Investigating the effects of biochar and activated carbon amendment on the microbial community ...

266 Pages·2016·5.31 MB·English

Investigating the effects of biochar and activated
carbon amendment on the microbial community
response in a volatile petroleum hydrocarbon -
contaminated gravelly sand

George Mangse
BSc. (Hons), MSc.

Thesis submitted to Newcastle University in partial fulfilment of the
requirements for the award of degree of Doctor of Philosophy

January, 2016

Environmental Engineering Group
School of Civil Engineering and Geosciences

Investigating the effects of biochar and AC on the microbial community response in a volatile petroleum hydrocarbon contaminated gravelly
sand
Abstract
Laboratory batch and long-term column experiments were conducted to investigate the effects
of wood-chip biochar and coal-derived activated carbon amendment on the microbiology of a
volatile petroleum hydrocarbon (VPH) - contaminated gravelly sand. First, a stable isotope-
labelled mono aromatic compound – toluene was used as a model VPH to gain insights into
the mineralization of VPHs by soil microorganisms in the presence and absence of biochar or
activated carbon. The biodegradation of a mixture of 12 VPHs was subsequently monitored in
batch microcosms over a duration of 6-19 days by measuring headspace CO concentration.
2
Further analysis was carried out by characterizing changes in the soil microbial community
composition using next generation sequencing techniques – 454 pyrosequencing and Ion
torrent sequencing. Increases in the levels of headspace CO in contaminated soil batches as
2
compared to live and abiotic controls to which no VPHs were added indicated a stimulation of
microbial activity in the batches through VPH addition. By fitting a maximum specific growth
rate of 0.6 h-1 (in line with published rates), it was possible to match model predictions of
45CO and 44CO concentrations with the experimentally determined data. Half-saturation
2 2
constants of 4.06 x 103 mgL-1, 7.76 x 102 mgL-1 and 1.83 x 102 mgL-1 were predicted for soil,
soil & BC and soil & AC respectively, much higher than values reported in the literature.
Differences in the half-saturation constant suggests that sorbent amendment affects the
microbial ecology, by making microorganisms which can utilize substrates at lower
concentrations more competitive. Yield coefficients (g biomass-C relative to g (biomass-C +
CO -C)) compared more closely in the nutrient (N & P) amended soils ranging from
2
4.83±0.46 in soil and biochar to 7.86±0.72 in unamended soil, than in the batches without
nutrients, 4.1±3.1 in soil & BC, 17.7±5.2 in soil and 13.7±4.6 in soil & AC. Sorbent
amendment thus reduced yield coefficients, thereby slowing the growth of VPH degrading
biomass. Microbial community structure analysis revealed an increase in the relative
abundance ranking of members of the genera Pseudomonas, Pseudoxanthomonas, and
Arenimonas by up to 32 folds and in the families Nocardioidaceae and Pseudomonadaceae
by at least 32 folds in sorbent amended and unamended soil batches and columns compared to
their initial soil conditions. Consequently, amending soils with 2% BC or AC changed the
biokinetics of VPH degradation by rendering VPHs less bioavailable, but did not appear to
have any detrimental effects on the VPH degrading bacteria both in the short- and long-term,
and may serve as a sustainable, cost-effective approach for enhancing the natural attenuation
of VPHs in soil, thus addressing the challenge of petroleum hydrocarbon contamination.

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Investigating the effects of biochar and AC on the microbial community response in a volatile petroleum hydrocarbon contaminated gravelly
sand
Dedication
To the glory of God the Father for giving me this extremely rare opportunity and to my
beloved mum (of blessed memory) for believing in me and for investing the very best in me. I
love you mum.
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Investigating the effects of biochar and AC on the microbial community response in a volatile petroleum hydrocarbon contaminated gravelly
sand
Acknowledgements
My profound gratitude goes to my primary supervisor Dr. David Werner for his unwavering
support, patience and understanding throughout this project. This research would not have
come to this point but for your professional supervision and guidance. I truly appreciate you.
My secondary supervisors, Dr. Russell Davenport and Dr. Paola Meynet have been great in
their support. Thank you for giving out of your wealth of experience, and for the training and
instructions you provided during the course of this work. I would also like to thank other
members of staff within the School of Civil Engineering and Geosciences, Bernie Bowler,
Paul Dunohoe, Donna Swan, Sarah Smith and David Race for your technical support
throughout my research. You guys are simply great. And to all other staff who contributed in
one way or the other to the success of this project, to my colleagues and friends, I say thank
you.
I also would like to appreciate the critique of my thesis examiners Professor Patrick
HÖHENER and Dr. Neil Gray. I did find your comments and feedback very valuable.
My appreciation equally goes to my sponsors, The Petroleum Technology Development Fund
(PTDF), Nigeria for this very rare opportunity given to me. Indeed this has been a worthy
experience and will always be cherished. More power to your elbow.
Finally, I would like to use this opportunity to acknowledge every member of my family for
your patience and support. To my Dad, and siblings - Favour, Hope, Grace, Love and Justice
for all of the encouragements and understanding throughout my time in School, I want to say
a big thank you. The best is yet to come for you.

