Microbial – deposit feeder aquaculture bioremediation systems

289 Pages·2017·5.21 MB·English

Microbial – deposit feeder
aquaculture bioremediation systems

A Thesis Submitted By

Georgina Robinson

For the Degree of Doctor of Philosophy

School of Marine Science and Technology
Newcastle University
February 2017

Abstract

Land-based intensive aquaculture produces large volumes of particulate organic waste
that can be upcycled into high value secondary biomass. In this research, the application of
two key principles underpinning low-cost bioremediation technologies, namely the addition
of rate limiting (i) electron acceptors (oxygen), and (ii) donors (carbon) is investigated in a
sediment-based aquaculture effluent treatment system integrating the sea cucumber,
Holothuria scabra. Growth trials of H. scabra, combined with next generation sequencing
(NGS) technologies, were used to examine the response of sea cucumbers and sediment
bacterial communities under contrasting redox regimes, describing fully oxic and redox-
stratified sediments. The oxic system resulted in high taxonomic and functional diversity of
bacteria with a range of dissimilatory metabolisms required for successful bioremediation of
aquaculture wastes; however, the final biomass of H. scabra was significantly lower than the
redox-stratified sediments (449.22 ± 14.24 g m-2 versus 626.89 ± 35.44 g m-2). Improving the
resource quality of aquaculture waste through carbon supplementation was investigated.
Increasing the carbon/nitrogen ratio from 5:1 to 20:1 with soluble starch significantly
increased the biomass production of H. scabra on redox-stratified sediments compared to
controls (1011.46 ± 75.58 g m-2 versus 702.12 ± 35.93 g m-2). A benthic flux incubation
study, combined with NGS, demonstrated that carbon supplementation did not change the
pathway of nitrogen cycling by mediating a shift from net release of ammonium to net
assimilation, as hypothesised. A final study elucidated the critical role of the sea cucumber
microbiome during aquaculture waste bioremediation, demonstrating that endogenous
bacteria are primed, at ecological and genomic levels, to respond to nitrogen - a key nutrient
limiting deposit feeder growth. Deposit feeder–microbial aquaculture bioremediation systems
have the potential to rectify current inefficiencies of nitrogen use in the aquaculture
production chain by offering a more economically and environmentally sustainable alternative
to closing the nitrogen cycle loop.

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We’ve got to learn to
Reduce, Reuse, Recycle
- Jack Johnson, “The 3 R’s”
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Acknowledgements

