Table Of ContentTowards In situ extraction of fine chemicals and biorenewable fuels
from fermentation broths using Ionic liquids and the Intensification of
contacting by the application of Electric Fields
BY
Satya Aravind Gangu,
B.Tech., Chemical Engineering, Osmania University, India, 2003
Submitted to the Department of Chemical and Petroleum Engineering and the
Graduate Faculty of the University of Kansas in partial fulfillment of the
requirements for the degree of Doctor of Philosophy
Committee Members:
____________________________
Aaron M. Scurto (Chair)
____________________________
Laurence R. Weatherley
____________________________
Bala Subramaniam
____________________________
Cory J. Berkland
____________________________
Belinda M. Sturm
Date defended: ______________
The Dissertation Committee for Satya Aravind Gangu certifies that this is the approved version
of the following dissertation:
Towards In situ extraction of fine chemicals and biorenewable fuels from
fermentation broths using Ionic liquids and the Intensification of contacting by the
application of Electric Fields.
Committee Members:
____________________________
Aaron M. Scurto (Chair)
____________________________
Laurence R. Weatherley
____________________________
Bala Subramaniam
____________________________
Cory J. Berkland
____________________________
Belinda M. Sturm
Date approved: ______________
ii
ABSTRACT
Cost-effective synthesis of fine chemicals/biorenewable fuels via fermentation invariably
hinges upon efficient separations from the dilute broth. Biphasic/in situ extraction can positively
impact both upstream fermentation and the downstream product separations. For developing
environmentally sustainable processes, ionic liquids are touted as greener alternative to organic
solvents not only because of their relatively low volatility but also due to the ability to tune their
properties and design new ionic liquids for task specific needs. Solvent selection for in situ
fermentation is depended on high solute partitioning and their biocompatibility with the
microorganisms. Such information for these new set of solvents, ionic liquids that comprise of
ions is very limited compared to organic solvents. Here, a new methodology to enable successful
use of ionic liquids in in situ extraction is developed by focusing on two model systems: 1. Fine
chemical - (1R,2S)-1,2-napthalene dihydrodiol (NDHD), produced from the biotransformation of
naphthalene by Escherichia coli strain JM109(DE3) pDTG141 and 2. Biorenewable fuel -
Biobutanol (along with Acetone and Ethanol), produced from the Fermentation of sugars by
Clostridia.
The partitioning of each of the solutes, NDHD, Acetone, 1-butanol and ethanol between
water and ionic liquid has been measured in the range of concentrations typical of actual
fermentations. Different cationic classes of ionic liquids including 1-alkyl-3-methylimidazolium
([RMIm]), trialkylmethylammonium ([TRMAm]) and trihexyltetradecylphosphonium ([P ])
6,6,6,14
were investigated along with anions such as halides and hydrophobic
bis(trifluoromethylsulfonyl)imide [Tf N] and (trifluorotris(perfluoroethyl)phospate [FAP]. The
2
model ionic liquid, [HMIm][Tf N] (an IUPAC/IACT standard), demonstrated the highest affinity
2
for NDHD (K = 2.8) while phosphonium and ammonium ionic liquids with bulkier alkyl side
C
chains had the lowest extractability (K < 0.5). Incorporating polar functional groups by
C
replacing one of three octyl side chains in the trioctylammonium cation with side chains
containing functional groups such as carbonyl and cyano increased the partition coefficients (K
C
> 2) illustrating the role of molecular design for improvement.
Ternary diagrams for the extraction of ABE solutes from water using model ionic liquid,
[HMIm][Tf N] were developed. Acetone and 1-butanol solutes were favorably extracted from
2
water with high selectivities while its affinity for ethanol was low. NRTL activity coefficient
iii
model was used to model the ternary data and a regression program was written to obtain the
binary interaction parameters for the ternary system. Simulation of 1-butanol extraction using
[HMIm][Tf N] was conducted using process simulator, Aspen Plus. Energy analysis was
2
performed on an optimized flowsheet and these results along with equipment costs were
compared with traditional organic solvent extraction and distillation.
