Department of Chemical and Petroleum Engineering
The University of Kansas
Comprehensive Examination
Continuous Enzymatic Peracetic Acid Production for in situ Wastewater Disinfection
Victor Kumar Sharma
Presented to the graduate research committee:
Alan M. Allgeier, Ph.D.
Bala Subramaniam, Ph.D.
Kyle Camarda, Ph.D.
Juan Bravo Suarez, Ph.D.
Justin M. Hutchison, Ph.D.
August 31, 2022
Project Summary
Overview: Wastewater management is a crucial component of several sustainable development goals of
the United Nations, which pledge to increase wastewater recycling and safe reuse globally substantially.
Established methods and practices have several technical, economic, and environmental challenges, which
can be overcome by employing peracetic acid-based disinfection systems. However, limited production
capacity globally and issues related to safety, transportation, storage, and chemical stability of peracetic
acid are some major deterrents behind its wide application in municipal wastewater disinfection systems.
Literature accounts of biocatalytic method of peracetic acid are mostly limited to trivial biocatalyst
characterization in lab-scale batch systems. Continuous enzymatic peracetic acid production has not yet
been demonstrated on a preparative scale. Moreover, mechanistic aspects of potential enzyme inhibition or
deactivation by peracetic acid are also not yet well-understood.
The proposed research hypothesizes that a continuous biocatalytic system can be developed by
immobilizing the biocatalyst onto functionalized polymeric resins to produce peracetic acid at targeted
concentrations for in situ wastewater disinfection applications. This goal will be accomplished by (1) Using
an optimization-based approach to covalently immobilize a selected perhydrolase enzyme onto
commercially available primary amine functionalized ion-exchange resin via glutaraldehyde activation and
characterize it to understand the catalyst-support interactions; (2) Studying and comparing thermodynamic
and kinetic stability of the free and immobilized biocatalyst; (3) Using the immobilized biocatalyst in a
continuous packed bed reactor and optimize the system for the production of peracetic acid at a targeted
concentration; and (4) Demonstrating the effectiveness of this continuous system for disinfection of
secondary effluents resourced from local municipal wastewater treatment facilities. The project will be
implemented over a three-year period, along with the educational training of one graduate student
researcher and three undergraduate researchers at the University of Kansas. Facilities are available across
STEM departments in KU, including but not limited to research centers such as the Vaccine Analytics and
Formulation Center (VAFC), Center for Environmentally Beneficial Catalysis (CEBC), and the Institute
for Bioengineering Research (IBER). Collaborations will be made with Fort Lewis College, Durango, CO,
for enzyme synthesis and purification expertise. Holistically, this project will also pioneer a
multidisciplinary approach to developing a novel, sustainable wastewater disinfection strategy.
Intellectual Merits: The proposed research will reveal previously uncharacterized yet fundamental
insights into the enzyme’s catalytic mechanism. Thermodynamic and kinetic studies on the free and
immobilized enzyme will reveal latent cognizance of the stabilization mechanism of the enzyme upon
immobilization. The study will also demonstrate optimization-based approaches for (1) evaluating ideal
immobilization parameters for this enzyme-support pair and (2) production of a targeted concentration of
peracetic using this immobilized enzyme. This study will also consider the aspect of mass transfer
limitations crucial to continuous reactor systems, yet largely unexplored in the realm of biocatalysis. In
addition, the effects of practical wastewater conditions, like pH, dissolved ions, residual particulates, etc.,
on the catalytic activity of the free and immobilized enzyme will also be assessed.
Broader Impacts: Through promoting a novel approach to wastewater disinfection method with the
direct application of biocatalyst, the proposed research addresses a vital aspect of the overall well-being of
contemporary society. Immobilized biocatalysts will aid in achieving a long-term sustainability strategy by
avoiding a demand for chemically produced peracetic acid and enabling continuous in situ wastewater
disinfection. The proposed method can be easily integrated with existing municipal wastewater treatment
systems with minimal or no modifications to the established process. The actions anticipated to carry out
this proposal would have wider effects at several levels. By employing a biocatalytic route and using
renewable feedstock like glycerol derivatives, concepts of green chemistry are infused into this study,
inspiring thoughtful ideas on developing sustainable wastewater management systems. The P.I. will be
responsible for leading the project and managing technical and professional mentorship programs for
graduate and undergraduate researchers using KU-based initiatives to engage students from diverse
backgrounds.
Table of Contents
1. Project Description
1
1.1. Background Information
1.1.1. Motivation
1.1.2. Peracetic acid for wastewater disinfection
1.1.3. Status/challenges of PAA production and application in wastewater disinfection
systems
1.1.4. Enzymatic PAA generation
1.1.5. Enzyme immobilization overview
1.1.6. Review of Immobilization of esterase enzymes
1
1
1
3
1.2. Hypotheses and Research Goals
6
1.3. Objectives and Research methodologies
1.3.1. Objective 1: Synthesis and immobilization of recombinant AXE from B. subtilis
and characterization of the immobilized enzyme
1.3.2. Objective 2: Evaluation of thermodynamic and kinetic stability of BsAXE and
BsAXE@GA/PA110
1.3.3. Objective 3: Continuous PAA production using immobilized BsAXE in packed
bed reactor
1.3.4. Objective 4: Disinfection studies and optimization of target PAA concentration
and contact time
6
6
3
4
5
9
12
13
1.4. Project Timeline
14
1.5. Broader Impacts
1.5.1. Outreach and Dissemination
1.5.2. Personnel Development
1.5.3. Diversity, Equity, Inclusion, and Belonging
15
15
15
15
2. References
16
3. Facilities, Equipment and Other Resources
21
4.1. Proposed Budget
22
4.2. Budget Justification
30
1. Project Description
1.1. Background Information
1.1.1.
