Stable Isotope Systematics of Abiotic Nitrate and Nitrite Reduction Coupled With Anaerobic Iron ...

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Stable Isotope Systematics of Abiotic Nitrate and
Nitrite Reduction Coupled With Anaerobic Iron
Oxidation: the Role of Reduced Clays and Fe-
Bearing Minerals
Citation
Grabb, Kalina C. 2015. Stable Isotope Systematics of Abiotic Nitrate and Nitrite Reduction
Coupled With Anaerobic Iron Oxidation: the Role of Reduced Clays and Fe-Bearing Minerals.
Bachelor's thesis, Harvard College.
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Stable isotope systematics of
abiotic nitrate and nitrite reduction coupled with
anaerobic iron oxidation:
The role of reduced clays and Fe-bearing minerals

A thesis presented
by
Kalina Cozette Grabb
to
the School of Engineering and Applied Sciences
and the Department of Earth and Planetary Sciences
in partial fulfillment of the requirements
for a degree with honors
of Bachelor of Arts

April 2015
Harvard College

ABSTRACT

Under anaerobic conditions, it is widely assumed that nitrate (NO -) and
3
nitrite (NO -) reduction is primarily the result of microbial respiration (Burgin and
2
Hamilton, 2007; Granger et al., 2008). However, it is also proven that the abiotic
reduction of nitrate and nitrite by reduced iron(II) (Fe(II)), whether mineral-bound
or surface-associated, may also occur under certain environmentally relevant
conditions (Chao and Kroontje, 1966; Straub et al., 1996; Picardal, 2012,). With a
range of experimental conditions, nitrogen and oxygen stable isotope systematics
of abiotic nitrite reduction by Fe(II) were investigated in an effort to characterize
the biotic and abiotic processes in the environment. While homogenous reactions
between NO - and Fe(II) in artificial seawater (ASW) showed little reduction,
2
heterogeneous reactions involving Fe-containing minerals showed considerable
nitrite loss. Specifically, rapid nitrite reduction was observed in experiments that
included reduced nontronite clay and an Fe(II)-Fe(III) hydroxide mineral, termed
green rust. These iron oxides and clay minerals offer both a source of reduced
iron in the mineral matrix as well as a surface for Fe(II) activation. Additional
control experiments with corundum as a non-Fe containing mineral surface
showed little NO - loss, implicating a more dominant role of structural iron in the
2
clays during nitrite reduction. In experiments with nontronite and green rust the
isotope effects (15ε and 18ε) ranged from 3 to 16‰ for 15ε and 2 to 6‰ for 18ε.
Nitrite reduction rates and 15ε values within an experimental condition were
directly correlated with slower reactions having higher isotopic fractionation. The
apparent 18ε was affected by oxygen atom exchange with water, which lowered
the isotope effect. Although little data exist for comparison with the dual isotopes
of microbial NO - reduction, these data serve as a benchmark for evaluating the
2
role of abiotic processes in nitrogen (N) reduction, particularly in sediment
systems low in organic carbon and high in reduced iron.

  i
TABLE OF CONTENTS

ABSTRACT ............................................................................................................ i
TABLE OF CONTENTS ..................................................................................... ii
LIST OF FIGURES ............................................................................................. iv
ACKNOWLEDGEMETNS ................................................................................. v
1. INTRODUCTION ............................................................................................ 1
2. BACKGROUND ............................................................................................... 4
2.1. Abiotic nitrate (NO -) and nitrite (NO -) reduction coupled with Fe(II)
3 2
oxidation ........................................................................................................................ 4
2.1.1. Abiotic nitrate (NO -) reduction .............................................................................. 4
3
2.1.2. Abiotic nitrite (NO -) reduction .............................................................................. 5
2
2.1.3. Abiotic nitrite (NO -) reduction by Fe(II)-Fe(III) hydroxide minerals, green rust . 6
2
2.1.4. Abiotic nitrate (NO -) and nitrite (NO -) reduction by Fe(II)-bearing clay minerals
3 2
........................................................................................................................................... 7
2.2. Biotic nitrate (NO -) and nitrite (NO -) reduction coupled with Fe(II)
3 2
oxidation ........................................................................................................................ 8
2.2.1. Microbial nitrate (NO -) and nitrite (NO -) reduction by denitrifying bacteria ...... 8
3 2
2.2.2. Microbial nitrite (NO -) reduction by anaerobic ammonium oxidation (Anammox)
2
........................................................................................................................................... 9
2.2.3. Microbial nitrate (NO -) and nitrite (NO -) reduction by nitrate-reducing iron-
3 2
oxidizing bacteria .............................................................................................................. 9
2.2.4. Microbial nitrate (NO -) reduction coupled with Fe(II) oxidation in Fe(II)-bearing
3
clay minerals ................................................................................................................... 10
2.3. Stable isotope systematics of NO - and NO - in nitrate and nitrite reduction 11
3 2
2.3.1. Stable isotope systematics overview ..................................................................... 11
2.3.2. Stable isotope systematics in nitrate (NO -) and nitrite (NO -) reduction by
3 2
microorganisms in the environment ................................................................................ 13
3. METHODS ...................................................................................................... 16
3.1. Nitrate (NO -) and nitrite (NO -) reduction experiments ................................. 16
3 2
3.1.1 Overview of experiments ....................................................................................... 16
3.1.2. Experiments analyzing the homogeneous reaction of abiotic nitrite (NO -)
2
reduction with aqueous Fe(II) ......................................................................................... 17
3.1.3. Experiments analyzing the heterogeneous reaction of abiotic nitrite (NO -)
2
reduction with Fe(II)-bearing clay minerals ................................................................... 19
3.1.4. Experiments analyzing the heterogeneous reaction of abiotic nitrite (NO -)
2
reduction with Fe(II) adsorbed onto non-Fe(II)-bearing minerals .................................. 20

