 2002).

During this experiment, the optimum extraction for geosmin and 2-mib was obtained using 6.25 g

and 3.125 g salt.

The higher area values of geosmin and 2-mib by salt addition can be due to two reasons. First, the

salting-out agents improve extraction efficiency by decreasing the solubility of geosmin and 2-mib

in solution, thus increasing the amount of absorbed analytes on the fibre (Wu, et al., 2000). A

second reason will be limitation of headspace. In order to increase the extraction efficiency, the

volume of headspace in the vial should be minimized (Yang & Peppard, 1994; Pawliszyn, 1997).

The addition of extra salt reduces the volume of headspace, which promotes accumulation of

compounds and extraction to the fibre (Figure 49).

During this study, using 6.25 g salt and 3.125 g salt does not make a significant difference towards

the extraction efficiency of geosmin and 2-mib. When comparing the area of geosmin with the use

of 6.25 g salt and 0 g salt, × 15 efficiency was observed for 6.25 g. For 3.125 g salt compared with

0 g salt, × 11 efficiency was identified. This was similar for 2-mib, 6.25 g was × 15 efficient whereas

3.125 g was × 12 efficient compared to extraction without salt. For that reason, 6.25 g salt was

used to ensure optimum extraction during further experiments. Several studies were done to

select optimum salt content for the SPME GC-MS analysis of geosmin and 2-mib. Du, et al., (2017),

tested salt addition of 0.3 g mL-1 and no salt. They concluded an improvement in the extraction

efficiency of about 1.9 and 2.1 times for geosmin and 2-mib, respectively. Sung, et al., (2005)

reported that salt addition of 30%, compared to no salt added, offered an improvement in the

extraction efficiency of about 2.6 – 3.2 times. Yuan, et al., (2013) studied the effect of ionic strength

by adding NaCl ranging from 0 to 30% (w/v) and observed the improvement about 4.8 – 9.6 times.

Therefore, they used 30% of NaCl for their experiments.

91

Figure 49: Addition of more salt reduces the volume of head space which allows the efficient extraction

5.2.4. Sample storage time and stability

This experiment was done to identify the stability of geosmin, 2-mib and TCA after the preparation

of the samples. It should be noted that the GC-MS, complete analysis time per sample was about

40 min. Therefore, frequent analysis cannot be accomplished within few hours intervals to identify

the effect of bioflocs on geosmin and 2-mib. Due to that, different periods of times were studied

to identify the effect of storage time which may be useful for subsequent experiments.

According to the results of the experiment, storage of samples for 0 h, 48 h, 72 h and 96 h did not

produce significantly different areas of geosmin (Figure 27) or areas of 2-mib (Figure 28). Agreeing

to the results, at 0 h, SD is less than 48 h, 72 h and 96 h. When considering the results, 48 h storage

gives ×1.7 area of geosmin and ×1.9 area of 2-mib as compared to storage for 0 h, while 72 h

storage produces ×1.3 area of geosmin and ×1.5 area of 2-mib as compared to 0 h. The storage of

geosmin for 96 h produce ×2.1 area of geosmin and ×2.0 area of 2-mib than 0 h. This increase may

occur due to the geosmin and 2-mib concentration to the headspace during longer periods.

Therefore, considering these results, storage of the prepared samples was not done during the

experiments. Always immediate analysis after preparation of samples was done.

5.2.5. Assessment of the repeatability and reproducibility of SPME GC-MS

Repeatability is the degree of agreement among individual test results when the procedure is

carried out repeatedly (Zhang, et al., 2006). This is normally expressed as the RSD (Relative

Standard Deviation). To assess the repeatability of the analytical technique (SPME GC-MS), the

92

same samples were analysed 3 times in sequence with same concentrations of geosmin and 2-mib

(Villas-Boas, et al., 2011).

Experiment 1 was designed to analyse the repeatability of the SPME GC-MS protocol developed

for geosmin and 2-mib. During this experiment, for area of geosmin (Table 9) RSD1= 43%, RSD2 =

50% and RSD3 = 42% was obtained for 1 µg L-1 concentration of geosmin. When considering about

data for 2-mib (Table 9), RSD1= 51%, RSD2 = 51% and RSD3 = 44% were found. The acceptable limit

of %RSD value was 15% (Magdic, et al., 1996). Hence, for geosmin and 2-mib analysis repeatability

was not achieved considering the acceptable limit. In addition, when making repeated injections

from the same vial to assess quantitative repeatability, volatility of the compound should be taken

into consideration. Once the vial septum is pierced, there is a possibility for evaporation of

geosmin/2-mib/TCA, which can obviously affect the sample concentration.

According to the results, both geosmin and 2-mib show significantly higher values for the third

injection. This can be due to several reasons with the major one being TCA volatilization. If the

internal standard escapes through volatilization more than geosmin and 2-mib, the relative are

value increases during the second and third injections. Table 16 illustrates the area of TCA, geosmin

and 2-mib during the consecutive injections and their ratio values reference to the first injection.

It clearly shows that the area of TCA is more drastically declining for the third injection, than

geosmin and 2-mib.

Table 16: The area of geosmin, 2-mib, TCA and their ratio reference to the first injection

No. of

injection

area

of TCA

TCA ratio with

first injection

area of

geosmin

geosmin ratio

with first

injection

area of

2-mib

2-mib ratio with

first injection

1 73149 1.00 98421 1.00 55142 1.00

2 25292 0.35 45867 0.47 26253 0.48

3 8840 0.12 27985 0.28 16892 0.31

This may be the major reason to obtain higher values of GSM/TCA and 2-mib/TCA. Therefore, the

achievement of accurate results for geosmin and 2-mib analysis cannot be assured upon use of

TCA as an internal standard.

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The internal standard (TCA), geosmin and 2-mib should have the same chemical and physical

properties. Furthermore, their behaviour during extraction should be identical (Yoo, et al., 2009).

As a result, the use of an internal standard essentially eliminates the need to reach equilibrium of

analyte partitioning between the fibre and the sample (Potter & Pawliszyn, 1994). Additionally,

any change in extraction conditions, including the change of the fibre properties due to irreversible

adsorption of some of the matrix components is compensated using internal standard (Pawliszyn,

et al., 1997).

Korth, et al., (1991) suggested the use of geosmin-d3 and 2-mib-d3 (deuterated internal standards)

as internal standards for geosmin and 2-mib GC-MS analysis. These standards still indicated the

correct initial concentration of geosmin and 2-mib, even when < 10% of the original geosmin and

2-mib remained in solution. They explained further that accurate results for the initial

concentrations of geosmin and 2-mib can still be obtained when these internal standards were

added at the time of sampling and the sample was analysed within 2 weeks (stored at room

temperature) or 3 - 4 weeks (stored under refrigeration). These standards are insensitive to losses

by volatilization, adsorption etc. since their behaviour is virtually identical with geosmin and 2-mib.

Experiment 2 was designed to evaluate the reproducibility of newly developed analysis method

for geosmin and 2-mib. Reproducibility was determined by calculating the %RSD value. The

GSM/TCA and 2-mib/TCA were obtained on three days during three successive injections.

According to the RSD values of three days (Table 10), %RSD was 66%, 32% and 29% at 0 h, 24 h

and 96 h, respectively for geosmin analysis. For 2-mib analysis (Table 11), %RSD was 66%, 53% and

52% at 0 h, 24 h and 96 h, respectively. According to these results %RSD value for both compounds

were exceeding the acceptable limit (15%). Thus, this analysis method does not produce better

reproducibility and repeatability for geosmin and 2-mib.

