assessing the biocompatibility of oligomers and amine

108 Pages·2015·1.65 MB·English

ASSESSING THE BIOCOMPATIBILITY OF OLIGOMERS AND AMINE-
FUNCTIONALIZED POLYMERS FOR USE IN TWO-PHASE
PARTITIONING BIOREACTORS

by

Jesse Harris

A thesis submitted to the Department of Chemical Engineering
in conformity with the requirements for
the degree of Masters of Applied Sciences

Queen’s University
Kingston, Ontario, Canada
October, 2015

Copyright ©Jesse Harris, 2015
Abstract
Two-phase partitioning bioreactors (TPPBs) are a bioprocessing tool that limits inhibition of cell
growth by toxic compounds in bioreactors by utilizing an immiscible secondary phase to sequester
compounds that inhibit microbial cell growth or function. The earliest TPPBs used organic solvents as the
sequestering phase despite challenges identifying solvents that were inexpensive and biocompatible,
although recent efforts have utilized polymers as the sequestering phase in TPPBs, as most commodity
polymers are inexpensive and biocompatible.
Research on the selection of polymers for use as the sequestering phase in TPPBs has focused on
predicting solute uptake by first principles in order to maximize the partitioning coefficient of a target
solute. Some research has shown that low molecular weight (MW) polymers have improved partitioning
coefficients, although their biocompatibility is in need of further study; this thesis investigates the effect
of polymer MW on microbial biocompatibility. Trends in biocompatibility were assessed for
polypropylene glycol to Saccharomyces cerevisiae and Pseudomonas putida, which were selected due to
their prior use in TPPBs. Given that log P has been previously used as an important physiochemical
property for predicting biocompatibility, with higher log P values associated with improved
biocompatibility, experiments were performed to determine the average log P of polymers. Polymer
samples were also water-washed to shift the log P upwards by removing low MW polymer chains.
Average log P determination experiments showed that as the polymer MW increased, the measured log P
also increased. Biocompatibility, as measured by the change in optical density of the cultures after 24
hours of exposure to the polymers, also improved with increasing MW and log P, and that polypropylene
glycols possessing MWs of 1000 and higher were all found to be biocompatible. It was also shown that
water-washing noticeably improved biocompatibility by removing low MW polymer from samples.
Initial research was also undertaken to examine the use of amine-functionalized reactive polymers
for extracting organic acids in TPPB in terms of their efficacy and their biocompatibility. An amine
functionalized polyacrylate was synthesized which was stable under acidic conditions, and did not inhibit
cell growth over 24 hour exposure. It was also shown to extract 60-85% of organic acids from aqueous
solution over 2 hours with polymer concentrations of 10 g/L, and acid concentrations of 2.5 g/L.
This work addresses two major areas. The first is improving the understanding of the
biocompatibility of low molecular weight PPGs. Second is improving the understanding of the
biocompatibility and acid extraction of amine-functionalized hydrogels, and investigates the potential for
such materials for use in acid producing TPPBs.

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Co-Authorship
Dr. Andrew Daugulis was a major contributor to all chapters of this thesis. His contributions were
both technical and editorial in nature. Dr. Scott Parent also made substantial technical contributions to
chapter 5.

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Acknowledgements
I would like to acknowledge Dr. Andrew Daugulis. Without his resources and technical advice
this research would have been impossible.
I would like to acknowledge my wife Brittany. Without her love, support and encouragement I
would have lost my focus and drive.
I would like to acknowledge my son Reid. Without his company the writing process would have
been much more lonely.

