RAPID HARVEST OF ALGAE FOR BIOFUEL PRODUCTION WITH THE AGGREGATING ...

232 Pages·2014·4.14 MB·English

ABSTRACT

Title of Dissertation: RAPID HARVEST OF ALGAE FOR BIOFUEL
PRODUCTION WITH THE AGGREGATING
BACTERIUM BACILLUS SP. STRAIN RP1137

Ryan J. Powell, Doctor of Philosophy, 2014

Directed By: Dr. Russell T. Hill, Professor,
University of Maryland Center for
Environmental Science

Algal biofuels represent one of the most promising means of sustainably replacing
liquid fuels. However significant challenges remain before algal based fuels become
competitive with fossil fuels. One of the largest challenges is the ability to harvest the
algae in an economic and low energy manner. In this dissertation I describe the isolation
of the bacterium, Bacillus sp. strain RP1137, which can rapidly aggregate several algae
that are candidates for biofuel production. This bacterium aggregates algae in a pH
dependent and reversible manner and retains its aggregation ability after
paraformaldehyde fixation. The optimal ratio of bacteria to algae is described as well as
the robustness of aggregation at different salinities and temperatures. Aggregation is
dependent on the presence of calcium or magnesium ions and likely occurs via charge
neutralization through binding of calcium ions to the cell surface of both algae and
bacteria. I show charge neutralization occurs at least in part through binding of calcium to
negatively charged teichoic acid residues. A comparison of the aggregation efficiency of
RP1137, Bacillus megaterium QM B1551 and Bacillus subtilis SMY showed that
RP1137 and B1551 are equally efficient at aggregating algae while SMY does not
aggregate algae. The genome of RP1137 was sequenced to understand the molecular
underpinning of the mechanism of aggregation. The difference in aggregation phenotypes
between the three bacilli was used to inform a genomic comparison which revealed two
putative proteins that are predicted to be bound to the cell wall and are found only in
RP1137 and B1551 but not SMY. This work characterizes the conditions under which
Bacillus sp. RP1137 aggregates algae and the mechanism by which that aggregation
occurs.
RAPID HARVEST OF ALGAE FOR BIOFUEL PRODUCTION WITH THE
AGGREGATING BACTERIUM BACILLUS SP. STRAIN RP1137

By
Ryan Joseph Powell

Dissertation submitted to the Faculty of the Graduate School of the
University of Maryland, College Park, in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
2014

Advisory Committee:
Professor Russell T. Hill, Chair
Professor Allen Place
Professor Feng Chen
Professor Harold Schreier
Professor Paul Behrens
© Copyright by
Ryan Joseph Powell
2014
Dedication
I dedicate this work to my wife Kerry for her love and support and to my parents
John and Connie Powell who taught me how to work hard and persevere through the
most difficult of times.
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Acknowledgments
I am forever in debt to my parents who taught me how to work hard and to strive
to do good for those around me. These lessons prepared me for life better than anything
else they could have done. Focusing on the good I can do for others is the prime
motivating force behind the work within this dissertation and the work that I will
continue after my Ph.D. For this motivation and for their love and support I thank my
parents.
I thank my advisor Dr. Russell Hill for bringing me into his lab after a rocky
initial three years starting my Ph.D. in another lab and for finding ways to support me.
Working with Russell has been a pleasure; he has allowed me the freedom to investigate
what interests me and the guidance to turn those interests into papers and patents. Russell
has been a staunch ally who has fought to help me get to my goals and has provided
excellent guidance on the path to achieving these ambitions.
There are many great people in the Hill lab and within IMET that have helped me
complete this dissertation. I thank Leah Blasiak for many scientific discussions, expert
editing of manuscripts and the reminder to check the literature. I thank Jan Vicente for
accompanying me on over 500 runs in the rain, snow and heat of Baltimore. Jan has been
a constant source of support and great friend. Jeanette Davis has also been a great friend
and confidant in the toughest of times for me. I thank Jindong Zan for many great
scientific discussions and for being a great friend. Within IMET I thank Joel Graham for
great scientific advice. I thank the Place lab, particularly Ernest and Grant for always
being very helpful and somehow always knowing where to find that one key chemical or
piece of equipment that I needed. Finally, I thank Matt Moore for always finding a way to
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get things done, whether that was helping me find parts for my next prototype or wading
through the paperwork needed to hold my wedding at the institute.

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Statement of contribution
Taylor Carter helped gather data for the bioinformatics analyses presented in
Chapter 4. Diane Sumutka, Donald Belle and Li Zheng helped with the isolation of the
bacteria from the HTB1 culture, testing those bacteria for their effect on the growth of
HTB1 and testing the isolates for AHL production respectively.

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Table of contents
Chapter 1 Introduction ..................................................................................................................... 1
1.1 Motivation for studying biofuels............................................................................................ 2
1.2 Introduction to biofuels .......................................................................................................... 5
1.3 First generation biofuels ........................................................................................................ 6
1.4 Second generation biofuels .................................................................................................... 7
1.5 Third generation biofuels ....................................................................................................... 8
1.6 Algal biofuel production systems .......................................................................................... 9
1.7 Focus and Objectives ........................................................................................................... 14
Chapter 2: Rapid Aggregation of Biofuel Producing Algae by the Bacterium Bacillus sp. RP1137.
....................................................................................................................................................... 15
2.1 Abstract ................................................................................................................................ 16
2.2 Introduction .......................................................................................................................... 17
2.3 Experimental procedures ..................................................................................................... 18
2.3.1 Strains and culture conditions. ...................................................................................... 18
2.3.2 16S rRNA gene based strain identification. .................................................................. 20
2.3.3 Filtration aggregation assay. ......................................................................................... 20
2.3.4 Fluorescence microscopy. ............................................................................................. 22
2.3.5 Temperature dependence. ............................................................................................. 22
2.3.6 Salinity dependence. ..................................................................................................... 23
2.3.7 pH dependence. ............................................................................................................. 23
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2.3.8 Viability dependence. ................................................................................................... 24
2.3.9 Cell ratio........................................................................................................................ 24
2.3.10 Aggregation of other algae. ......................................................................................... 25
2.3.12 Proteinase K digestion assay. ...................................................................................... 25
2.3.13 Carbohydrate inhibition assay. .................................................................................... 26
2.3.14 Ca2+ & Mg2+ dependence assay. .................................................................................. 26
2.4 Results .................................................................................................................................. 26
2.4.1 Isolation of an algae-aggregating bacterium. ................................................................ 26
2.4.2 Determination of the optimal bacterium:alga ratio. ...................................................... 31
2.4.3 Characterization of the physical conditions that affect algal aggregation. ................... 34
2.4.4 Aggregation of candidate biofuel producing algae. ...................................................... 41
2.4.5 Characterization of the mechanism of aggregation. ...................................................... 41
2.5 Discussion ............................................................................................................................ 47
Chapter 3 Mechanism of algal aggregation by Bacillus sp. strain RP1137 ................................... 55
3.1 Abstract ................................................................................................................................ 56
3.2 Introduction .......................................................................................................................... 57
3.3 Experimental Procedures ..................................................................................................... 57
3.3.1 Strains and culture conditions. ...................................................................................... 57
3.3.2 Filtration aggregation assay. ......................................................................................... 58
3.3.3 Bacterial aggregation efficiency time course. ............................................................... 58
3.3.4 Algal aggregation efficiency time course. .................................................................... 59
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