During the study of Wee, et al., (2015) %RSD of geosmin determination at 1 ppt showed variable

RSD values of 1.7 – 6.9% for different working days. Several authors have reported different RSD

values during their studies such as RSD of, 3 – 12 % (Bao, et al., 1999) and RSD of 5.3 – 6.8 %

(Watson, et al., 2000).

94

When considering the area of each compound during the injections, Figure 50 illustrates the area

of geosmin, 2-mib and TCA. This also clearly depicts that the behaviour of TCA does not

corresponding with the variation of geosmin and 2-mib during the injections.

Different types of internal standards were used by different authors for their experiments to

analyse geosmin and 2-mib, such as deuterated internal standards (Sung, et al., 2005; Palmentier

& Taguchi, 2001; Yuan, et al., 2013), fluorobenzene (Shin & Ahn, 2004; Wu & Duirk, 2013), TCA

(Pestana, et al., 2014) and 2-isobutyl-3-methoxypyrazine (IBMP) (Ding, et al., 2014). However,

during this experiment TCA was used as an internal standard due to its cost effectiveness.

Figure 50: The area of geosmin, 2-mib and TCA of three successive injections during 0 h, 24 h and 96 h (spiked with1 µg L-1 geosmin, 2-mib and TCA)

A; First injection, B; Second injection, C; Third injection

0

50

100

150

200

250

300

0 50 100

Are

a va

lues

Tho

usa

nd

s

Time

Area of TCA

Area of geosmin

Area of 2-mib

A

0

20

40

60

80

100

120

0 50 100

Are

a va

lues

Tho

usa

nd

s

Time

Area of TCA

Area of geosmin

Area of 2-mib

B

0

5

10

15

20

25

30

35

40

0 20 40 60 80 100

Are

a va

lues

Tho

usa

nd

s

Time

Area of TCA

Area of geosmin

Area of 2-mib

C

95

5.2.6. Limit of detection (LOD)

LOD is the lowest quantity of a substance that can be distinguished from the absence of that

substance (a blank value) with a stated confidence level (generally 99%) (Long, et al., 1983).

Concentration levels of geosmin and 2-mib at 10 ng L-1 can be detected under optimal conditions

of GC-MS (Lloyd, et al., 1998). During this experiment according to the results (Figure 29), geosmin

can be detected at 0.01 µg L-1 (10 ng L-1) and 2-mib (Figure 30) can be detected only up to 0.05 µg

L-1 (50 ng L-1). Parinet, et al., (2010) stated that 10 ng L−1 limit of detection was sufficiently low for

water quality monitoring. Although the repeatability and reproducibility was not assured, this

method was used as the optimized method for geosmin and 2-mib analysis for further

experiments.

5.3. Section C: Experiments done to determine the suppression of geosmin and 2-

mib by bioflocs

This section explained the results of experiments done to determine the suppression of geosmin

and 2-mib by bioflocs.

5.3.1. Experiment 1: Identification of naturally occurring geosmin and 2-mib in experimental

water and carryover effect of GC-MS

Presence of taste and odour compounds, mainly geosmin and 2-mib, in drinking water/ fresh water

is well described in literature (Cook, et al., 2001; Juttner & Watson, 2007; Suffet, et al., 1999; Lin,

et al., 2003). There are some reports available in the literature discussing that marine

Actinobacteria produce geosmin and 2-mib in aquaculture (Klausen, et al., 2005) and even in RAS

system (Guttman & van Rijn, 2008). Therefore, this experiment was done to ensure the absence of

geosmin and 2-mib in the sea water which was used for the experiments. According to the results

(Table 12), natural sea water sample (SW_1) did not contain geosmin and 2-mib down to the

lowest detection limit.

Secondly, the sample carryover effect between runs of SPME GC-MS was studied during this

experiment. Carryover is the appearance of an analyte in a run when a blank containing no analyte

is injected (Prosen & Zupančič-Kralj, 1999). During this experiment, possible carryover effect was

96

monitored by injecting a blank sea water sample immediately after injecting a sample with high

concentration of geosmin and 2-mib. The most common type of carryover results from a tiny

residue of sample that is left over from a previous injection (Lord & Pawliszyn, 2000). As shown in

Table 12 blank sea water sample (SW_2) injected after the sample spiked with geosmin and 2-mib

(SW+), did not contain any detectable residue of geosmin and 2-mib.

5.3.2. Experiment 2: Bioflocs spiked with geosmin and 2-mib with aeration

This experiment was designed to identify the effect of bioflocs on geosmin and 2-mib. Surprisingly,

the geosmin and 2-mib spiked to the experimental setup disappeared. According to the analysis, 1

µg L-1 geosmin and 2-mib became undetectable from the experimental setup less than 48 h.

Therefore, subsequent experiments were designed to identify the reasons for the volatilization of

geosmin and 2-mib. The same incident was reported by several authors. In an experiment done by

Hsieh, et al., (2010) to identify the biodegradation of geosmin and 2-mib with slow sand filters,

they observed that the geosmin concentration in the negative control was reduced by 47%, even

when there was no observable colony growth. They suggested that, since the negative control was

aerated during the experiment, the reduction may be attributed to volatilization loss for geosmin.

During studies about the determination of system losses of geosmin and 2-mib in a bench-scale

filtration apparatus by Elhadi, et al., (2004), both geosmin and 2-mib losses were 42% and 30%,

respectively, based on target concentrations of 200 ng L-1.

Therefore, the aeration may be the major reason for depletion of these compounds from the

experimental setup. This was investigated further in the experiments without adding bioflocs.

Another important remark is that the same concentrations of geosmin and 2-mib were not

achieved in the treatments (SW+, BF+ and SBF+) although they were spiked with the same

concentrations. At 0 h, 1 µg L-1 geosmin and 1 µg L-1 2-mib were spiked to SW+, BF+ and SBF+.

However, analysis of the GC-MS results showed different values for GSM/TCA and 2-mib/TCA.

This difference at the starting point of the experiment was suspected to be due to the pipetting

error or due to unknown reason. During the studies of Schram, et al., (2018), they also observed

that most geosmin was lost from the system between t = 0 and t = 1.8 h. During this period, the

geosmin concentration in the water showed a strong decline which did not result in a

corresponding increase in the geosmin concentration in the fish. They stated that the strong initial

97

decline of the geosmin concentration in the water cannot be attributed to uptake in the fish and

other, unknown geosmin sinks also seem to play a role. Therefore, they excluded the observed

geosmin concentrations in water at t = 0 and used the observed levels at t = 1.8 h as the initial

geosmin concentrations in water.

5.3.3. Experiment 3: Effect of aeration for geosmin and 2-mib using different aeration

techniques

Considering the results obtained from experiment 2, this experiment was testing the effect of

aeration for evaporation of geosmin and 2-mib. Air stone (AS), air pipette (AP) and no aeration

(NA) was used as different treatments. The concentration of geosmin and 2-mib was increased ×50

times (50 µg L-1 geosmin and 2-mib) to clearly identify the effect of aeration.

After 24 h of the experiment (Table 14), the significantly highest GSM/TCA and 2-mib/TCA values

were obtained from NA. There was no significant difference between AP and AS treatments. After

24 h, 48% in NA (reference to 0 h value), 92.7% in AP (reference to 2 h value), and 100% in AS of

geosmin was loss when compared with 0 h. Also for loss of 2-mib, 58% in NA (reference to 0 h

value), 93.32% (reference to 2 h value) in AP and 100% in AS was evidenced. These results clearly

suggested that aeration is the major factor to volatilize geosmin and 2-mib from the experimental

setup.