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Table of Contents
Abstract…………………………………………………………………………………………….. i
Co-Authorship……………………………………………………………………………………… iii
Acknowledgement…………………………………………………………………………………. iv
Table of Contents…………………………………………………………………………………... v
List of Figures……………………………………………………………………………………… viii
List of Tables………………………………………………………………………………………. ix
List of Abbreviations………………………………………………………………………………. x
Chapter 1 - Introduction…….………………………………………………………………........... 1
1.1 Background…………………………………………………………………………….......... 1
1.2 Objectives…………………………………………………………………………………… 2
1.3 References………………………………………………………………………................... 3
Chapter 2 - Literature Review…….……………………………………………………………….. 5
2.1 Two-phase partitioning bioreactors…………………………………………………………. 5
2.2 Selection of non-aqueous phase……………………………………………………………. 6
2.2.1 Biocompatibility………………………………………………………………………. 7
2.2.2 Non-biodegradability…………………………………………………………………. 8
2.2.3 Low-cost………………………………………………………………………………. 8
2.3 Predicting biocompatibility………………………………………………………………… 9
2.3.1 Molecular Weight …………………………………………………………………….. 9
2.3.1 Critical Log P…………………..……………………………………………………… 10
2.4 Polymers in two-phase partitioning bioreactors…………………………………………….. 14
2.4.1 Polymer selection in TPPB: partitioning coefficient of the target molecule…………… 16
2.4.1 Effect of a molecular weight on the physical properties of polymers………………….. 19
2.5 Methods in determining log P………………………………………………………………. 20
2.5.1 Estimation of log P by software……………………………………………………….. 20
2.5.2 Shake flask method……………………………………………………………………. 21
2.5.3 Slow stir method………………………………………………………………………. 22
2.5.4 HPLC method…………………………………………………………………………. 23
2.6 Reactive extraction of organic acids by reactive polymers………………………………… 25
2.6.1 Organic acid production……………………………………………………………….. 25
2.6.2 Reactive extraction…………………………………………………………………….. 27
2.6.2 Reactive polymers…………………………………………………………………….. 29

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2.7 Microorganisms……………………………………………………………………………. 30
2.7.1 Pseudomonades putida……………………………………………………………….. 31
2.7.2 Saccharomyces cerevisiae……………………………………………………………. 31
2.8 Scope of Thesis..…………………………………………………………………………... 31
2.9 References…………………………………………………………………………………. 33
Chapter 3 - Biocompatibility of Polymers for TPPB…………………………………………….. 49
3.1 Preface……………………………………………………………………………………… 50
3.2 Abstract…………………………………………………………………………………….. 52
3.3 Introduction………………………………………………………………………………… 53
3.4 Materials and methods…………………………………………………………………….. 55
3.4.1 Log P determination of PPGs of different MWs…………………………………….. 55
3.4.2 Cultures and media…………………………………………………………………… 56
3.4.3 Critical log P determination…………………………………………………………. 56
3.4.4 Polymers and Solvents………………………………………………………………… 57
3.4.5 Toxicity of polymers of different MW……………………………………………….. 57
3.4.6 Effect of removing low MW polymer fractions……………………………………… 57
3.4.7 Analytical methods…………………………………………………………………… 58
3.5 Results……………………………………………………………………………………... 58
3.5.1 Critical log P………………………………………………………………………….. 58
3.5.2 Log P of different MW PPGs………………………………………………………… 59
3.5.3 Biocompatibility of PPG……………………………………………………………... 62
3.5.4 Washed PPG experiments……………………………………………………………. 65
3.6 Discussion…………………………………………………………………………………. 67
3.7 References…………………………………………………………………………………. 70
Chapter 4 - Acid extraction by amine-functionalized polymers……………….………………... 74
4.1 Preface…………………………………………………………………………………….. 75
4.2 Abstract……………………………………………………………………………………. 77
4.3 Introduction………………………………………………………………………………... 78
4.4 Materials and methods…………………………………………………………………….. 81
4.4.1 Ester-linked hydrogel preparation……………………………………………………. 81
4.4.2 Amide-linked hydrogel preparation………………………………………………….. 81
4.4.3 Acid extraction……………………………………………………………………….. 82
4.4.4 Time course of acid extraction……………………………………………………….. 82
4.4.5 Cell Growth Experiments..…………………………………………………………... 83