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Investigating the effects of biochar and AC on the microbial community response in a volatile petroleum hydrocarbon contaminated gravelly
sand

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Investigating the effects of biochar and AC on the microbial community response in a volatile petroleum hydrocarbon contaminated gravelly
sand
Table of Contents
Abstract ...................................................................................................................................... i
Dedication .................................................................................................................................. ii
Acknowledgements .................................................................................................................. iii
Table of Contents ...................................................................................................................... v
List of tables .............................................................................................................................. x
List of figures .......................................................................................................................... xii
Glossary of abbreviations .................................................................................................... xvii
Chapter 1: Introduction, aim and thesis scope ................................................................ 1
Introduction .......................................................................................................... 1
Research motivation ............................................................................................. 3
Research questions ............................................................................................... 4
Aim and objectives ............................................................................................... 4
Thesis scope and structure .................................................................................... 5
Chapter 2: Literature review ............................................................................................ 7
Petroleum hydrocarbon degrading microbial communities .................................. 7
Metabolism of petroleum hydrocarbons ............................................................... 8
Environmental risk assessment: Effects of pollutants versus concentration of
pollutants in the environment ................................................................................................. 12
Chemo dynamics of organic pollutants in the environment ............................... 12
Growth dynamics of heterotrophic microorganisms – The Monod and Contois
models 19
The Nitrogen cycle ............................................................................................. 20
Modelling the fate of organic contaminants in the environment ........................ 21
Current remediation strategies ............................................................................ 22
In situ sorbent amendment – a novel technique for the remediation of volatile
petroleum hydrocarbon contaminated soils and sediments ...................................................... 24
Sorbent amendment effect on soil biota ............................................................. 25
Chapter 3: Effects of biochar and activated carbon amendment on the bio-kinetics of
toluene degradation in gravelly sand .................................................................................... 30
3.1 Introduction ........................................................................................................ 30
3.2 Aim ..................................................................................................................... 31
3.2.1 Objectives ........................................................................................................... 31
3.2.2 Hypotheses ......................................................................................................... 31
3.3 Materials and methods ........................................................................................ 33
3.3.1 Soil, biochar and activated carbon ...................................................................... 33
3.3.2 Chemical pollutant .............................................................................................. 33
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Investigating the effects of biochar and AC on the microbial community response in a volatile petroleum hydrocarbon contaminated gravelly
sand
3.3.3 Batch experiments .............................................................................................. 33
3.3.4 CO2 analysis by GC-MS analysis, microbial respiration ................................... 34
3.3.5 CO2 leakage experiments ................................................................................... 35
3.3.6 Determination of carbon isotope signatures ....................................................... 36
3.3.7 GC-FID analysis, headspace VPH measurements ............................................. 36
3.3.8 Sample clean up and quantification of strongly sorbed residuals ...................... 36
3.3.9 Determination of microbial degradation of poorly available substrate after
pollutant source removal ................................................................................................... 37
3.3.10 Determination of cation (Ca2+ and Mg2+) concentrations in soil systems, ICP-
OES analysis ..................................................................................................................... 37
3.3.11 Batch modelling ................................................................................................. 38
3.4 Results and Discussion....................................................................................... 39
3.4.1 Effects of biochar and activated carbon amendment on biodegradation and
sorption of 13C7-Toluene ................................................................................................... 39
3.4.2 Effects of substrate availability on the mineralisation of soil organic matter in a
batch system ...................................................................................................................... 42
3.4.3 Relationship between VPH biodegradation and SOM mineralisation in batch
systems 45
3.4.4 Quantitative estimation of CO2 loss from batch microcosms ............................ 47
3.4.5 Predicting biochar and activated carbon amendment effects on the
biodegradation of stable-isotope labelled toluene (13C7) in gravelly sand ....................... 51
3.4.6 Predicting the biodegradation of poorly available substrate (13C7 - toluene) by
microorganisms in different soil systems after evacuation of pollutant source ................ 59
3.5 Conclusions ........................................................................................................ 61
Chapter 4: A comparison of the varying effects of different petroleum hydrocarbon
classes on the bacteria community response in gravelly sand ........................................... 63
4.1 Introduction ........................................................................................................ 63
4.2 Aim..................................................................................................................... 64
4.2.1 Objectives........................................................................................................... 64
4.2.2 Hypotheses ......................................................................................................... 65
4.3 Materials and methods ....................................................................................... 66
4.3.1 Soil and chemical pollutant mixtures ................................................................. 66
4.3.2 Batch experiments .............................................................................................. 66
4.3.3 Microbial respiration .......................................................................................... 66
4.3.4 Sample collection and storage ............................................................................ 66
4.3.5 DNA extraction and PCR amplification (454 pyrosequencing library
preparation) ....................................................................................................................... 67