These results are in accordance with Bellu, et al., (2008) who suggested that an increase in aeration

rate has an effect on the volatilization of geosmin. This effect is most pronounced when comparing

the aeration rates of 30 mL min-1 and 150 mL min-1 flow rate (Figure 51). Geosmin losses were 34%

and 67% for the aeration rate of 30 mL min-1 and 150 mL min-1, respectively, after 25 minutes.

98

Figure 51: Influence of aeration rate of 150 mL min-1, 120 mL min-1, 60 mL min-1, 30 mL min-1 on geosmin in dark

condition (Bellu, et al., 2008)

Schrader & Blevins, (1993) observed that a bubbled air aeration method enhanced loss of geosmin

by volatilization. Rescorla, (2012) observed a loss of 18% and 5% of geosmin concentrations over

a two hour period which was attributed to volatilization. Therefore, to determine volatilization

rates, Rescorla, (2012) conducted three experiments and the results are shown in Figure 52.

Accordingly, the rate of volatilization is dependent on concentration. The initial concentration in

the experiments ranged from 56 to 78 ng L-1. At higher geosmin concentrations, the increased mass

transfer driving force would result in a higher rate of volatilization. Similarly, the rate of

volatilization would be lower at lower geosmin concentrations.

Figure 52: Volatilization rates of three different concentrations of geosmin (Rescorla, 2012) Experiment #1: Concentration 56.3 ng L-1, Temperature 21.5 °C; Experiment #2: Concentration 77.8 ng L-1,

Temperature 21.0 °C; Experiment #3: Concentration 73.4 ng L-1, Temperature 22.0 °C,

99

Another remark of this experiment concerns unreasonable values for some samples during the

SPME GC-MS. In Table 14 and Table 15, GSM/TCA and 2-mib/TCA value for AP treatment at 0 h

was relatively lower than other two treatments although they were spiked with the same

concentration of geosmin and 2-mib. This may be due to an analysis error.

Therefore, the observations of this experiment were in accordance with other studies which

concluded aeration will be one of the major factors for volatilization of geosmin and 2-mib from

the experimental setup. Next experiment was designed to ensure that aeration was the major

factor for volatilization of geosmin and 2-mib, but not the salinity.


experimental bottles

From the previous experiment it was concluded that the aeration is one of the major factors which

affect the volatilization of geosmin and 2-mib from the experimental setup. Hence, this experiment

was designed to determine the effect of salinity and aeration for the volatilization of geosmin and

2-mib.

For GSM/TCA results at the beginning of the experiment (0 h) (Figure 32), FW a- has a significantly

lower value than the other three treatments, even though they were spiked with same

concentration. At 2 h, FW a- treatment shows a higher value of GSM/TCA indicating that this may

be due to an analysing error. After 24 h of the experiment, results indicated higher GSM/TCA

(Figure 32) and 2-mib/TCA (Figure 33) values for SW a- and FW a- than for FW a+ and SW a+. For

both ratios, SW a- had the highest value. This clearly indicates that the aerated sea water and

aerated freshwater experimental setups have a tendency to volatilize geosmin and 2-mib more

than their non-aerated counterparts. Therefore, only aeration can be considered as a major factor

which facilitates volatilization of geosmin and 2-mib.

Nam-Koong, et al., (2016) used aquaculture fresh water from an experimental RAS with a salinity

of 0.2% and aquaculture sea water from an experimental RAS stocked with a salinity of 26%. Each

type of water sample was spiked with geosmin (5 μg L-1) and 2-mib (100 ng L-1). An Approximately

equal reduction level of geosmin and 2-mib during ultrasound treatment in different water

100

matrices (Figure 53) demonstrated no significant differences between the off-flavour degradation

in sea and fresh water samples.

Figure 53: Mean reduction [%] ± confidence interval (p < 0.05) of 2-MIB and GSM in tap water, RAS fresh water

and RAS sea water.

According to this experiment, FW a+ has lost 95.7% of geosmin and 95.23% of 2-mib. SW a+

treatment has lost 98.9% geosmin and 98.8% 2-mib within 24 h while FW a- and SW a- did not

evidenced any loss. In view of these results, a next experiment was designed without aeration.

5.3.5. Experiment 5: Effect of bioflocs on geosmin and 2-mib without aeration

This experiment was then designed to check the suppression of geosmin and 2-mib using bioflocs.

The setup was not aerated during the experiment. According to the results, at 0 h (Figure 34),

GSM/TCA value of BF+, SBF+ and SW+ were not significantly different. BF treatment had the lowest

value for GSM/TCA. This indicates that when aeration was not supplied, the spiked concentrations

at 0 h of the experiment did not deviate from each other. The 2-mib/TCA value at 0 h (Figure 35),

for SW+ was significantly lower than of BF+ and SBF+. This may be due to an experimental error.

After 24 h of the experiment, the highest GSM/TCA and 2-mib/TCA was obtained for BF+

treatment. Values of SBF+ treatment and SW+ treatment were significantly lower than of BF+.

These higher GSM/TCA and 2-mib/TCA values in BF+ treatments may be due to the death of

organisms in bioflocs, which can produce and store geosmin and 2-mib. In the experimental setups,

all the suspended bioflocs settled down and piled up after half an hour, due to no aeration causing

101

low oxygen concentrations in the bottom of the bottles. Furthermore, the absence of aeration in

biofloc systems can lead to a reduction of dissolved oxygen to lethal levels after approximately

30 min (Vinatea, et al., 2010). This induces the lysis of cells and release of geosmin and 2-mib

produced within bacterial cells (Wu & Juttner, 1988; Bafford, et al., 1993; Rosen, et al., 1992). This

may be the reason for BF+ giving significantly higher values than other treatments.

Therefore, during the experiment it was concluded that the experimental setup for geosmin and

2-mib should be tightly covered (no air exchange) to reduce volatilization. Secondly, the bioflocs

should be kept alive in suspension during the experimental period to observe the effect of bioflocs

on geosmin and 2-mib.

Therefore, a next experiment was designed to observe the behaviour of bioflocs in sealed vials.

The effectiveness of a rotator and a shaker was tested to keep the flocs in suspension without

dying.


placed on a rotator versus shaker

The water quality parameters of bioflocs were studied in sealed vials during this experiment and

the usefulness of a rotator and a shaker were tested to keep bioflocs in suspension throughout the

experimental period.

According to the results (Figure 36, 37, 38 and 39), temperature, DO%, pH, ammonium and nitrite

concentrations of the bioflocs were similar in the vials stored on the rotator and shaker. Based on

their results, it was concluded that for next experiments either rotator or shaker can be used to

keep the bioflocs in suspension.

The lowest DO% of bioflocs was 48.5% on the 5th day of the experiment. Microorganisms consume

dissolved oxygen to maintain metabolic activities (Avnimelech, 2009). In this sense, the aeration

system must be sufficient to supply dissolved oxygen to the target species and microorganisms in

bioflocs (Van Wyk, et al., 1999; De Schryver, et al., 2008). However, in some studies, the DO

concentrations were maintained below 3 mg L−1 (36.3%), especially in conditions of high

concentrations of suspended solids (Ray, et al., 2010; Gaona, et al., 2011; Krummenauer, et al.,

2011). Therefore, this concentration was assumed to be sufficient for the bacteria living in the

bioflocs.

102

The lowest pH of bioflocs was 6.51 at day 5 of the experiment. The pH was gradually reaching acidic

conditions due to the low oxygen concentration in the vials. Ammonium concentration of bioflocs

reached up to 5 mg L-1 upon day 3, which is the highest detection limit of the test kit. During the

4th and 5th day, ammonium concentrations can be higher than the indicated value. Nitrite

concentration was 0.3 mg L-1 during all 5 days of experimental period. Ammonia is produced as a

major end product of protein catabolism (Anthonisen, et al., 1976).