First and foremost, I would like to thank Gavin Johnston for providing the initial
impetus for the research at HIK Abalone Farm Pty and Bert, Louise and Roger for supporting
the project. Throughout my time at HIK, I received tremendous support from Greg and his
technical team, including Raphael, Gustav, Deon, Graham, Shorty, Sidelo, Nevado, and
Tembeko, to build and maintain my recirculation system. I still have my melted plastic plaque
from the oven and still occasionally enjoy playing underwater music to my sea cucumbers. I
owe a great deal of thanks to Matt and Nelly for providing research support, to all the SASC
volunteers and Rhodes students, especially Ann and Chris, for their assistance with
experiments.
My supervisors have been instrumental in my development and I thank them for
giving me free reign and occasionally turning a blind eye to my extracurricular activities.
Firstly, I would like to thank Selina, for believing me capable of a PhD all those years ago
when we sat on bus driving around Zanzibar. You have supported me throughout this journey
as a close friend and supervisor – thank you for looking after me, putting me up and feeding
me during my time in Newcastle. I would like to thank Matthew Slater for his supervision
during the first year of my PhD – having you on the end of skype to answer my questions
when starting my first experiment was awesome. Cliff for teaching me statistics…I have
come a long way from a girl who didn’t know how to calculate a standard error and fully
admit that I am now a bit of a data geek. Also – thanks to you – I am slowly learning to
develop a bit of OCD! Finally, to Gary who took me on part way through my PhD. You have
been instrumental in helping me to write and publish my research and have always been there
for me, thank you.
I would like to acknowledge and thank the commercial hatcheries: Indian Ocean
Trepang (formerly Madagascar Holothurie S.A.) and RIA 3 in Vietnam that supplied the
juvenile sea cucumbers for this research. Thank you to Igor Eeckhaut for your initial guidance
and input on my PhD proposal and to Jaco Chan Kit-Waye, Oliver Meraud and Olivier Avalle
for assisting with the shipment from Madagascar. I would also like to thank Duy Nguyen
Dinh Quang, Ty and Minh for their epic effort in successfully shipping live animals from
Vietnam to South Africa and Ampie Van Wyk and Michael Falls for your valuable help and
assistance with the red tape.
I would like to acknowledge several people who assisted with specific components of
the research: Margot for teaching me to use a pipette and Gwyneth for teaching me Mothur;
Andrew Free for denoising my 454 data in QIIME; Matthew Wade for patiently helping with
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R scripts and Andrew Nelson for advising on Illumina workflows. The experiment in Chapter
5 was performed jointly with Thomas MacTavish with assistance and support from Candida
Savage (Otago University, New Zealand), Trevor Probyn (DAFF, South Africa), Iain
Alexander and Bradley Eyre at Southern Cross University, Australia. The experiment in
Chapter 6 was performed at NAQUA in the Kingdom of Saudi Arabia. I am grateful to all the
staff in the sea cucumber department, particularly Peter, Rory, Dylan, Wang, Melvin, Claro,
Froilan, Risi and Monir for running the experiment and for their help during sampling.
Sincere thanks to Ismail and his staff at CAS for sample analysis and to Shuaib and his team
in microbiology, particularly Peter, for helping with the DNA extraction and sample
shipment. Finally, I would like to thank Steve Rushton and Will Reid at Newcastle University
for their superb assistance with the modelling of the sea cucumber microbiome.
I gratefully acknowledge the financial support from the Biotechnology and Biological
Research Council (BBSRC) who funded my Industrial CASE studentship (Grant Code
BB/J01141X/1), the industrial partner in South Africa, HIK Abalone Farm Pty, and the
Technology and Human Resources Program of the National Research Foundation, South
Africa (grant number TP2011070800007).
Finally, and most heartfelt, to my family, thank you for all your support during the
four years that I have lived overseas - I am very fortunate to have a loving home to return to
every year.
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Table of contents
Abstract ........................................................................................................................................ i
Acknowledgements ................................................................................................................... iii
Table of contents ........................................................................................................................ v
List of figures .......................................................................................................................... xiii
List of tables ............................................................................................................................ xvi
Chapter 1. General Introduction ................................................................................................. 1
1.1 Aquaculture ...................................................................................................................... 1
1.1.1 Current trends in global aquaculture production ....................................................... 1
1.1.2 Marine land-based aquaculture .................................................................................. 2
1.1.3 Treatment of dissolved inorganic nitrogen ................................................................ 3
1.1.4 Treatment of particulate organic wastes .................................................................... 4
1.1.5 Sustainability issues in land-based intensive aquaculture ......................................... 5
1.1.6 Ecologically-based systems ....................................................................................... 6
1.1.7 Technological developments ..................................................................................... 6
1.1.8 Particulate organic waste – an increasing problem ................................................... 7
1.2 Integration of deposit feeding sea cucumbers into land-based aquaculture systems ....... 8
1.2.1 Use of particulate organic matter for the production of secondary organisms .......... 8
1.2.2 Deposit feeding sea cucumbers as candidate species for aquaculture bioremediation
............................................................................................................................................ 8
1.2.3 Holothuria scabra – a candidate species for aquaculture bioremediation ................. 9
1.3 Marine sediments ............................................................................................................ 12
1.3.1 Biogeochemical cycling of carbon and nitrogen ..................................................... 12
1.3.2 Deposit feeding sea cucumbers as components of the benthic recycling system .... 14
1.3.3 Food resources and limitation of deposit feeder growth ......................................... 15
1.3.4 Trophic transfer efficiencies in benthic food webs ................................................. 16
1.3.5 Impact of organic loading on marine sediments ...................................................... 16
1.4 Application of bioremediation technologies ................................................................... 18
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1.4.1 Factors controlling microbial growth ...................................................................... 18
1.4.2 Biostimulation ......................................................................................................... 20
1.4.3 Combining integrated multi-trophic aquaculture, microbial-based systems and
bioremediation technologies ............................................................................................ 20
1.5 Outline of thesis ............................................................................................................. 21
Chapter 2. Determination of key physico-chemical parameters in the design of sediment-
based aquaculture bioremediation systems .............................................................................. 23
2.1 Introduction .................................................................................................................... 23
2.2 Materials and methods ................................................................................................... 24
2.2.1 Study site ................................................................................................................. 24
2.2.2 Experimental animals .............................................................................................. 25