The toxicity of twenty different ionic liquids to the mutant strain of E. coli was tested with
results varying from biocompatible to antimicrobial evidenced from EC values of growth rates.
50
Here, the molecular toxicity was measured and EC refers to concentration of the ionic liquid
50
that reduces cell growth rate by 50% at molecular level; the EC was compared with water
50
solubilities, to determine if the ionic liquid was toxic at molecular level. As widely known, the
increase of alkyl-chain length increased the toxicity. However, even highly alkylated cations
may become biocompatible by the choice of anion, for e,g, trioctylmethylammonium bromide
was antimicrobial while with [Tf N] anion, it was biocompatible. The mechanism of growth
2
inhibition in presence of ionic liquids was studied through imaging and initial explanations of
possible inhibition mechanisms includes the effect of dissociation of these ionic liquids on how
the cations/anions interact with the cell membrane.
Fermentation broths can be viscous and exhibit non-Newtonian behavior and efficient liquid-
liquid contacting is required for higher mass transfer rates and hence faster extractions.
Intensification of contacting of non-Newtonian rheology fluids was studied by the application of
electric fields. Continuous phase was Mineral oil containing a rheological modifier while
aqueous carboxymethylcellulose (CMC) solutions were used as dispersed phase with the
apparent viscosities varying between 1 cP to > 1000 cP. Significant reduction in drop size was
observed when the applied voltages were varied between 0 to 15 kV; viscosity of the continuous
phase resulted in lower terminal velocities while dispersed phase viscosity affected the droplet
formation times. Empirical correlations for droplet diameter as a function of physical properties,
nozzle dimensions and electric field strength were developed and discussed here.
Keywords: in situ extraction, ionic liquids, NDHD, Acetone-Butanol-Ethanol (ABE), toxicology / biocompatibility,
phase equilibrium, energy analysis, liquid-liquid contacting, non-Newtonian rheology, charged droplets, DC electric
fields
iv
ACKNOWLEDGEMENTS
I would like to express my sincerest gratitude to my advisor Professor Aaron Scurto and Co-
advisor Professor Laurence Weatherley for their guidance, motivation, continuous
encouragement and patience through the course of my research and thesis writing process. I
would like to thank my committee members, Professors Cory Berkland, Bala Subramanian and
Belinda Sturm, for their cooperation and for serving on my committee.
I would like to thank Professor Stevin Gehrke, for providing constant access to his lab
equipment critical for biocompatibility studies and Professor Michael Detamore & his lab
members for being cooperative while conducting these studies. I would like to acknowledge the
CPE TORP group for allowing me to use their rheometers to characterize the non-Newtonian
fluid rheology and the Microscopy & Analytical Imaging Laboratory for their help with imaging
of the bacteria.
I would like to acknowledge the help from the Center for Biocatalysis and Bioprocessing at
the University of Iowa; Professor Tonya Peeples and her group members for sharing bacterial
strains; Professor Jack Rosazza for arranging for NDHD stock samples; frequent interactions
about cell culture techniques with various group members were helpful. I would like to
acknowledge Professor Jerzy Petera, University of Lodz, Poland for his interest and interactions
along with inputs on the modeling of charged droplet trajectories in non-Newtonian systems.
I would also like to acknowledge the financial support from KU, CEBC and DuPont;
members of CEBC, especially Claudia Bode, for her constant encouragement; CPE staff
including the lab technicians.
I would like to acknowledge the companionship with various former CPE graduate students:
Zheyan Qiu, during process intensification lab setup; Wei Ren, Azita Ahosseini and Sylvia
Nwosu during Ionic liquid studies. I would like to acknowledge the work performed by Nameer
and Wei Ren on developing overall ternary diagrams for ABE solutes. I would like to
acknowledge all my roommates who made my adjustment to graduate student life easier and fun,
especially Milind Singh, Sagar Sarsani, and Prakash Manikwar.