4.3.6 454 pyrosequencing and Ion torrent sequencing................................................ 67
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Investigating the effects of biochar and AC on the microbial community response in a volatile petroleum hydrocarbon contaminated gravelly
sand
4.3.7 Microbial community structure analysis ............................................................ 68
4.3.8 Statistical analysis .............................................................................................. 69
4.4 Results and Discussion ....................................................................................... 70
4.4.1 Biodegradation of volatile petroleum hydrocarbon mixtures in batch systems . 70
4.4.2 Microbial diversity and species richness ............................................................ 72
4.4.3 Taxa distribution across samples ........................................................................ 76
4.4.4 OTU functions in petroleum hydrocarbon degradation ...................................... 80
4.4.5 Inter-relationship between bacterial communities within different soil samples86
4.4.6 General analysis of 454 pyrosequencing and Ion torrent sequencing data output
91
4.5 Conclusions ........................................................................................................ 93
Chapter 5: Short-term effects of sorbent amendment on the attenuation of volatile
petroleum hydrocarbons in gravelly sand - a nutrient limiting perspective ..................... 95
Introduction ........................................................................................................ 95
Aim ..................................................................................................................... 96
Objectives ........................................................................................................... 96
Hypotheses ......................................................................................................... 97
Materials and methods ........................................................................................ 98
Soil, biochar and activated carbon ...................................................................... 98
Chemical mixture ............................................................................................... 98
Batch experiments .............................................................................................. 98
Microbial respiration .......................................................................................... 98
Sample collection and storage ............................................................................ 98
Total cell counts and biomass yield determination ............................................ 99
DNA extraction and PCR amplification ............................................................. 99
454-pyrosequencing ........................................................................................... 99
Microbial community structure analysis ............................................................ 99
Statistical analysis ............................................................................................ 100
Results and Discussion ..................................................................................... 101
Microbial respiration and total cell counts ....................................................... 101
Analysis of pyrosequencing-generated dataset ................................................ 107
Microbial diversity and species richness .......................................................... 107
Effects of sorbent and nutrient amendment on phylum level taxa distribution
across soil samples .......................................................................................................... 111
OTU functions viz-a-viz petroleum hydrocarbon degradation......................... 114
Relationship between microbial communities in different samples ................. 120
Conclusions ...................................................................................................... 123
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Investigating the effects of biochar and AC on the microbial community response in a volatile petroleum hydrocarbon contaminated gravelly
sand
Chapter 6: Effects of volatile petroleum hydrocarbon vapour migration on the
microbial community composition in a biochar and activated carbon amended sand – a
long term study ..................................................................................................................... 125
6.1 Introduction ...................................................................................................... 125
6.2 Aim................................................................................................................... 126
6.2.1 Objectives......................................................................................................... 126
6.2.2 Hypotheses ....................................................................................................... 127
6.3 Materials and methods ..................................................................................... 128
6.3.1 Soil, biochar and activated carbon ................................................................... 128
6.3.2 Chemical mixture ............................................................................................. 128
6.3.3 Column experiments ........................................................................................ 128
6.3.4 Sample collection and storage .......................................................................... 129
6.3.5 Total cell counts and biomass growth .............................................................. 129
6.3.6 DNA extraction and PCR amplification .......................................................... 129
6.3.7 454-pyrosequencing ......................................................................................... 130
6.3.8 Microbial community structure analysis .......................................................... 130
6.3.9 Statistical analysis ............................................................................................ 130
6.4 Results and Discussion..................................................................................... 131
6.4.1 Summary of chemical monitoring results from previous column studies
(Bushnaf, K., 2014) ........................................................................................................ 131
6.4.2 Total cell counts ............................................................................................... 133
6.4.3 Preliminary analysis of pyrosequencing dataset .............................................. 134
6.4.4 Microbial diversity and species richness ......................................................... 135
6.4.5 Taxa distribution across different samples ....................................................... 141
6.4.6 Relationship between microbial communities in different samples ................ 144
6.4.7 Microbial functions, petroleum hydrocarbon degradation and nitrogen cycling
147
6.5 Conclusions ...................................................................................................... 156
Chapter 7: Overall conclusions and recommendations for future work .................. 158
7.1 Effects of biochar and activated carbon amendment on pollutant toxicity to soil
VPH degrading bacteria ......................................................................................................... 158
7.2 Effects of biochar and activated carbon amendment on the growth of VPH
degrading bacteria .................................................................................................................. 158
7.3 Effects of biochar and activated carbon amendment on microbial ecology in
VPH-contaminated gravelly sand .......................................................................................... 159
7.4 Broader implications of the current research ................................................... 160
7.5 Recommendations for future work .................................................................. 161
References ............................................................................................................................. 163
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