2.2.3 Experimental design ................................................................................................ 25
2.2.4 Experimental system and rearing conditions .......................................................... 25
2.2.5 Photo-identification and growth of Holothuria scabra ............................................ 28
2.2.6 Water quality ........................................................................................................... 29
2.2.7 Sediment characteristics .......................................................................................... 29
2.2.8 Behavioural observations ........................................................................................ 30
2.2.9 Proximate composition analysis ............................................................................. 31
2.2.10 Statistical analyses ................................................................................................ 31
2.3 Results ............................................................................................................................ 32
2.3.1 Water quality ........................................................................................................... 32
2.3.2 Sediment characteristics .......................................................................................... 32
2.3.3 Growth and survival ................................................................................................ 33
2.3.4 Behaviour ................................................................................................................ 35
2.3.5 Proximate composition ........................................................................................... 36
2.4 Discussion ...................................................................................................................... 40
2.5 Conclusion ..................................................................................................................... 43
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Chapter 3. Taxonomic and functional profiling of sediment microbial communities under
contrasting redox regimes ......................................................................................................... 45
3.1 Introduction .................................................................................................................... 45
3.2 Materials and methods .................................................................................................... 46
3.2.1 Study site ................................................................................................................. 46
3.2.2 Experimental animals .............................................................................................. 46
3.2.3 Experimental design ................................................................................................ 46
3.2.4 Experimental system and rearing conditions ........................................................... 47
3.2.5 Growth of Holothuria scabra ................................................................................... 47
3.2.6 Water quality, sediment quality, and environmental variables ............................... 47
3.2.7 Deoxyribonucleic acid extraction ............................................................................ 48
3.2.8 Polymerase chain reaction amplification of the variable region 4-5 of the 16S
ribosomal ribonucleic acid gene ....................................................................................... 49
3.2.9 Pyrosequencing and sequence analysis ................................................................... 49
3.2.10 Statistical and bioinformatics analysis .................................................................. 50
3.3 Results ............................................................................................................................ 53
3.3.1 Water and sediment quality ..................................................................................... 53
3.3.2 Holothuria scabra survival and growth .................................................................... 54
3.3.3 Sequencing and quality control ............................................................................... 55
3.3.4 Species richness, diversity and evenness of bacterial communities ........................ 55
3.3.5 Relative abundance of prominent taxa .................................................................... 58
3.3.6 Phylum-level taxa with statistical differences between treatments ......................... 58
3.3.7 Comparison of bacterial community composition between treatments .................. 60
3.3.8 Environmental factors driving bacterial community composition .......................... 60
3.3.9 Microbial biomarker discovery and visualization ................................................... 63
3.3.10 Predicted metagenome ........................................................................................... 64
3.4 Discussion ....................................................................................................................... 68
3.5 Conclusion ...................................................................................................................... 76
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Chapter 4. Effect of resource quality on growth of Holothuria scabra during aquaculture
waste bioremediation ............................................................................................................... 78
4.1 Introduction .................................................................................................................... 78
4.2 Methods .......................................................................................................................... 80
4.2.1 Study site ................................................................................................................. 80
4.2.2 Experimental animals .............................................................................................. 80
4.2.3 Experimental design ................................................................................................ 80
4.2.4 Carbon sources ........................................................................................................ 81
4.2.5 Aquaculture waste collection .................................................................................. 82
4.2.6 Experimental system and rearing conditions .......................................................... 83
4.2.7 Water quality, sediment quality, and environmental variables ............................... 85
4.2.8 Growth of Holothuria scabra .................................................................................. 85
4.2.9 Statistical analyses .................................................................................................. 85
4.3 Results ............................................................................................................................ 86
4.3.1 Water quality, sediment quality and environmental variables ................................ 86
4.3.2 Sediment characteristics .......................................................................................... 88
4.3.3 Growth and survival ................................................................................................ 91
4.3.4 Multiple regression analysis.................................................................................... 94
4.3.5 Economic analysis .................................................................................................. 94
4.4 Discussion ...................................................................................................................... 97
4.5 Conclusion ................................................................................................................... 102
Chapter 5. Effect of carbon supplementation on benthic nitrogen cycling during aquaculture
waste bioremediation incubations .......................................................................................... 104
5.1 Introduction .................................................................................................................. 104
5.2 Materials and methods ................................................................................................. 105
5.2.1 Study site ............................................................................................................... 105
5.2.2 Experimental animals ............................................................................................ 105
5.2.3 Experimental design .............................................................................................. 105
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