Motivation
With rapidly escalating demands on water resources globally, adequate and appropriate wastewater
management is paramount to the overall environmental sustainability.1 Disinfection is one of the most
critical aspects of overall wastewater management which ensures its effective reuse in crucial sectors like
agriculture.2 In fact, the United Nation’s sustainable development goal 6.3 is to substantially increase the
recycling and safe reuse of wastewater globally.3 Briefly, wastewater treatment is done in two sequential
stages- primary and secondary treatment, often with the addition of a third or tertiary treatment stage.4
Primary treatment targets mostly coarse solids, settleable solids, and suspended particulate solids. The
secondary treatment targets pollutants like fine particulate biological oxygen demand (BOD), dissolved
solids, nutrients, and pathogens. Following secondary treatment, disinfection of the treated wastewater is
crucial because it contains harmful pathogens like bacteria, parasite cysts and eggs, viruses and fungi which
can spread diseases like cholera, typhoid, tuberculosis, dysentery, and hepatitis, among other water-borne
diseases.5 Inadequate and/or improper disinfection of wastewater can endanger human lives pathogens may
end up contaminating freshwater bodies, which also pose huge threats to aquatic lives due to a potential
increase in the biological oxygen demand.6 Disinfection is done after secondary treatment prior to the
release of the water into water bodies using disinfecting agents like chlorine, ozone, hydrogen peroxide,
performic acid, peracetic acid, or by physical methods like UV irradiation, ultrasound, membrane
separation, or a combination of both chemical and physical methods.7,8
Chlorine in the form of calcium hypochlorite, sodium hypochlorite, chlorinated lime, or chlorinated
water is very commonly used for disinfection application and is considered to be the most cheapest
disinfection method.7 However, chlorine-treated wastewater is found to have formed
mutagenic/carcinogenic compounds like tricholoromethanes, trichloroamines and chloroacetic acid,9 which
increase with the chlorine dosage.10 In addition, certain viruses, bacterial spores, and protozoa oocysts are
not entirely eliminated on chlorine application.7 Ozonation is also effective method of disinfection which
facilitates the decimation of microorganisms by decomposing into reactive oxygen species like superoxide,
hydroperoxyl, and hydroxyl radicals.11 However, some studies have found that ozone treatment can also
lead to the formation of trihalomethanes and haloacetic acids.12 UV (ultraviolet radiation) based
disinfection systems are quite effective compared to chlorine, ozone, and peracetic acid in reducing
microorganisms, as observed by measuring the total coliform as a function of contact time. 13 However, it
requires high recurring costs owing to periodic replacement of the lamps and is unsuitable for large
wastewater treatment facilities. Moreover, there are challenges associated with the design and construction
of UV reactors, eliminating particle-bound microorganisms as well as ambiguity regarding regrowth, repair
and photoreactivation of bacterial DNA.14 In essence, despite the existence of established chemical and
physical methods like chlorination, ozonation, and UV, each of these methods have unique challenges
associated with their design, construction and operations. Since liquid chemical disinfection methods are
advantageous in terms of capital cost, operational ease and rapid startup, there is a compelling need to seek
for alternative and more potent methods like peroxy-compound (hydrogen peroxide, performic acid,
peracetic acid, etc.) based disinfection systems that could reach the target coliform levels at competitive
contact times compared to existing technologies while also tackling the aforementioned challenges.15
1.1.2.
Peracetic acid for wastewater disinfection
Peroxy-compounds like hydrogen peroxide (H2O2) and peracetic acid (PAA) have been known for their
antimicrobial capabilities and application as effective antiseptics and sterilizers.16 Compared to H2O2, PAA
is recognized to be a substantially more potent disinfecting agent based on its low dosage requirement for
the same target coliform levels in treated wastewater. To put into context, dosage required for PAA could
1
approximately be 175-475 times lower than that of H2O2.15 Due to its extensive anti-microbial capability,
low capital cost, ease of integration with existing municipal wastewater treatment facilities, absence of toxic
or mutagenic byproducts, short contact time, and low relative dependence on pH, PAA is deemed to be a
highly promising candidate for disinfection of municipal wastewater.15,17
PAA is an effective oxidant with an oxidation potential of 1.959 V vs standard hydrogen electrode and
a second order rate constant for oxidation of organic compounds up to the order of 10-5 M-1s-1.18 Due to its
highly electrophilic nature, PAA can carry out electrophilic attack on the electron-rich functional groups
present in organic compounds. Malchesky suggested that PAA attacks the S-H (sulfhydryl) and S-S
(disulfide) bonds present in the microbial cell membranes which inactivates the microbial cells through
oxidative disruption,19 possibly by producing reactive oxygen species like superoxide and hydroxyl
radicals.20 PAA was found to be effective against a range of microbes like bacteria, viruses, bacterial spores,
and protozoan cysts.21 Although PAA has lesser tendency of forming halogenated byproducts compared to
chlorine, there is potential of some halide ions to get oxidized to its hypohalous acid form. Typically, it
requires 30 minutes of contact time at a PAA concentration of 2-7 mg/ml for 3 log reduction in total
coliform for secondary effluent.22 Using commercially available PAA at 2 mg/L, Flores et al. were able to
demonstrate 99.9% inactivation (~4 log reduction) of E. coli in just 5 minutes (Figure 1).23 A recent pilot
scale study by Metropolitan Sewer District of Greater Cincinnati and the Office of Research and
Development in Cincinnati, Ohio, established that 2 mg/L (2 ppm) dosage of PAA was sufficient for up to
1.5 and 1.8 log reductions of E. coli and
fecal coliform, respectively.24 Another
pilot study at Miller st. Wastewater
Treatment Plant in Orange Park, Florida,
established that 9 mg/L dosage of PAA
was sufficient for up to 3.3 log reduction
of fecal coliform.25 The highest dosage of
PAA reported so far in the literature was
480 mg/L (480 ppm) which was required
in municipal sludge disinfection, and not
treated wastewater.26 Contact time and
PAA dosage are mutually inversely
proportional for a target level of coliform
removal. For effective disinfection based
on a target coliform removal, in addition
to dosage and contact time, the
Figure 1. Decrease in colony forming units (CFU) as a function of
2
wastewater quality and composition is
time employing the commercial mixture of PAA. (Slope and R for
also an important factor. Table 1 provides
those plots that show features corresponding to a straight line in a
a summary of log reduction results from
significant portion of their trajectory). Reprinted from Ref. 23
different published studies.