According to the results, both rotator and shaker can be used for the next experiments to keep

bioflocs in suspension and live.

5.3.7. Experiment 7: Effect of bioflocs for suppression of geosmin and 2-mib without aeration

on a shaker

This experiment was designed to identify the effect of bioflocs on geosmin and 2-mib using BF,

BF+, SW+ as treatments spiked with 50 µg L-1 of geosmin and 50 µg L-1 of 2-mib.

At 0 h, SW+ and BF+ treatment did not significantly differ from each other for GSM/TCA (Figure

40) and 2-mib/TCA (Figure 41), but still they did not indicate the same values although the same

concentrations were spiked. After 72 h of the experiment, both compounds had disappeared from

the experimental setup. This may be due to the use of parafilm to cover the Erlenmeyer flasks

during the experiment. Parafilm is a plastic paraffin film. A number of bio-filtration studies for the

removal of geosmin have been reported with usage of alternative media such as glass beads,

porous ceramic and plastic media (Namkung & Rittmann, 1987; Egashira, et al., 1992; Hrudey,

et al., 1995; Terauchi, et al., 1995; Sugiura, et al., 2003). Therefore, geosmin and 2-mib may be

absorbed by or volatilized through the parafilm. In addition, frequent sampling through piercing of

parafilm allows more volatilization.

During the study of Sklenar & Horne (1999), on the effect of the cyanobacterial metabolite geosmin

on the growth of a green alga, they used parafilm to cover the Erlenmeyer flasks with a

concentration of 480 ng L-1. However, they did not report any loss of this compound during their

experiment.

103


This experiment was finally designed considering all the observations and results from the previous

experiments. Four types of treatments were used, BF, BF+, SBF+ and SW+ spiked with 50 µg L-1 of

geosmin and 50 µg L-1 of 2-mib. Airtight vials were used with enough headspace to allow air

exchange during the experimental period. In addition, to ensure similar concentrations at 0 h of

the experiment, a stock solution was made and distributed in to vials. A rotator was used to keep

the bioflocs in suspension.

The analysis results at 0 h, for GSM/TCA (Figure 42) and 2-mib/TCA (Figure 43) in SW+, BF+, and

SBF+ did not show the same value although they result from the same stock solution. However,

these values were not significantly different form each other. This can be explained only by

unknown geosmin and 2-mib sinks as explained by Schram, et al., (2017) or may be due to the

analysis error.

At the end of the experiment (240 h), the highest GSM/TCA and 2-mib/TCA values were obtained

for SW+. The BF+ and SBF+ treatments were significantly lower than SW+ for both geosmin and 2-

mib. For GSM/TCA, SBF+ and BF+ were not significantly different, while for 2-mib/TCA, SBF+ was

significantly higher than BF+. According to this experiment it can be suggested that geosmin and

2-mib are reduced by bioflocs. The removal percentage of geosmin from the BF+ was 15.1%, for

SBF + 9.1% and for SW+ 2.5%. When considering 2-mib, BF+ reduces 56% of 2-mib, SBF+ 36.4% and

SW+ 37%. The decay of geosmin and 2-mib in the BF+ treatment may be a result of both

biodegradation and chemical/physical adsorption, while the decay of geosmin and 2-mib in the

SBF+ may be only due to chemical/physical sorption as the bacteria in the bioflocs are inactive.

Therefore, it can be concluded that the bioflocs may be able to reduce the geosmin and 2-mib

present in the water, but it can take a longer time period. This longer time may be due to the use

of a high concentration of geosmin and 2-mib during the experiment.

Based on previous studies, biodegradation is considered as a highly effective method to treat

geosmin and 2-mib in water and aquaculture systems (Ho, et al. 2012). According to an experiment

done by Ma, et al., (2016), they observed a rapid decrease in the geosmin and 2-mib concentrations

in the aqueous phase in all containers with bioflocs during the first 24 h (Figure 54). It was

estimated that only 4.58% of the geosmin and 8.49% of the 2-mib were biologically removed during

104

this period. But during this experiment, after 24 h period a considerable decline of geosmin and

2-mib was not observed in BF+ treatment. Because SBF+ and BF+ treatments were similar to each

other this period.

Figure 54: Geosmin (left) and 2-mib (right) concentrations in water spiked with these two compounds and

incubated for 2 weeks in beakers containing sterilized bioflocs, non-sterilized bioflocs, no bioflocs, no geosmin or

2-mib. Data are given as mean ± SD (Ma, et al., 2016)

Ma, et al., (2016) concluded from their study that biodegradation was less important than

chemical/physical sorption in the overall removal of geosmin and 2-mib. There was almost no

obvious biological removal observed. Therefore, the decay curves for sterilized and non-sterilized

bioflocs were very similar. Ho & Newcombe (2010) made a similar observation. They found that

only 20% of the geosmin and 2-mib was removed by biodegradation after six months of operation

in pilot and laboratory-scale activated carbon columns.

This statement is in agreement with the current study, for geosmin, which shows that SBF+ and

BF+ treatments were not significantly different after 240 h. However, for 2-mib, BF+ treatment was

significantly lower than SBF+. Since 2-mib is less hydrophobic than geosmin (Cook, et al., 2001), it

is assumed that the removal kinetics of geosmin are similar to or not superior to those of 2-mib.

Microbial degradation of geosmin and 2-mib has been demonstrated with several microorganisms

(Izaguirre, et al., 1988; Tanaka, et al., 1996; Lauderdale, et al., 2004; Guttman & van Rijn, 2012).

Geosmin and 2-mib were found to support the growth of bacteria (Guttman and van Rijn, 2012),

when present as sole carbon source at high ambient concentrations (>1 mg L-1). In the present

study, although the geosmin and 2-mib concentrations are higher than those found in the natural

105

environment (up to 2000 ng L-1) (Juttner & Watson, 2007), bioflocs was not operated under

optimum conditions. Because bioflocs were stored in closed vials throughout the experimental

period.

106

Chapter 6: General conclusions

In the present study the influence of bioflocs for the suppression of geosmin and 2-mib was

investigated. In order to analyse the geosmin and 2-mib in the laboratory, an optimized method

was developed using SPME GC-MS.

According to the experiments, 60 °C of incubation temperature and 20 min extraction time was

selected for the analysis. The salting out effect clearly increases the extraction of geosmin and 2-

mib. When considering about the amount of salt, the use of 3.125 g and 6.25 g have no significant

difference for extraction of odour compounds. Therefore using 3.125 g is more cost effective for

the commercial analysis of geosmin and 2-mib. In addition, the storage of geosmin and 2-mib was

not recommended, because the standard deviation of the analytical results is greater when the

sample storage time is longer. Therefore, when the geosmin and 2-mib samples were prepared for

analysis, an immediate analysis is recommended. Repeatability and reproducibility of the

developed method for geosmin and 2-mib analysis by SPME GC-MS was exceeding the acceptable

RSD limit (15%). This indicates further development is needed for the analysis method. Moreover,

the use of TCA as an internal standard was found to be less convenient for the accurate analysis of

geosmin and 2-mib. This newly developed method was able to detect 0.01 µg L-1 concentration of

geosmin and 0.05 µg L-1 concentration of 2-mib in the laboratory.

Water quality parameters of the bioflocs were optimum during the experimental period although

the TSS, SVI and ash values are higher than the recommended levels for the RAS. The organisms

living in the bioflocs, visual observations and water quality parameters indicate that healthy

bioflocs were used for the experiments and the natural sea water used to grow the bioflocs does

not contain geosmin or 2-mib above the detection limit.