Finally, I am indebted to my parents Sastry and Lakshmi for instilling a strong sense of
purpose, values and spirituality. I would like thank my in-laws, Murthy, Nirmala and Harish, for
their faith in me and constant encouragement. I am ever grateful to my brother Prashanth and
v
sister-in-law, Jyothi, for their love and support; their children Vikram and Varsha for bringing a
smile. Most importantly, I offer special thanks to my wife, Sirisha, for enduring the hardships of
a tough graduate life, patiently pushing me to achieve my goals and for her unending love for
me.
vi
Title Page
Acceptance Page
Abstract
Acknowledgements
Table of Contents
1. INTRODUCTION ............................................................................................................ 1
1.1. Overview ............................................................................................................ 1
1.1.1. Introduction to Biocatalysis .......................................................................................... 1
1.1.2. Types of biocatalysis .................................................................................................... 2
1.1.3. Downstream processing ................................................................................................ 3
1.1.4. In situ product removal ................................................................................................. 5
1.1.4.1. Overview .................................................................................................................. 5
1.1.4.2. Extractive or biphasic biocatalysis ........................................................................... 7
1.1.4.3. Solvent extractability ................................................................................................ 8
1.1.4.4. Toxicity .................................................................................................................... 9
1.1.4.5. Solvent selection criteria ........................................................................................ 10
1.1.4.6. Ionic liquids ............................................................................................................ 12
1.1.5. Outline of the document ............................................................................................. 13
1.2. Review of downstream processing of model biocatalytic systems ................................. 14
1.2.1. NDHD model system.................................................................................................. 14
1.2.1.1. Overview ................................................................................................................ 14
1.2.1.2. Literature review on NDHD extraction .................................................................. 15
1.2.2. Introduction to ABE model system ............................................................................ 19
1.2.2.1. Overview ................................................................................................................ 19
1.2.2.2. Production of ABE solutes ..................................................................................... 19
1.2.3. Literature review of down-stream processing of 1-butanol from fermentation systems
.................................................................................................................................... 25
1.2.3.1. Ex situ solvent extraction ....................................................................................... 26
vii
1.2.3.2. In situ extraction ..................................................................................................... 29
1.2.3.3. Other methods ........................................................................................................ 32
1.2.3.4. Economic and energy analysis ............................................................................... 34
1.3. Application of ionic liquids to whole-cell biocatalysis .................................................... 39
1.3.1. Literature review of biphasic biocatalysis using ionic liquids ................................... 39
1.3.1.1. Asymmetric reduction reactions ............................................................................. 39
1.3.1.2. Hydrogenation reactions ......................................................................................... 59
1.3.1.3. Scale up studies ...................................................................................................... 63
1.3.1.4. Application of Ionic liquids to Clostridia fermentation ......................................... 64
1.3.2. Toxicity of Ionic liquids ............................................................................................. 66
1.3.3. Summary ..................................................................................................................... 72
1.4. Electric fields .......................................................................................................... 73
1.4.1. Product separation techniques .................................................................................... 73
1.4.1.1. Liquid-liquid extraction for bioseparations ............................................................ 73
1.4.1.1.1. Mixing ............................................................................................................. 74
1.4.1.1.2. Rheology .......................................................................................................... 74
1.4.1.2. Solvent extraction equipment ................................................................................. 75
1.4.1.3. Intensification of liquid-liquid contacting .............................................................. 77
1.4.2. Background ................................................................................................................. 77
1.4.2.1. Types of electric fields ........................................................................................... 78
1.4.2.2. Applications ............................................................................................................ 79
1.4.3. Electrostatic spraying ................................................................................................. 80
1.4.3.1. Theory .................................................................................................................... 80
1.4.3.2. Selection of continuous phase and dispersed phase ............................................... 83
1.4.3.3. Space charge migration .......................................................................................... 85
1.4.4. Effect of electric field on key properties .................................................................... 87
1.4.4.1. Drop charge ............................................................................................................ 87
1.4.4.2. Droplet formation and droplet diameters ............................................................... 90
1.4.4.3. Droplet velocities – terminal velocities .................................................................. 94
1.4.5. Literature review......................................................................................................... 99
viii
1.4.5.1. Presence of broth components .............................................................................. 100