Table 1. Summary of dosage, contact time, wasterwater type, organism removed, and log reduction data from
several published studies.
PAA dosage
Contact time
Wastewater type
Organism
Log reduction
Reference
5-10 mg/L
5-7 mg/L
6 mg/L
2 mg/L
9 mg/L
1.5 mg/L
2 mg/L
15 min
60 min
5 min
20 min
60 min
53 min
30 min
Secondary effluent
Secondary effluent
Secondary effluent
Secondary effluent
Secondary effluent
Tertiary effluent
Treated wastewater
Fecal coliform
Total coliform
Thermotolerant coliform
E. coli
E. coli
E. coli
E. coli
3.7
3.7
1.5
1.5
3.4
1.5
1
27
2
28
29
24
25
30
31
1.1.3.
Status/challenges of PAA production and application in wastewater disinfection systems
PAA is commercially available as an equilibrium mixture containing PAA, acetic acid, H2O2 and water
where PAA concentration is generally within the range of 8-13.5 % by weight.32 The preferred route of
PAA production is oxidation of acetic acid with H2O2 according to equation (1), which can be accelerated
by an acid catalyst like sulfuric acid:33
CH3C(O)OH + H2O2 ↔ CH3C(O)OOH + H2O.
(1)
PAA is thermodynamically unstable and might cause explosions when highly concentrated. So, in
typical industrial processes, 37% PAA is obtained with 61% water, which is further diluted to lower
concentration and stabilized with dipicolinic acid34 or other proprietary stabilizers. In aqueous solution,
PAA spontaneously decomposes to acetic anion, oxygen, and H+. The rate of PAA decomposition increases
with pH up to a maximum at pH 8.2, which is a challenging aspect to implementation in wastewater
disinfection. It was found that dilute PAA solution (1% by weight) could lose up to half of its potency over
6 days.35 Furthermore, dissolved cations present in wastewater can catalytically decompose PAA36,37 along
with other wastewater matrix components like dissolved organic carbon, phosphate anions, peptones, total
suspended solids, salinity, hardness, etc.18 Other challenges like low storage temperature and highly
corrosive nature of PAA solution are also limiting factors behind PAA application for wastewater
disinfection.
For unstable chemicals like PAA, on-site production can be a viable option to overcome challenges
related to storage and transportation and alleviate safety concerns. However, process safety is still a
challenge on-site with existing technology. Moreover, on-site PAA production would require huge capital
investments and might not be a feasible option for existing wastewater treatment facilities. Despite the
concerns related to process safety, storage, and transportation, PAA is advantageous over other methods in
several ways as discussed in section 1.1.2.
1.1.4.
Enzymatic PAA generation
Challenges related to PAA production could be overcome using safer and more sustainable methods like
enzymatic PAA production. Certain enzymes having perhydrolase activity belonging to the subgroup of
serine hydrolases, are able to produce PAA at concentrations adequate for disinfection
applications.38,39There are several benefits of enzymatic synthesis over chemocatalytic methods, like high
selectivity, use of aqueous reaction medium, room temperature and pressure operation, and replenishable
source of catalyst production.40 Biocatalytic methods can operate efficiently only at dilute concentrations
as high substrate/product concentrations can induce inhibitory effects on the enzyme, often leading to
irreversible loss of catalytic activity.41 For industrial processes requiring bulk chemical products which are
of low to moderate cost, biocatalysis therefore are not generally considered to be an economically viable
option. However, in the context of wastewater disinfection application, PAA dosage is apparently required
at concentrations up to 25 mg/L or 0.33 mM, which can easily
be achieved in situ with an enzyme catalyzed system. In a
proprietary method, Dicosimo et al. demonstrated that up to
9g/L of PAA could be produced using a carbohydrate esterase
as catalyst and esters of alcohols and glycerols as the substrate
in 5 to 30 minutes in presence of H2O2.42 This concentration is
much higher than what is required for disinfection applications
as evident from previous literature studies (Table 1). Several
perhydrolase enzymes, belonging to the enzyme class acetyl
Figure 2. Stabilization of peroxygen donor
xylan esterases (AXEs), have been identified, which can
at the catalytic triad of a perhydrolase
produce peracetic acid from acetyl esters of glycerol in
enzyme. (Reprinted from Ref. 44)
presence of H2O2.38,39,43
3
Esterase enzymes having perhydrolase activity, contain an active site made up of a Ser-His-Asp catalytic
triad, in which a carboxylic acid forms an acyl-enzyme intermediate with the serine group, followed by
reaction with H2O2 to generate a peracid.44 It was suggested that a strong hydrogen bond was formed
between the peroxy hydroxyl group with the carbonyl oxygen of a proline residue in the vicinity of the
active site catalytic triad which facilitated the peroxide attack on the acyl-enzyme intermediate (Figure 2).