According to the experiments with bioflocs, geosmin and 2-mib are involved in rapid volatilization

with the supply of aeration. When consider about the effect of salinity for the volatilization of

geosmin and 2-mib, sea water or fresh water does not matter, but the rate of aeration can change

the amount of geosmin and 2-mib retained in the water. When aeration was not supplied to the

bioflocs, it shows the production of geosmin and 2-mib. This may be due to the death of the

bacterial cells in bioflocs and release of these odour compounds stored in the cells. During the final

107

experiment the bioflocs spiked with geosmin and 2-mib were found to result in a decrease of 15.1%

and 56% of geosmin and 2-mib levels after 10 days. Sterile bioflocs also showed a reduction of

geosmin and 2-mib content by 9.5% and 36.4%. This observation suggests that there may be a

possibility to decrease the geosmin and 2-mib content using the bioflocs but biodegradation could

be less important than chemical/physical sorption in the removal of geosmin and 2-mib.

Additionally, a longer experimental period is required to identify the effect of bioflocs for the

removal of geosmin and 2-mib.

Finally, although biodegradation of geosmin and 2-mib using bioflocs was not assured, this

experiment proved that the bioflocs did not produce geosmin and 2-mib when they were kept in

suspension and alive.

108

Chapter 7: Recommendations for further research

The SPME GC-MS analysis method for geosmin and 2-mib in the laboratory needs to be developed

further in order to achieve better repeatability and reproducibility. During this research, TCA was

used as internal standard due to its cost effectivity. However, more suitable internal standards

such as deuterated analogues (d3-geosmin, d3-2-MIB) are recommended for the biofloc

experiments.

The centrifugation was done during these experiments to obtain water from the experimental

setup for GC-MS analysis, which may not be 100% effective to remove the bioflocs and bacteria

from the water. These remaining biological particles could be interfere with the SPME GC-MS

analysis process. In addition, filtration or freezing is not effective to separate bioflocs and bacteria

from the water due to the volatilization nature of the compounds (geosmin and 2-mib). The

experiments should be performed in completely closed systems to minimize the volatilization loss.

Aeration can be studied as a geosmin and 2-mib removal method from water but further

experiments are necessary to study the compounds’ behaviour in fish muscles.

The possible unknown geosmin and 2-mib sink at the beginning of the experiment should be

studied further, and immediate sampling is not recommended after spiking. Also, the bacteria and

other organisms living in the bioflocs used for this research were not characterized. The bacteria,

which have the ability to degrade geosmin and 2-mib in the bioflocs, has to be investigated further.

109

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135

9: Appendices

Appendix I: Water quality parameters of the biofloc reactor tank

Day Temperature

(°C)

Dissolved oxygen (mg.L-1)

Dissolved oxygen (%)

pH Salinity (PSU)

NH4+

(mg.L-1) NO2

-

(mg.L-1)

1 18.97 6.79 91.9 8.18 32.03 0.25 0.300

2 23.57 6.46 92.6 7.60 32.29 0.00 0.300

3 24.40 6.44 94.1 8.06 32.18 0.00 0.300

4 24.41 6.21 91.0 8.06 32.53 0.00 0.300

5 24.94 5.65 84.8 8.10 32.70 0.00 0.300

6 25.00 6.40 97.1 7.98 32.72 0.00 0.300

7 24.55 5.56 84.5 8.02 32.40 0.00 0.300

8 25.67 5.38 81.2 7.69 32.72 0.00 0.300

9 26.38 5.95 90.5 7.85 33.00 0.00 0.300

10 26.94 6.40 98.2 7.94 33.45 0.00 0.300

11 26.46 6.19 92.7 7.95 33.77 0.00 0.300

12 26.10 6.10 92.4 7.95 34.29 0.00 0.010

13 25.87 5.89 89.9 7.85 34.72 0.00 0.010

14 26.23 5.70 86.4 7.58 35.01 0.25 0.025

15 26.19 6.31 94.2 7.93 35.68 0.00 0.025

16 26.34 7.12 105.2 8.10 34.83 0.00 0.025

17 26.48 6.89 103.5 8.11 34.65 0.25 0.025

18 26.54 6.58 100.3 8.27 34.69 1.50 0.025

19 25.77 6.60 98.4 8.15 33.09 0.25 0.025

20 21.65 7.13 105.9 8.20 34.67 0.25 0.025

21 25.78 7.24 105.7 8.15 33.17 0.00 0.010

22 26.55 6.71 101.0 8.12 33.19 0.00 0.100

23 26.69 6.60 101.5 8.26 35.45 0.25 0.025

24 26.17 6.71 102.5 8.24 34.42 1.50 0.050

25 25.24 6.25 95.4 8.34 36.36 1.50 0.025

26 25.75 6.38 97.2 8.39 37.24 1.50 0.050

27 25.58 6.64 97.7 8.37 37.09 1.50 0.050

28 26.24 6.66 102.6 8.25 35.52 0.00 0.025

29 26.09 6.30 97.2 8.12 36.08 0.00 0.025

30 25.75 6.71 100.4 8.18 33.75 0.00 0.025

31 25.43 5.73 85.3 8.18 34.28 1.50 0.025

32 25.21 5.57 82.0 8.07 32.17 0.25 0.050

33 25.36 5.41 79.3 8.00 32.51 0.25 0.300

136

34 25.34 6.72 100.2 8.22 33.49 0.00 0.300

35 25.20 6.66 95.8 7.98 30.05 0.25 0.300

36 25.17 6.45 93.3 8.01 30.54 0.00 0.300

37 25.24 5.90 84.4 8.05 31.10 0.00 0.300

38 25.01 5.46 75.8 7.95 32.85 0.25 0.300

39 24.48 5.70 80.0 8.12 32.39 0.25 0.300

40 24.68 7.32 104.5 8.09 32.94 0.25 0.300

41 24.86 5.72 82.6 7.95 33.75 0.25 0.300

42 24.03 6.76 95.8 7.82 32.40 0.25 0.300

43 24.12 7.13 99.8 8.03 32.24 0.25 0.300

44 23.97 7.06 98.6 8.10 30.23 0.25 0.300

45 24.50 6.83 97.6 8.02 30.31 0.25 0.300

46 24.22 6.13 85.7 7.87 31.78 0.25 0.300

47 24.63 6.63 96.5 8.03 32.39 0.25 0.300

48 24.13 7.09 100.9 8.05 31.38 0.25 0.300

49 24.03 7.33 101.0 8.12 30.71 0.25 0.300

50 23.88 5.77 82.1 7.50 29.45 0.25 0.300

51 24.16 6.27 88.9 8.10 30.58 0.25 0.300

Appendix II: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib with different incubation temperatures (40 °C, 60 °C, 80 °C)

Temperature (°C)

Replicate GSM/TCA 2-mib/TCA Area of

TCA Area of

geosmin Area of 2-mib

40 1 1.44 0.77 69542 100185 53812 40 2 1.55 0.78 62139 96087 48520 40 3 1.49 0.74 55939 83392 41635

60 1 1.76 0.80 82539 145480 65940 60 2 2.04 0.96 76924 156865 73907 60 3 1.69 0.77 78367 132746 60143

80 1 2.15 0.66 79511 170621 52646 80 2 1.83 0.50 90134 164627 44982

80 3 1.75 0.50 86682 151529 42992

137

Appendix III: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib with different extraction times (5 min, 10 min, 15 min, 20 min, 40 min)



TCA Area of


5 1 2.38 1.03 46553 110608 47739 5 2 1.57 0.60 63329 99529 38161 5 3 1.46 0.59 68846 100455 40395

10 1 1.57 0.65 90906 142511 59443 10 2 1.53 0.70 105234 160902 73936

10 3 1.51 0.64 103054 155541 66262

15 1 1.58 0.74 116335 183317 85575 15 2 1.64 0.72 99025 162021 71089 15 3 1.57 0.63 97528 153409 61153