 ii
3.1.5. Experiments analyzing the heterogeneous reaction of abiotic nitrite (NO -)
2
reduction with green rust ................................................................................................. 20
3.1.6. Experiments analyzing abiotic nitrate (NO -) reduction coupled with Fe(II)
3
oxidation .......................................................................................................................... 21
3.2. Anoxic Protocol .................................................................................................... 21
3.3. Chemical Analysis ................................................................................................ 22
3.3.1. Iron (Fe) concentration measurements .................................................................. 22
3.3.2. Nitrite (NO -) concentration measurements .......................................................... 23
2
3.3.3. Nitrate (NO -) concentration measurements ......................................................... 23
3
3.3.4. Nitrous oxide (N O(g)) concentration measurements ........................................... 23
2
3.4. Nitrite (NO -) isotope measurements ................................................................. 24
2
3.5. Clay reduction and clay treatment ..................................................................... 24
3.6. Iron sorption isotherms ....................................................................................... 25
3.7. Laboratory synthesis of “green rust” ................................................................ 25
4. RESULTS ........................................................................................................ 27
4.1. Nitrite (NO -) reduction coupled with Fe(II) oxidation .................................... 27
2
4.1.1. Homogenous reactions (experiment 1 and 2) ........................................................ 27
4.1.2. Heterogeneous Reactions (experiment 3, 4, and 5) ............................................... 28
4.1.2.a. Nitrite (NO -) reduction with Fe(II)-bearing minerals ......................................... 28
2
4.2.1.b. Nitrite (NO -) reduction by Fe(II) adsorbed to non-Fe-bearing minerals
2
(experiment 3, 4, and 5) .................................................................................................... 31
4.1.2.c. Nitrite (NO -) reduction by green rust (experiment 6, 7, 8, 9, and 10) ................ 32
2
4.2. Nitrate (NO -) reduction coupled with Fe(II) oxidation (experiment 11 and
3
12) ................................................................................................................................. 34
4.3. Nitrous oxide (N O(g)) concentration within experiments .............................. 36
2
4.4. Iron sorption isotherms ....................................................................................... 37
4.5. Nitrite (NO -) reduction isotope effects (15ε and 18ε) ........................................ 38
2
4.5.1. Isotopic fractionation during nitrite (NO -) reduction by nontronite .................... 38
2
4.5.2. Isotopic fractionation during nitrite (NO -) reduction by green rust ..................... 40
2
4.5.3. Relationship of δ18O and δ15N during nitrite (NO -) reduction, 18ε:15ε ................. 42
2
5. DISCUSSION .................................................................................................. 44
5.1. Factors controlling nitrate (NO -) and nitrite (NO -) reduction within the
3 2
natural environment ................................................................................................... 44
5.1.1. Conditions in which Fe(II)-minerals will catalyze abiotic nitrate (NO -) and nitrite
3
(NO -) reduction over biotic processes ........................................................................... 44
2
5.1.2. The effects of pH on abiotic nitrite (NO -) reduction within the environment ..... 48
2
5.2. Factors controlling the stable isotope systematics of nitrate (NO -) and nitrite
3
(NO -) reduction within the natural environment ................................................... 51
2
5.2.1. Analysis of the change in isotope effect during abiotic nitrite (NO -) reduction .. 51
2
5.2.2. Relation between reduction rate kinetics and isotope effects ............................... 53
5.2.3. Effects of oxygen isotope exchange on 18ε:15ε of nitrite (NO -) reduction ........... 54
2
6. CONCLUSIONS ............................................................................................. 56
7. WORKS CITED ............................................................................................. 59