It was also suggested that the kinetic mechanism could be explained by a 2-substrate ping-pong model. Tao
et al. hypothesized that a similar mechanism was responsible behind the catalytic activity of an AXE
enzyme isolated from Bacillus subtillis CICC 20034, which was demonstrated to produce up to 138 mM of
PAA at pH 8 and 20° C using triacetin as the substrate. However, the authors determined the kinetic
parameters of the B. subtilis with different acetyl donor substrates and H2O2 using the single substrate
Michaelis Menten model (Table 2) by varying each substrate concentration in turn while keeping the other
in turn.39
Table 2. Kinetic properties AXE from B. subtillis39
Varied
substrate
KM
(H2O2)
[mM]
KM (acetyl donor)
[mM]
Kcat/KM (H2O2) [s−1
M−1]
Kcat/KM (acetyl donor) [s−1
M−1]
H 2O 2
1.11 ± 0.19
-
3298
-
Triacetin
-
140.70 ± 33.94
-
2534
Ethyl acetate
-
409.12 ± 48.73
-
89
There are enough choices of enzymes with perhydrolase activity, which could catalyze the production
of PAA from esters of alcohols, diols, and glycerols, which could be leveraged for in situ PAA production.
Cell free biocatalysis has proven to be beneficial over the whole cell process in the context of several
industrial applications.45 However, for large-scale application with very low margin of profit like that of
wastewater treatment, biocatalytic methods have not been implemented widely due to cost-related
concerns.46 The notion that enzymes suffer from irreversible loss of catalytic activity driven by factors like
high temperature, pH, and high concentration of organic substrates is the root cause behind these concerns.
Recyclability of the enzymes is also a limiting factor behind the scale-up of enzymatic processes. A
common strategy of minimizing the impact of both these challenges is by immobilizing the enzyme on
inert, solid supports. Immobilization has been found to stabilize the enzyme by making it less susceptible
to inhibition by the organic molecules and allows the recyclability by retaining them within the reactor.
Pertaining to this study, immobilized AXEs will facilitate the continuous generation of H2O2 by enzyme
retention within the reactor and minimize potential inhibition from H2O2 and acetyl donor like triacetin.
1.1.5.
Enzyme immobilization overview
In the context of large-scale and/or industrial applications, limited operational stability of free enzymes
often preclude their use as catalysts despite excellent catalytic properties. Operational stability issue may
arise from high temperature, pressure, presence of organics and/or high salt concentration in the process
fluids, all of which could be detrimental to enzyme proteins.47 In general, protein destabilization can be
analyzed from a thermodynamic or a kinetic aspect.48 Denaturation or unfolding under thermally induced
conditions which is related to its thermodynamic stability can lead to partial to total loss of enzymatic
activity. Kinetic stability can be reduced due to presence of inhibitors which can also be the reactant or
product at high concentrations, which form unreactive intermediates leading to loss of catalytic activity.
Immobilization is generally referred to the process of supporting enzymes on to a solid material to
transform it into a heterogeneous catalyst, which enhances the stability sometimes up to 60,000 times for
some enzymes.49 Immobilization of enzymes on suitable solid support helps retaining the enzyme and
enables the flexibility of using them in different reactor configurations like in different kinds of flow
reactors or reused in batch and fed batch reactors. Attachment of enzymes to solid supports promote
4
thermodynamic stability by making them
less susceptible to conformational
changes and denaturation.50 In most
cases, enzyme activity is found to slightly
decrease upon immobilization, however,
in certain lipases, it increases due to
promotion of open conformation, which
is the catalytically active form of the
enzyme.51
Broadly,
enzyme
immobilization strategies can be
categorized into four main types: (i)
adsorption,
(ii)
Figure 3. Major types of enzyme immobilization strategy.
entrapment/encapsulation,
(iii)
(Reprinted from Reference 52)
crosslinking, and (iv) covalent bonding
(Figure 3).52 Adsorption is based on weak attractive forces like hydrogen bond between the enzyme and the
support, which allows easy leaching of the enzyme. Entrapment and encapsulation are effective in
protecting the enzyme from rapid conformational changes; however, it introduces high mass transfer
resistance. Entrapment and encapsulated enzymes are useful for therapeutic and drug delivery
applications.53 Crosslinking usually generates aggregated enzyme molecules around the solid support and
is often used to compliment covalently bonded or encapsulated/entrapped enzymes. The degree of
crosslinking highly influences the conformational integrity of the enzymes 54 and is not a widely
implemented method for large-scale syntheses. Covalent bonding for multi-point attachment of enzymes to
amine functionalized supports is well studied in the literature and is proven to be suitable for several
enzymatic processes.55 Glutaraldehyde (GA) is widely used for multi-point covalent attachment of enzyme
to the support. The aldehyde-amine interaction on one end creates the imine linkage between the GA and
the support, while the same linkage on the other end is formed between the non-ionized primary amine
group of the enzyme.
1.1.6.