20 1 1.67 0.77 130821 218825 100285 20 2 1.74 0.91 149543 259855 135767 20 3 1.74 0.75 117464 204307 87774

40 1 1.79 0.69 130741 234053 90001 40 2 1.92 0.77 149704 287309 114730 40 3 1.82 0.79 136584 248751 107629

Appendix IV: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib with different salt amounts (0 g, 3.125 g, and 6.25 g)

Salt (g)


TCA Area of

geosmin Area of 2-

mib

0 1 0.69 0.35 13981 9636 4857 0 2 0.59 0.28 17068 10064 4701 0 3 0.66 0.28 15088 9957 4279 0 4 0.51 0.27 14324 7277 3919

3.125 1 2.65 1.23 27040 71675 33206 3.125 2 1.93 1.08 60831 117181 65827 3.125 3 1.92 1.13 64828 124418 73542 3.125 4 1.99 1.10 49234 98056 54398

6.25 1 2.52 1.29 55795 140413 72052 6.25 2 2.48 1.30 63160 156860 82390 6.25 3 2.60 1.14 36327 94332 41367 6.25 4 2.51 1.31 64896 162609 85158

138

Appendix V: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib with different storage times (0 h, 48 h, 72 h and 96 h)

Storage Time (h)

Replicate

GSM/ TCA

2-mib/ TCA

Area of TCA

Area of geosmin

Area of 2-mib

0 1 1.24 0.62 44635 55188 27831 0 1 1.34 0.60 32342 43424 19364 0 1 1.26 0.57 34833 44060 19906 0 1 1.32 0.59 33075 43718 19630 0 1 1.33 0.59 33007 44045 19370

48 2 1.50 0.74 39598 59542 29361

48 2 1.49 0.76 50783 75432 38818 48 2 1.67 0.97 81417 136083 78906 48 2 1.46 0.66 31362 45854 20677 48 2 1.37 0.70 50192 68928 35232

72 3 1.88 0.95 27874 52402 26491 72 3 1.98 1.18 35766 70801 42045 72 3 1.92 0.96 30481 58445 29331 72 3 1.92 1.00 26637 51158 26577 72 3 1.80 1.01 36612 65959 36910

96 1 1.27 0.49 79297 100329 38936 96 2 1.05 0.45 76806 80787 34501

96 3 1.18 0.45 89821 106021 40634 96 4 1.16 0.54 105124 121632 56754 96 5 0.88 0.43 96204 84659 41171

Appendix VI: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib during the

analysis for repeatability of the GC-MS method.

Sample Injection Time GSM/TCA 2-mib/TCA Area of

TCA Area of


1 1 1.34 0.79 88569 118310 70293 2 1 1.45 0.75 55060 79861 41488 3 1 1.28 0.71 75817 97092 53645

1 2 1.88 1.11 24966 46916 27824

2 2 1.68 1.00 31605 53164 31535 3 2 1.94 1.00 19305 37520 19401

1 3 3.09 2.09 10740 33228 22466 2 3 3.47 1.93 7093 24598 13709 3 3 3.01 1.67 8688 26128 14500

139

Appendix VII: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib during the analysis for reproducibility of the GC-MS method.

Time (h)

injection Replicate GSM/TCA

2-mib/ TCA

Area of TCA

Area of geosmin

Area of 2-mib

0 1 1 1.88 0.76 111269 209299 84280 0 1 2 1.85 0.89 126237 233626 112027 0 1 3 1.82 0.70 95097 173492 66809 0 1 4 1.74 0.77 123780 215277 95172 0 1 5 1.74 0.76 125966 218804 95305

0 2 1 2.18 1.28 44698 97618 57313 0 2 2 2.50 1.67 36146 90352 60234 0 2 3 2.48 1.47 48278 119611 71050 0 2 4 2.25 1.38 37543 84577 51622 0 2 5 2.57 1.65 36459 93656 60169

0 3 1 4.21 3.26 8005 33714 26085 0 3 2 3.64 3.12 6914 25141 21551 0 3 3 4.04 2.51 10720 43269 26878 0 3 4 4.09 3.08 8857 36238 27257 0 3 5 4.21 3.46 11430 48143 39557

24 1 1 1.73 0.88 148292 257236 130510

24 1 2 1.81 1.26 226973 410084 285016 24 1 3 1.77 0.75 108097 191736 81404 24 1 4 1.74 0.83 133282 232170 110491 24 1 5 1.70 0.76 120725 204790 91752

24 2 1 2.53 1.48 25205 63700 37234 24 2 2 2.12 1.16 32607 69160 37769 24 2 3 2.46 1.49 32992 81271 49094 24 2 4 2.46 1.76 97776 240638 172324 24 2 5 2.42 1.57 30104 72922 47307

24 3 1 4.29 3.66 6213 26644 22743 24 3 2 2.83 2.17 14724 41649 31999

24 3 3 3.26 2.80 10023 32722 28057 24 3 4 3.01 2.25 6976 20965 15682 24 3 5 3.32 2.31 7868 26158 18174

96 1 1 1.27 0.49 79297 100329 38936 96 1 2 1.05 0.45 76806 80787 34501 96 1 3 1.18 0.45 89821 106021 40634 96 1 4 1.16 0.54 105124 121632 56754 96 1 5 0.88 0.43 96204 84659 41171

96 2 1 1.46 0.92 31024 45356 28410 96 2 2 1.80 1.06 18686 33654 19760

140

96 2 3 1.83 1.12 27479 50388 30894 96 2 4 1.72 1.09 25543 43934 27742 96 2 5 1.93 1.05 20024 38732 21072

96 3 1 1.80 1.51 8600 15486 13000 96 3 2 2.32 1.79 6557 15240 11769 96 3 3 1.87 1.36 7831 14630 10675 96 3 4 2.02 1.56 6388 12889 9938 96 3 5 1.88 1.29 8030 15135 10361

Appendix VIII: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib during the analysis for limit of detection of the GC-MS method

Concentration

(µg L-1) Replicate GSM/TCA 2-mib/TCA

Area of TCA

Area of geosmin

Area of 2-mib

0.100 1 0.11 0.05 57413 6171 3031 0.100 2 0.10 0.05 80221 8236 4313

0.100 3 0.10 0.05 6078 2830 0.103

0.050 1 0.05 0.03 79235 3869 2139 0.050 2 0.05 0.03 79051 4325 2546 0.050 3 0.06 0.03 2663 1194 0.057

0.010 1 0.03 0.00 71934 2194 0

0.010 2 0.01 0.00 64220 751 0 0.010 3 0.01 0.00 728 0 0.010

0.001 1 0.00 0.00 63874 0 0 0.001 2 0.00 0.00 63846 0 0 0.001 3 0.00 0.00 0 0 0

Appendix IX: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib during the analysis for NA, AS and AP treatments (0 h, 2 h, 6 h, 24 h)

Time (h)