 iii
LIST OF FIGURES

Figure 1: Marine Nitrogen cycle ............................................................................ 2
Figure 2: Abiotic nitrite reduction coupled with Fe(II) oxidation ......................... 2
Figure 3: Rayleigh fractionation curve ................................................................ 13
Table 1: Nitrite reduction experiments conducted ............................................... 18
Table 2: Nitrate reduction experiments conducted .............................................. 19
Figure 4: Laboratory synthesis of green rust ....................................................... 26
Figure 5: Homogeneous nitrite reduction ............................................................ 27
Figure 6: Heterogeneous nitrite reduction- Na-montmorillonite and illite .......... 29
Figure 7: Heterogeneous nitrite reduction- nontronite ........................................ 30
Figure 8: Heterogeneous nitrite reduction- Fe(II)-adsorbed minerals ................. 31
Figure 9: Heterogeneous nitrite reduction- green rust ......................................... 32
Figure 10: pH pH during nitrite reduction ........................................................... 34
Figure 11: Abiotic nitrate reduction ..................................................................... 35
Figure 12: Fe(II) sorption isotherm ..................................................................... 37
Table 3: 15ε, 18ε, and 18ε:15ε values- nontronite and green rust ............................ 38
Figure 13: Isotopic fractionation- nontronite ....................................................... 39
Figure 14: Isotopic fractionation- green rust ....................................................... 41
Figure 15: Dual nitrite N and O isotopic compositions ....................................... 43
Figure 16: Isotopic fractionation versus the reaction rate constant ..................... 54

 iv
ACKNOWLEDGEMETNS

I would like to thank my entire advising team for the opportunity to
conduct this thesis and the endless support throughout the entire process. First off,
I would like to express my gratitude to Dr. Scott Wankel and Dr. Colleen Hansel
at Woods Hole Oceanographic Institution for advising me throughout the year and
providing me with the opportunity to conduct research in their lab during the
summer. I would also like to thank Dr. Carly Buchwald for teaching me nearly
everything I know about working in a glove box and being there to skype with me
to answer any and all questions. I also want to extend an enormous thank you to
Dr. David Johnston at Harvard for introducing me to geochemistry and advising
me since the beginning of my research. The guidance, knowledge, and enthusiasm
that this group of advisors has shed on me made this thesis possible and also
reminds me daily how much I love this subject at hand and how appreciative I am
to be surrounded by such an inspirational group of researchers.
I also owe a huge thank you to…
Zoe Sandwith for helping me in the lab with my plethora of samples and
explaining to me patiently the secrets about the mass spec.
Net Charoenpong for teaching me the ins and outs about nitrogen isotopes
and sharing your knowledge during inspiring chats in the lab.
The entire HWP team for the support, belief, and encouragement that kept
me working even after long days of travel and games as well as all of the thought
and effort you put into reading and editing my thesis.
To all of my professors within the EPS and ESE departments that have
shed a bit of their knowledge on me and taught me to love science through the
enthusiasm they share for their subjects.
Chenoweth Moffatt, Patrick Ulrich and the entire EPS and ESE
departments for endless support, unselfish funding, ceaseless smiles and laughs,
and even more cookies and tea.

  v

1. INTRODUCTION

Since the invention of the Haber-Bosch process, nitrogen (N) levels in the
environment are increasing due to elevated levels of fertilizer production, fossil
fuel combustion, and industrial emissions (Rice and Herman, 2012). Levels of
reactive nitrogen (N) in natural biospheres are rising due to anthropogenic
r
pollution, and this increase has instigated negative impacts on human health,
restrictions on the natural ecosystems biodiversity, and alterations to geochemical
cycling. While N has many forms, nitrate (NO -) is more soluble in water than
r 3
other forms and is the leading global pollutant in groundwater. Nitrate pollution
hinders the ability to use ground water as drinking water and also leads to algal
blooms that cause eutrophication. Eutrophication degrades the water quality and
harms natural ecosystems (Burgin and Hamilton, 2007; Galloway et al., 2008).
The overabundance of nitrate in nature has led to investigations into processes
that remove nitrate from aqueous ecosystems.
In the subsurface biosphere, where oxygen is limited, anaerobic nitrate
reduction is responsible for removing nitrate through reduction to other forms of
nitrogen, including nitrogen gas (N (g)), nitrous oxide (N O(g)), nitrite (NO -),
2 2 2
and ammonium (NH +) (Burgin and Hamilton, 2007) (Figure 1). While microbial
4
nitrate reduction by denitrifying bacteria has been studied for decades, this
process only accounts for less than half of the total nitrate reduction that occurs
within natural ecosystems (Burgin and Hamilton, 2007). A greater understanding
of the complex N cycle has promoted further discoveries about biotic and abiotic
processes that lead to nitrate reduction, including the coupling of nitrate reduction
with abiotic and biotic iron (Fe) oxidation (Burgin and Hamilton, 2007) (Figure
2).
1