Review of Immobilization of esterase enzymes
Several accounts of esterase enzymes with hydrolase activity56–58 and at least three instances of AXEs
with perhydrolase activity39,54,59 were reported in relevant literature with regards to immobilization on solid
supports. For esterases with hydrolase activity, different immobilization techniques like adsorption
technique on glass fiber membranes,56 encapsulation in calcium alginate hydrogel beads,57 and covalent
adsorption on magnetic cornstarch.58 These studies demonstrated that immobilized enzymes exhibited
decent residual activity, higher thermostability and recyclability. Immobilization of AXEs with
perhydrolase activity were limited to covalent bonding on modified graphite oxide59 or acrylate amino
resin39 and crosslinking with GA on chitosan modified iron oxide nanoparticles.54 . With regards to
hydrolase esterase, Malik et al. laid out the experimental strategy for characterizing kinetic parameters in a
packed bed configuration, considering mass transfer limitations.57 However, with regards to perhydrolase
enzymes like AXEs, published studies were limited to trivial activity measurements and kinetic studies
using the candid single-substrate Michaelis-Menten model.60 Using this model, where each substrate is held
constant and the other is varied, Tao et al. derived the kinetic constants of a recombinant AXE of B. subtilis
demonstrated a covalent bonding immobilization strategy using commercially available primary amine
functionalized polymer resins.39 It was shown that immobilization could facilitate biocatalyst reuse in batch
reaction and produce PAA at concentrations of up to 11000 ppm within 5 minutes.
1.2. Hypotheses and Research Goals
The comprehensive discussion in the preceding section suggests that a continuous enzymatic PAA
synthesis strategy could mitigate the challenges associated with PAA production and enable effective in
5
situ wastewater disinfection. Proprietary work on enzymatic PAA synthesis has shown that process
conditions can be modulated to produce a “target” concentration of PAA required for the corresponding
application.38 Based on the literature presented in Table 1, it can be safely assumed that no more than 500
ppm of PAA dosage would be required for efficient wastewater disinfection application, which is
approximately 4.5% of the amount of PAA that could be produced using AXE from B. subtilis (BsAXE).39
Immobilization of BsAXE can be leveraged for continuous PAA production at targeted concentration for
effective disinfection application, which is yet largely unexplored. The proposed study will consider key
aspects like continuous reactor configurations, inhibition effects of substrates, products and/or H2O2, and
effect of treated wastewater conditions on the immobilized enzyme, are paramount to in situ PAA
disinfection. Based on the understandings of (i) effectiveness of PAA as a disinfectant for wastewater, (ii)
capability of AXEs as suitable biocatalysts for PAA synthesis, (iii) enhancement of enzyme stability upon
immobilization, and (iv) advantages of continuous reactor systems towards large scale applications,61 the
following hypotheses are proposed:
(a) Covalent immobilization of BsAXE on amine functionalized support will enhance the
thermodynamic and kinetic stability of the enzyme in practical wastewater conditions (pH,
dissolved salts, etc.) and facilitate in situ production of PAA.
(b) Low target concentration of PAA (<500 ppm) can be achieved in a continuous reactor like a packed
bed reactor by modulating the amount of catalyst in the reactor, feed flow rate and molar
concentration of substrates in the feed.
(c) Target concentration and contact times can be further optimized based on coliform reduction
studies of the treated water stream.
Therefore, the over-arching research goal of the proposed work is to develop and investigate a
continuous reactor system for in situ peracetic acid generation using covalently immobilized acetyl xylan
esterase enzyme on commercially available primary amine functionalized polymer resins, with the intended
application towards wastewater disinfection. To accomplish this research goal, evaluation of the hypotheses
proposed above will be achieved through four specific objectives which are described along with requisite
and relevant research methodologies in the following section.
1.3. Objectives and Research methodologies
1.3.1.
Objective 1: Synthesis and immobilization of recombinant AXE from B. subtilis and
characterization of the immobilized enzyme
Recombinant AXE from B. subtilis will be utilized in this study because it is the perhydrolase enzyme
so far reported with the highest yield of PAA. Covalent immobilization of the purified AXE will be done
using Purolite® A110 (PA110) as the support, which is a commercially available primary amine
functionalized macroporous polystyrene resin crosslinked with divinyl benzene. Hydrophobic resins from
Purolite® is known to have successfully used for immobilization of lipase enzyme with higher activity than
native state.62 Furthermore, existing collaboration of Purolite® with the University of Kansas will facilitate
any potential customization needs. Prior to and after immobilization, the purified enzyme activity
measurements will be done using standard assay as described by Tian et al.63 It is expected that enzyme
loading on the support can be estimated by appropriate characterization techniques and immobilization
1.3.1.1.
Expression and Purification of recombinant AXE from B. subtilis: The Cah gene which
encodes the AXE enzyme of interest will be amplified using PCR technique described by Tao et al.39 The
amplified gene will be overexpressed in E. coli and crude enzyme will be purified using the technique
described by Tian et al.63 The purified enzyme (BsAXE) is expected to exist in a trimer form with molecular
weight of 107 kDa (each subunit of 36 kDA). For gene isolation and amplification, B. subtilis culture (CICC
6
20034) will be obtained from China Center of Industrial Culture Collection.64 Requisite chemicals,
enzymes, and other supplies will be obtained from commercial vendors.
1.3.1.2.
Specific activity measurement of BsAXE: Protein concentration measurement will be
done spectrophotometrically with the Bradford assay,65 using Coomassie blue dye as the protein binding
agent. Activity measurement will be done spectrophotometrically at 405 nm using the standard assay
described by Tian et al. with p-nitrophenyl acetate as the substrate at 40 °C in 50 mM potassium
phosphate buffer (pH 7).63 One unit of enzyme activity (U) is defined as the amount of p-nitrophenol in
μmol produced by 1 mg of enzyme per minute, under the assay conditions described above.
1.3.1.3.