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4.4.6 Analytical Methods…………………………………………………………………... 83
4.5 Results……………………………………………………………………………………... 83
4.5.1 Extraction of butyric and benzoic acid………………………………………………. 83
4.5.2 Stability Time Course………………….…………………………………………….. 84
4.5.3 Extraction of succinic, acetic, and formic acid ………………………………………. 85
4.5.4 Biocompatibility……………………………………………………………………... 86
4.6 Discussion and Conclusions….…………………………………………………………… 86
4.8 References………………………………………………………………………………… 89
Chapter 5 - Conclusions and Future Work……..……………………………………………….. 94
5.1 Conclusions……………………………………………………………………………….. 95
5.2 Future Work………………………………………………………………………………. 96

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List of Figures
Figure 2-1: Graph demonstrating critical log P phenomenon. Results show glucose utilization (%)
by Pseudomonas putida in the presence of various organic solvents. Graph taken from Vrionis et al.
2002……………………………………………………………………………………………….. 11

Figure 3-1: Biocompatibility versus log P of solvents after 24 hours of growth. a) Optical density at
600 nm of S. cerevisiae relative to control, b) Remaining glucose for S. cerevisiae relative to initial
concentration c) Optical density at 600 nm of P. putida relative to control d) Remaining glucose for
P. putida relative to initial concentration. Error bars indicate 1 standard deviation……….. 58

Figure 3-2: HPLC chromatograms of 1000 molecular weight polypropylene glycol (a) as received –
unwashed and (b) washed………………………………………………..……………………... 60

Figure 3-3: HPLC chromatograms of 1080 molecular weight polypropylene glycol standard (a) as
received – unwashed and (b) washed…………………………………………………………. 61

Figure 3-4: 24 hour growth of (a) S. cerevisiae and (b) P. putida relative to control in 50 mL culture
broth with 5 grams unwashed PPG over a range of molecular weights. Error bars have been given
to show 1 standard deviation……………………………………….…………………………. 62

Figure 3-5: 24 hour growth relative to control over a range of volumes (a) S. cerevisiae (b) P. putida.
( ) PPG 425 ( ) PPG 725 ( ) PPG 1000( ) dodecane and ( ) cyclohexane. Error bars
have been given to show 1 standard deviation…………………………………………………. 63

Figure 3-6: Cell growth after 24 hours in washed versus unwashed PPG. (a) S. cerevisiae with PPG
425 (b) P. putida with PPG 425 (c) S. cerevisiae with PPG 725 (d) P. putida with PPG 725 (e) S.
cerevesiae with PPG 1000 (f) P. putida with PPG 1000. (•) washed polymer (⧫) unwashed polymer.
Error bars have been given showing 1 standard deviation.…………………………………… 65

Figure 4-1: Synthesis of ester-linked amine-functionalized hydrogel………………………… 80

Figure 4-2: Synthesis of amide-linked amine-functionalized hydrogel……………………….. 81

Figure 4-3: Acid levels versus amount of polymer added. ■ Butyric acid with polyacrylate polymer,
× benzoic acid with polyacrylate polymer, ■ butyric acid with polyacrylamide polymer × benzoic
acid with polyacrylamide polymer. Error bars show one standard deviation…….………….. 83

Figure 4-4: Extraction of a 2.5 g/L acetic acid solution by 5 g/L polymer over 24 hours. ▲
polyacrylamide ▲ polyacrylate. Error bars show one standard deviation…….…………...… 84

Figure 4-5: Levels of organic acid when extracted by varying levels of polyacrylamide. ■ Butyric
acid, × benzoic acid, ▲acetic acid, • formic acid, + succinic acid. Error bars show one standard
deviation……………………………………………………………………………….………….. 85

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List of Tables
2-1: Critical log P of a selection of microbial species………………………………….………. 12

2-2: Top Value Added Chemicals from Biomass……………………………………………… 26
3-­1:
 Information
 for
 solvents
 used
 in
 critical
 log
 P
 experiments…………………………………
  54

3-2: Calculated average log P values for PPG samples………………………………………... 55

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