1.4.5.2. Mixing related ...................................................................................................... 101
1.4.5.3. Mass transfer ........................................................................................................ 102
1.4.5.4. Droplet coalescence .............................................................................................. 106
1.4.6. Design and Scale-up of electrical enhanced contactors ............................................ 109
1.5. References .................................................................................................................... 111
2. EXPERIMENTAL DETAILS ........................................................................................ 116
2.1. Synthesis of ionic liquids ............................................................................................. 116
2.1.1. Synthesis of Imidazolium based ionic liquids ...................................................... 116
2.1.2. Synthesis of Ammonium based ionic liquids ....................................................... 117
2.2. Toxicology studies ....................................................................................................... 120
2.2.1. Bacterial cell culturing ......................................................................................... 120
2.2.2. EC50 calculations ................................................................................................. 122
2.2.3. SEM microscopy .................................................................................................. 122
2.3. Partition studies ............................................................................................................ 123
2.3.1. Equilibrium/Partitioning ....................................................................................... 123
2.3.2. Solute concentration measurement using HPLC .................................................. 124
2.3.3. Solute concentration measurement using GC ...................................................... 125
2.3.4. Ionic liquid concentration measurement .............................................................. 127
2.3.5. Karl-Fischer coulometer ....................................................................................... 129
2.4. DC Electric fields ......................................................................................................... 130
2.4.1. Synthesis of non-Newtonian continuous and dispersed phases ........................... 130
2.4.1.1. Preparation of Continuous phase ................................................................... 130
2.4.1.2. Preparation of Dispersed phase ..................................................................... 131
2.4.1.3. Equilibration of phases .................................................................................. 132
2.4.2. Experimental setup ............................................................................................... 132
2.4.2.1. Contacting column configuration and geometry ........................................... 132
2.4.2.2. High voltage electric field ............................................................................. 135
2.4.2.3. Electrodes ...................................................................................................... 135
2.4.2.4. Perfusor calibration ........................................................................................ 136
ix
2.4.2.5. Imaging .......................................................................................................... 137
2.4.2.6. Image analysis software................................................................................. 138
2.4.2.7. Droplet trajectory ........................................................................................... 138
2.4.2.8. Droplet diameter ............................................................................................ 139
2.4.3. Physical property measurement ............................................................................ 140
2.4.3.1. Density ........................................................................................................... 140
2.4.3.2. Interfacial tension .......................................................................................... 141
2.4.3.3. Rheology ........................................................................................................ 143
2.4.3.4. Electrical conductivity ................................................................................... 144
2.4.3.5. Dielectric constant ......................................................................................... 145
2.4.4. Description of a sample experimental run ............................................................ 146
2.4.5. Obtaining time dependent xyz data and droplet diameters .................................. 148
2.4.6. Experimental parameters studied ......................................................................... 149
2.5. Materials ....................................................................................................................... 150
3. TOWARDS IN SITU EXTRACTIVE OF FINCE CHEMICALS USING IONIC
LIQUIDS ........................................................................................................ 152
3.1. Evaluation of In situ extraction and fermentation techniques ...................................... 152
3.1.1. NDHD extraction using Organic solvents ............................................................ 152
3.1.2. In situ fermentation using a biocompatible organic solvents ............................... 155
3.1.3. NDHD extraction via Adsorption ......................................................................... 157
3.2. Ionic liquid as a potential in situ extractant .................................................................. 161
3.2.1. Relevance of partitioning data .............................................................................. 161
3.2.2. Relevance of toxicology ....................................................................................... 162
3.2.3. Problem statement ................................................................................................ 162
3.3. Partitioning ................................................................................................................... 162
3.3.1. NDHD .................................................................................................................. 162
3.3.2. Effect cations on the partitioning ......................................................................... 166
3.3.3. Effect of anions on the partitioning ...................................................................... 167
3.4. Toxicology ................................................................................................................... 168
3.4.1. Cell growth plot: Time vs. Optical Density (OD) ................................................ 168
x
Description:Cost-effective synthesis of fine chemicals/biorenewable fuels via fermentation invariably hinges upon efficient separations from the dilute broth. Biphasic/in situ extraction can positively impact both upstream fermentation and the downstream product separations. For developing environmentally