Immobilization of purified BsAXE: In general, immobilization procedure will be done
via the following steps:
(a) Purolite® A110 activation using glutaraldehyde in a suitable pH buffer to produce an
activated support (GA/PA110)
(b) Repeated washing and drying with buffer solution to remove excess unbound
glutaraldehyde
(c) Incubation with BsAXE dissolved in a suitable pH buffer for a specific amount of time
followed by buffer wash and freeze drying to obtain the immobilized enzyme
(BsAXE@GA/PA110)
Enzyme loading will be calculated by solution depletion method using the following mass balance
equation:
1
𝐸𝑖 = 𝑤 (𝑉𝐶𝑖 − 𝑉𝐶𝑓 − ∑𝑛𝑗=1 𝑉𝑗 𝐶𝑗 )
(2)
𝑆
Where Ei=enzyme loading (mg/g support), wS=weight of dry GA/PA110 (g), V=volume of
immobilization solution (mL), Ci= enzyme concentration of initial immobilization solution (mg/mL), Cf=
enzyme concentration of immobilization supernatant (mg/mL), Vj (mL) and Cj (mg/mL) are volume and
concentrations of enzyme in the supernatant of each washing process. Activity measurement of the
immobilized enzyme will be done via the same procedure described in section 1.3.1.2. Specific activity of
can be calculated by factoring in the enzyme loading per g of support (Ei).
1.3.1.4.
Optimization of immobilization conditions: Immobilization conditions directly affect
the enzyme loading on the support, which in effect, impact the specific activity of the immobilized enzyme.
In fact, the significance of optimization of experimental conditions with respect to enzyme immobilization
was demonstrated by Pizarro et. al using response surface methodology.66 As a part of hypothesis (b), it
was stated that amount of BsAXE@GA/PA110 in the reactor is one of the operating variables required to
control “target” PAA concentration. By optimizing the specific acitivity of BsAXE@GA/PA110, amount
of required BsAXE@GA/PA110 can be minimized for higher economic viability of the process. In the
proposed study, optimization of immobilization conditions will be done using the response surface
methodology. Specific activity of the BsAXE@GA/PA110 will be used as the only response variable. To
reduce complexity and any possible redundancy of the experimental design, three independent variables
will be used for optimization- (i) enzyme concentration of the initial immobilization solution, (ii) incubation
time, and (iii) glutaraldehyde concentration that will be used for Purolite A110 activation (section 1.3.1.3.,
step (a)). Minitab® Statistical Software will be used for design of experiment (DoE), analysis and numerical
optimization. Central composite method will be used for DoE with standard 20 runs spread into six axial,
eight factorial and six central points within the range of operability. A realistic range of operability will be
selected for all independent variables. A polynomial model like the following would be used for fitting the
experimental data:
𝑦 = 𝛼0 + ∑𝑖 𝛼𝑖 𝑋𝑖 + ∑𝑖 ∑𝑗 𝛼𝑖𝑗 𝑋𝑖 𝑋𝑗 + ∑𝑖 ∑𝑗 ∑𝑘 𝛼𝑖𝑗𝑘 𝑋𝑖 𝑋𝑗 𝑋𝑘 + ⋯
(3),
where 𝑦 is the response variable, 𝑋𝑖 , 𝑋𝑗, 𝑋𝑘 are the independent variables, and 𝛼0 , 𝛼𝑖 , 𝛼𝑖𝑗 , 𝛼𝑖𝑗𝑘 are model
constants. Non-linear regression will be used for evaluating the model constants and cubic terms might be
ignored if good fit is obtained with a quadratic model. ANOVA (analysis of variance) will be used for
further fine tuning of the model by removing insignificant terms and including additional terms if necessary
7
to obtain better response.67 For numerical optimization, initially, the response variable will be set to
maximum, which at a later time, might be revised to particular value based on experimental results from
objective 3 (discussed in section 1.3.3). Regarding constraints, immobilization time and enzyme
concentration will be set to minimize, and glutaraldehyde concentration will be set to a suitable range. It is
expected that this statistical DoE and optimization strategy will enable achieving the maximum possible
specific activity from a reasonable number of experiments and help understand the sensitivity of the specific
activity of the immobilized enzyme on the different immobilization conditions under consideration.
1.3.1.5.
Characterization of Purolite A110, GA/PA110 and BsAXE@GA/PA110: Qualitative
and quantitative analysis of surface and bulk characteristics of the native and glutaraldehyde activated resin,
and the immobilized enzyme will be achieved by employing appropriate analytical techniques.
(a) Surface area and pore volume of PA110, GA/PA110 and Bs@GA/PA110: Gas adsorption technique
will be used to evaluate the surface area and pore volume. Measurements will be taken using Micromeritics
ASAP 2020 using nitrogen as the adsorbate gas at 77 K. The samples will be freeze dried for 24 hours prior
to sorption experiments to remove any residual moisture from the samples. The expected isotherm is
supposed to be a type II isotherm,68 given the macroporous structure of the PA110 resins. For surface area
calculation BET method will be used,69 which is an extension of Langmuir monolayer. The mean pore
volume can be obtained using the pore radius at different pressures which will be calculated from the data
points of the isotherm using the BJH method.70 Total pore volume can be obtained from the total amount
of adsorbed gas at partial pressure ~0.99. Kitchin et al. used gas adsorption methods earlier to analyze
surface area and pore volume of similar macroporous primary amine resin which will be used as a reference
while analyzing the PA110.71 It is expected that changes in BET surface area and pore volume will be
rendered due to GA modification and/or BsAXE adsorption, which will be considered as direct evidence
of successful immobilization of BsAXE.