Aeration


TCA Area of


0 NA 1 34.69 33.24 53783 1865507 1787713

0 AS 1 30.09 28.67 56106 1688157 1608511

0 AP 1 5.64 7.39 53920 304102 398436

2 NA 1 0.95 1.61 84281 80176 135827 2 NA 2 5.14 5.57 52624 270449 292933 2 NA 3 11.39 10.36 63700 725816 660185

2 AS 1 0.29 0.60 54713 15800 32833 2 AS 2 1.13 1.89 17149 19432 32443 2 AS 3 1.20 1.92 53881 64733 103640

2 AP 1 35.54 28.14 22226 789904 625475

141

2 AP 2 23.36 13.43 54551 1274197 732698 2 AP 3 27.19 19.06 63073 1714949 1201872

6 NA 1 30.51 22.39 56181 1714071 1257905 6 NA 2 8.94 9.25 50902 455159 470741 6 NA 3 16.05 10.63 57096 916148 606990

6 AS 1 0.00 0.00 68209 0 0 6 AS 2 0.04 0.00 58783 2133 0 6 AS 3 0.00 0.00 57779 0 0

6 AP 1 17.53 10.80 60426 1059451 652664 6 AP 2 17.86 14.73 98485 1759165 1450317

6 AP 3 19.44 12.99 54278 1055329 705127

24 NA 1 24.12 17.10 69868 1685521 1194423 24 NA 2 15.17 13.84 70029 1062162 969362 24 NA 3 15.19 11.41 69468 1054968 792785

24 AS 1 0.00 0.00 82156 0 0 24 AS 2 0.00 0.00 90143 0 0 24 AS 3 0.00 0.00 66283 0 0

24 AP 1 1.64 0.37 73205 119750 27220 24 AP 2 2.41 1.93 83309 201173 161172 24 AP 3 2.26 1.76 54185 122324 95267

Appendix X: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib during the analysis for FW a+, FW a-, SW a+ and SW a- treatments (0 h, 2 h, 6 h, 24 h)

Time (h)

Treatment Replica

te GSM/TCA

2-mib/ TCA

Area of TCA

Area of geosmin

Area of 2-mib

0 FW a- 1 19.16 22.79 119520 2290339 2724351 0 FW a- 2 18.48 17.31 81219 1500923 1406118 0 FW a- 3 19.05 16.79 74028 1410511 1242951

0 FW a+ 1 23.97 21.95 147161 3527379 3229482 0 FW a+ 2 24.13 16.36 88944 2145986 1455018 0 FW a+ 3 25.87 14.39 77716 2010476 1118479

0 SW a- 1 25.56 24.45 132776 3394136 3246451 0 SW a- 2 28.24 20.94 77104 2177161 1614528 0 SW a- 3 23.69 17.68 70460 1669424 1245434

0 SW a+ 1 25.02 0.00 196017 4904852 0 0 SW a+ 2 26.14 22.68 101307 2648392 2298072 0 SW a+ 3 28.35 27.32 133026 3771505 3633946

2 FW a- 1 21.51 19.29 80247 1725785 1547671 2 FW a- 2 22.01 23.57 100658 2215359 2372626 2 FW a- 3 19.95 25.64 130479 2603462 3345875

2 FW a+ 1 21.66 14.94 90982 1970291 1359047

142

2 FW a+ 2 24.55 12.87 67286 1651610 866097 2 FW a+ 3 21.48 14.23 98996 2126409 1408383

2 SW a- 1 31.01 20.22 76153 2361749 1539446 2 SW a- 2 27.02 23.16 102011 2756674 2362926 2 SW a- 3 25.36 24.81 91397 2318157 2267362

2 SW a+ 1 22.43 0.00 84149 1887105 0 2 SW a+ 2 22.97 14.84 57765 1326907 857423 2 SW a+ 3 24.07 17.36 65980 1588287 1145252

6 FW a- 1 23.12 23.58 91397 2113091 2155375 6 FW a- 2 21.75 29.63 144042 3132780 4267469

6 FW a- 3 19.14 15.64 80012 1531117 1251700

6 FW a+ 1 13.97 10.04 76212 1064511 764907 6 FW a+ 2 12.08 7.58 83589 1009650 633448 6 FW a+ 3 16.54 8.72 67627 1118476 589663

6 SW a- 1 26.74 19.38 72968 1950817 1414172 6 SW a- 2 31.17 21.46 78831 2457112 1692042 6 SW a- 3 27.60 22.03 105352 2907462 2320475

6 SW a+ 1 8.69 0.00 85184 740647 0 6 SW a+ 2 12.85 8.34 66925 859840 558087 6 SW a+ 3 7.41 5.12 73812 547128 377720

24 FW a- 1 21.38 17.53 77458 1656116 1357871

24 FW a- 2 19.91 16.87 75014 1493601 1265190 24 FW a- 3 18.44 19.64 84298 1554432 1655305

24 FW a+ 1 0.50 0.34 97927 48868 33464 24 FW a+ 2 0.80 0.43 81074 65041 35050 24 FW a+ 3 1.87 1.74 159418 298594 277214

24 SW a- 1 30.27 19.90 64221 1943856 1278282 24 SW a- 2 29.38 27.01 84918 2494545 2293527 24 SW a- 3 23.59 26.43 92060 2171609 2432919

24 SW a+ 1 0.20 0.00 65514 13306 0 24 SW a+ 2 0.13 0.08 76874 9983 6375 24 SW a+ 3 0.48 0.48 94356 45062 45085

143

Appendix XI: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib during the analysis for BF, BF+, SBF+ and SW+ treatments (0 h, 2 h, 6 h, 10 h, 24 h)

Time (h)

Treatment

Replicate

GSM/TCA 2-mib/TCA Area of

TCA Area of

geosmin Area of 2-

mib

0 BF 1 0.00 0.00 44960 0 0 0 BF 2 0.05 0.00 47532 2589 0 0 BF 3 0.00 0.00 0 0 0

0 BF+ 1 33.12 29.38 26753 885955 785994 0 BF+ 2 29.07 22.43 32484 944303 728591 0 BF+ 3 32.74 34.11 31377 1027144 1070176

0 SBF+ 1 29.84 24.27 26560 792462 644643 0 SBF+ 2 25.60 37.21 49817 1275550 1853890 0 SBF+ 3 28.02 22.01 31418 880285 691659

0 SW+ 1 23.49 14.03 76502 1796681 1073327 0 SW+ 2 29.56 14.17 81454 2407529 1154319 0 SW+ 3 19.30 10.36 75846 1463794 785676

2 BF 1 0.09 0.00 34107 3104 0 2 BF 2 0.11 0.05 38531 4307 2050 2 BF 3 0.24 0.00 10388 2502 0

2 BF+ 1 32.82 29.96 25456 835365 762757 2 BF+ 2 32.57 31.79 31988 1041998 1017045

2 BF+ 3 26.27 25.96 43358 1138857 1125513

2 SBF+ 1 27.60 24.67 35438 977966 874126 2 SBF+ 2 23.58 26.81 56438 1330743 1512864 2 SBF+ 3 29.60 27.53 22372 662297 615919

2 SW+ 1 20.83 14.11 78641 1637713 1109454 2 SW+ 2 24.74 9.81 78532 1942859 770666 2 SW+ 3 20.28 20.99 171989 3488770 3610213

6 BF 1 0.10 0.04 37145 3720 1366 6 BF 2 0.14 0.10 37317 5094 3570 6 BF 3 0.08 0.12 40883 3316 4762

6 BF+ 1 36.84 41.44 35453 1305948 1469126

6 BF+ 2 34.06 34.22 32519 1107755 1112852 6 BF+ 3 32.39 35.26 30927 1001814 1090387

6 SBF+ 1 33.27 30.18 39111 1301042 1180521 6 SBF+ 2 27.34 35.71 48172 1316842 1720369 6 SBF+ 3 29.65 31.44 36140 1071403 1136167

6 SW+ 1 25.97 15.74 81478 2116134 1282691 6 SW+ 2 27.94 10.26 75043 2096358 770293 6 SW+ 3 22.41 13.35 77462 1736067 1034128

10 BF 1 0.08 0.00 40410 3055 0

10 BF 2 0.09 0.00 36365 3190 0

144

10 BF 3 0.08 0.00 31087 2553 0

10 BF+ 1 23.89 22.21 35089 838105 779371 10 BF+ 2 24.80 22.40 36673 909459 821440 10 BF+ 3 23.85 22.44 30370 724259 681532

10 SBF+ 1 25.02 19.25 29578 740130 569235 10 SBF+ 2 22.84 20.66 32980 753289 681278 10 SBF+ 3 27.31 22.73 28938 790159 657781

10 SW+ 1 20.73 19.86 112115 2323944 2226507 10 SW+ 2 26.15 10.21 64135 1677275 654713 10 SW+ 3 18.21 10.05 68267 1242859 686121

24 BF 1 0.14 0.00 27630 3744 0 24 BF 2 0.13 0.07 29488 3784 2068 24 BF 3 0.15 0.00 21034 3133 0

24 BF+ 1 40.99 34.57 18596 762210 642798 24 BF+ 2 42.82 51.64 33127 1418470 1710529 24 BF+ 3 58.69 58.79 14052 824776 826096

24 SBF+ 1 28.00 24.29 36148 1012315 877942 24 SBF+ 2 28.09 32.87 35612 1000367 1170617 24 SBF+ 3 28.13 24.74 34374 967082 850557

24 SW+ 1 23.24 15.25 72650 1688640 1108053 24 SW+ 2 26.83 13.08 83176 2231743 1087922

24 SW+ 3 22.14 13.34 74351 1646121 991872

Appendix XII: The changes in water quality parameters of bioflocs stored in closed vials placed on a rotator vs shaker for five days.

Method Day Temperature (°C) DO% pH Ammonium (mg L-1) Nitrate (mg L-1)

Rotator 1 28.2 67.1 7.28 0 0.3

Rotator 2 24.1 58 6.79 1.5 0.3

Rotator 3 25.9 56.8 6.6 5 0.3

Rotator 4 26.1 52.8 6.57 5 0.3

Rotator 5 26.9 48.5 6.51 5 0.3

Shaker 1 26.9 65 7.23 0 0.3

Shaker 2 25.6 59.6 6.84 0.25 0.3

shaker 3 27.2 57.9 6.62 5 0.3

shaker 4 27.5 51.9 6.52 5 0.3

shaker 5 27.9 51.2 6.51 5 0.3

145

Appendix XIII: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib during the analysis for BF, BF+ and SW+ treatments (0 h, 24 h, 48 h, 72 h)

Time (h)

Treatment

Replicate

GSM/TCA 2-mib/

TCA Area of

TCA Area of

geosmin Area of 2-

mib

0 SW+ 1 20.74 14.94 68588 1422786 1024432 0 SW+ 2 21.50 24.53 94511 2031632 2318318 0 SW+ 3 21.40 20.23 78861 1687621 1595611

0 BF+ 1 21.48 32.45 57539 1235729 1867194 0 BF+ 2 21.33 24.79 49958 1065605 1238677 0 BF+ 3 15.31 27.71 40275 616484 1115888

0 BF 1 0.05 0.00 61651 3187 0 0 BF 2 0.07 0.00 44818 3122 0 0 BF 3 0.07 0.00 52982 3736 0

24 SW+ 1 12.13 12.65 81557 989635 1031512 24 SW+ 2 13.31 12.14 77891 1037047 945632 24 SW+ 3 13.68 11.65 68853 942009 802350

24 BF+ 1 10.12 14.91 77643 785754 1157790 24 BF+ 2 11.56 15.89 72714 840462 1155688 24 BF+ 3 7.31 12.09 51090 373250 617870

24 BF 1 0.06 0.04 70724 4302 2830 24 BF 2 0.05 0.05 77394 3634 3850

24 BF 3 0.05 0.00 54486 2722 0

48 SW+ 1 1.55 0.81 83940 130195 68305 48 SW+ 2 1.55 0.74 74432 115141 54889 48 SW+ 3 1.56 0.65 66635 103815 43312

48 BF+ 1 1.29 0.65 69403 89302 45387 48 BF+ 2 1.17 0.49 52424 61149 25656 48 BF+ 3 0.95 0.76 64455 61248 48836

48 BF 1 0.00 0.00 73588 0 0 48 BF 2 0.00 0.00 87464 0 0 48 BF 3 0.00 0.00 65136 0 0

72 SW+ 1 0.27 0.00 71415 16991 0

72 SW+ 2 0.19 0.00 62438 14218 0 72 SW+ 3 0.26 0.00 73262 18099 0

72 BF+ 1 0.36 0.00 68684 22197 0 72 BF+ 2 0.32 0.00 62277 18343 0 72 BF+ 3 0.17 0.00 57451 12405 0

72 BF 1 0.00 0.00 74799 0 0 72 BF 2 0.00 0.00 68673 0 0 72 BF 3 0.00 0.00 88942 0 0

146

Appendix XIV: The GSM/TCA, 2-mib/TCA, area of TCA, area of geosmin and area of 2-mib during the analysis for BF, BF+, SBF+ and SW+ treatments at (0 h, 24 h, 240 h)

Time (h)

Treatment

Replicate

GSM/TCA 2-mib/TCA Area of

TCA Area of

geosmin Area of 2-

mib

0 BF 1 0.00 0.00 63735 0 0 0 BF 2 0.00 0.00 51894 0 0 0 BF 3 0.00 0.00 48114 0 0

0 BF+ 1 35.66 32.98 66546 2372713 2194824 0 BF+ 2 26.85 20.65 51972 1395595 1073339

0 SBF+ 1 24.37 28.06 84473 2058792 2370371 0 SBF+ 2 26.71 27.30 58429 1560649 1595305 0 SBF+ 3 38.90 48.89 91876 3574352 4491608

0 SW+ 1 40.24 29.85 71527 2877922 2135301 0 SW+ 2 54.87 46.20 70114 3847363 3238991 0 SW+ 3 46.44 33.74 62342 2895205 2103162

24 BF 1 0.00 0.00 88752 0 0 24 BF 2 0.00 0.00 60376 0 0 24 BF 3 0.00 0.00 62865 0 0

24 BF+ 1 33.74 30.13 98153 3311964 2957290 24 BF+ 2 33.36 16.48 49397 1647891 814023

24 BF+ 3 33.36 21.62 59148 1973281 1279071

24 SBF+ 1 29.00 19.64 61772 1791246 1212955 24 SBF+ 2 30.19 24.32 66233 1999472 1610666 24 SBF+ 3 1.00 2.87 76705 76650 219787

24 SW+ 1 34.07 27.26 107014 3646275 2917370 24 SW+ 2 1.14 2.90 138208 157270 400441 24 SW+ 3 33.51 29.65 135465 4538979 4016948

240 BF 1 0.00 0.00 42621 0 0 240 BF 2 0.00 0.00 31194 0 0 240 BF 3 0.00 0.00 37102 0 0

240 BF+ 1 21.84 10.22 29669 647871 303281

240 BF+ 2 32.88 11.93 20997 690440 250487 240 BF+ 3 24.87 13.27 25473 633424 338149

240 SBF+ 1 35.53 28.29 22276 791559 630245 240 SBF+ 2 0.65 1.40 14111 9143 19772 240 SBF+ 3 43.40 23.13 14947 648664 345667

240 SW+ 1 47.61 22.57 14611 695596 329821 240 SW+ 2 41.77 23.36 20352 850157 475487 240 SW+ 3 48.63 23.22 13759 669086 319423

SUPPRESSION OF GEOSMIN AND 2-MIB PRODUCTION IN RAS BY … · 2018-11-23 · v Acknowledgements...

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Transcript of SUPPRESSION OF GEOSMIN AND 2-MIB PRODUCTION IN RAS BY … · 2018-11-23 · v Acknowledgements...