(b) Total adsorption capacity of PA110: In the scope of the proposed study, the adsorption capacity of
PA110 is defined as millimoles of primary amine group per g of dry PA110. GA being a C-5 spacer arm,
it is possible that high density of amine groups on the resin might engage both aldehydic ends of some of
the GA molecules. This might result in some terminal aldehyde groups of GA being unavailable to interact
with the protein’s terminal amino groups. It is therefore important to quantify the adsorption capacity. In
the proposed study, adsorption capacity will be estimated using CO2 chemisorption by pulse technique.
Measurements will be taken using Micromeritics Autochem 2920, with a calibrated loop for pulsing and
coupled to an Agilent mass spectrophotometer detector. Helium will be used as the carrier gas and 10%
CO2 in He will be used as the adsorptive. The mass flow controller will facilitate the calibration of the MS
detector at various gas flow rates and plotting a calibration curve of CO2 concentration vs signal intensity.
This will be required to quantify total millimoles of CO2 adsorbed/g of GA/PA110 at saturation obtained
from the pulse chemisorption experiment. A schematic representation of the CO2 chemisorption system is
presented in Figure 4.
In
an
orthologous
experiment,
concentrated GA solution will be utilized for
determination of the adsorption capacity of
PA110 by saturating all primary amine sites.
The GA concentration of the initial and
supernatant solution will be analyzed using
an Agilent 6890 gas chromatograph with
Agilent J&W DB-WAXetr column-FID
assembly. Internal standard method will be
used for calibration and analysis. The
adsorption capacity calculated from the pulse
chemisorption experiment will then be used
to calculate the percentage of GA that will be
Figure 4. Schematic representation of a CO2 chemisorption
engaging both single as well as two primary
system coupled with mass spectrophotometric detector
8
amine sites using one or both aldehydic ends respectively, using the following equations:
2𝑄 −𝑄
𝑋𝑆 = 𝐺𝐶 𝐶𝑆
𝑄𝐺𝐶
(4)
𝑋𝐷 = 1 − 𝑋𝑆
(5)
where 𝑋𝑆 and 𝑋𝐷 are the fractions of GA engaging a single or two amine sites respectively, and 𝑄𝐶𝑆
and 𝑄𝐺𝐶 are adsorption capacities determined from the chemisorption and GC measurements, respectively.
It will be assumed that all primary amine sites on the resin will be occupied after treatment with GA for 24
h.
(c) Thermogravimetric Analysis: Thermogravimetric analysis (TGA) of the PA110, GA/PA110, and
BsAXE@GA/PA110 will be done in presence of excess nitrogen up to an adequately high temperature. It
is expected that using TGA analysis, degradation temperature of the resin can be evaluated. Additionally,
any moisture that could be bound to the resin under the storage conditions will be removed within a heating
ramp of 100 ˚C, which will facilitate the dry resin weight calculation that will be used to estimate enzyme
loading and specific activity of the immobilized AXEs.
(d) Fourier Transform Infrared spectroscopy (FTIR): It is common for covalent enzyme
immobilization studies to characterize the presence of relevant functional groups to confirm the presence
of enzymes on the support. Sample preparation will be done by grinding the PA110, GA/PA110 and
BsAXE@PA110 and incorporating them potassium bromide (KBr) pellets. IR spectra will be obtained in a
Perkin Elmer Spectrum 3 instrument between wave number 4000 m-1 and 500 m-1 and. It is expected that
FTIR studies will confirm the appearance of C=N signature peaks as a confirmation of enzyme binding to
the GA activated site of the GA/PA110 resin.
(e) Morphology Studies: Morphology studies will be performed with a Hitachi SU8230 Field Emission
Scanning Electron Microscope to capture high resolution SEM images of PA110, GA/PA110 and
BsAXE@GA/PA110. Morphology studies are commonly used for qualitative assessment of the dispersion
of the enzyme on the support surface and evaluate morphological changes due to enzyme immobilization.54
It can also be used an orthogonal study to determine average pore diameter to verify results from the gas
adsorption experiments.
1.3.2.
Objective 2: Evaluation of thermodynamic and kinetic stability of BsAXE and
BsAXE@GA/PA110
1.3.2.1. Thermodynamic Stability analysis: Enzymes are susceptible to conformational changes induced
by extreme or non-optimum process parameters. Protein unfolding parameters like free energy change of
unfolding (ΔGUF), secondary structure, and melting temperature of protein (Tm) are generally used as
measures of thermodynamic stability.48 In section 1.2., it was hypothesized that immobilization of BsAXE
onto a solid support will promote thermodynamic stability. To assess the hypothesis, it is necessary to
evaluate the effect of high concentration of substrates/products, pH, and dissolved salts atypical of treated
wastewater, on the protein secondary structure, melting temperature, and free energy change of protein
unfolding.
(a) CD spectroscopy: Circular dichroism (CD) spectroscopy is an analysis technique that uses the
interaction of circularly polarized light in the far UV region with the protein molecules to obtain an
absorption spectrum. The spectrum shows the molar ellipticity vs wavelength which can then be analyzed
using standard curve fitting methods to determine the percentage of α-helix, β-sheet, β-turn, and random
coil (Figure 5).72 For CD experiments, a JASCO J-810 CD Spectropolarimeter will be used over a
wavelength range of 190−260 nm. In general, free BsAXE samples will be treated with aqueous solutions
containing triacetin, H2O2, acetic acid and peracetic acid within a range of concentrations as well as different
proportions of their mixtures prepared with locally sourced secondary effluents. Desalting columns like
PD-10 from Cytiva Lifesciences will be used to extract the protein to ensure that all organics are salts are
removed from the sample before acquiring CD spectra to ensure minimum scattering and interference to
light absorption. Data analysis will be done using the open-source tool DichroWeb which has built-in
algorithms for multivariate regression to fit experimental ellipticity data.73 It is expected that the changes
9
Figure 5. Typical shapes of different
secondary structure of proteins (Reprinted
from ref 72)
induced to the BsAXE secondary structure upon treatment
with the above mentioned components which are integral to
the disinfection system, can be quantitatively assessed using
this technique. However, deployment of CD spectroscopy will
be limited to the assessment of free enzyme as it is not
considered suitable for immobilized enzyme due to light
scattering and absorption by the enzyme-support.
(b)
Differential scanning calorimetry (DSC):
Thermodynamic stability of the protein, as stated earlier, is
almost exclusively related to the free energy change of
unfolding (ΔGUF) and the melting temperature (Tm). Usually,
protein unfolding is characterized by two states- folded and
unfolded, and a transition state represented by a peak in the
molar heat capacity (CP) vs temperature (T) (Figure 6).74 As
the temperature increases, the Cp reaches a maximum value
characterized by the melting temperature Tm.
Figure 6. Experimental setup for a DSC experiment: the amount of heat required to increase the
temperature by the same increment (ΔT) of a sample cell (q s) is higher than that required for the reference
cell (qr) by the excess heat absorbed by the molecules in the sample (Δq). Reprinted from Ref. 74
For DSC experiments, aqueous solutions will be prepared under identical conditions as that discussed
in section 1.3.2.1 (a). Known amount of BsAXE@GA/PA110 will be added to these solutions and the
enzyme concentration will be calculated using enzyme loading. For the comparison, free enzyme of will be
analyzed separately by adding equivalent amount of free BsAXE to the solutions and GA/PA110 will be
added to match the weight of the resin in the BsAXE@GA/PA110 sample. DSC measurements will be done
using a TA Q200 DSC analyzer.
Enthalpy change of unfolding (ΔHUF) can be calculated as:
𝑇1
∆𝐻𝑈𝐹 = ∫𝑇0 𝐶𝑝 𝑑𝑇
(6),
which can be evaluated by integrating the area under the thermogram as depicted in Figure 6 (Δq).
Additional treatment will be done by Van’t Hoff treatment of the thermogram.75 Briefly, upper half width
of the thermogram at half height will be considered. Van’t Hoff enthalpy change (ΔHVH) can then be
calculated as:
𝐵′
∆𝐻𝑉𝐻 = 1 1
(7),
−
𝑇𝑚 𝑇2
10
where T2 is the upper temperature of the half-width under consideration, and B’ is a constant.76 The ratio of
ΔHVH/ΔHUF will provide insight to the proportion of the BsAXE structure that unfolds as a single
thermodynamic entity. For free energy change of unfolding (ΔGUF), the experimental Cp vs T can be
replotted as CP/T vs T by dividing the Cp values by T, which can then be integrated to obtain the entropy
(ΔSUF). ΔGUF can then be calculated with the general relationship ΔG=ΔH-TΔS. At lower temperature other
than Tm, Johnson proposed the following equation:
∆𝐻
𝑇
∆𝐺𝑈𝐹 = [∆𝐻𝑈𝐹 + ∆𝐶𝑃 (𝑇 − 𝑇𝑚 )] − 𝑇 [ 𝑇 𝑈𝐹 + ∆𝐶𝑃 𝑙𝑛 (𝑇 )]
(8)
𝑚
𝑚
From DSC studies, it is expected that for the BsAXE@GA/PA110, the peak will shift towards right,
yielding a higher Tm compared to free BsAXE, which will be regarded as an indicator of higher
thermodynamic stability. Also, the ΔGUF for the immobilized enzyme is expected to be lower than that of
the free enzyme as there could be partial unfolding or dissociation of the multimeric forms (trimer or
hexamer of BsAXE) during the immobilization process.
(c) Differential scanning fluorimetry (DSF): Protein unfolding can also be induced by high
concentration of organics, pH, etc. DSF or fluorescence dye-assisted thermal shift assay can be used to
analyze the unfolding of proteins using the RT-PCR method. Following a 24-hour incubation period under
conditions stated in section 1.3.2.1. (a), Sypro Orange probe (Thermofisher) will be added to well plates,
and 20 µL of the sample will be transferred to a 384 well block plate for DSF analysis. A Bio-Rad CFX384
RT-PCR will be used to collect fluorescence intensity data. The fluorescent probe associates with the
hydrophobic regions of a polypeptide chain as the protein unfolds, leading to an increase in fluorescence
intensity. It is expected that if the free BsAXE undergoes significant unfolding upon long incubation under
the relevant operating conditions. It could be evaluated using the fluorescent spectrum.
1.3.2.2. Kinetic stability and catalytic mechanism of BsAXE@GA/PA110: To illustrate the kinetic
stability and account for possible substrate/product inhibition, the catalytic mechanism of BsAXE needs to
be correctly understood. In their work, Tao et al. provided Michaelis constants of BsAXE for triacetin and
H2O2, merely illustrating the substrates’ relative ease of binding with the enzyme.39 Michaelis-Menten
model uses a rapid equilibrium treatment instead of a steady state treatment, which renders an inaccurate
picture when applied to multi-substrate kinetics. An implicit assumption of Michaelis-Menten model is that
the total enzyme concentration is the sum of free enzyme and the enzyme-substrate complex.77 It does not
consider non-productive substrate binding to the enzyme.77 Moreover, information regarding BsAXE
inhibition by triacetin, PAA, or acetic acid, which is a by-product of the reaction is not available in the
literature. Hydrogen peroxide seemed to negatively affect the yield of the reaction,39, but a comprehensive
kinetic model is still lacking. Bernhardt et al. suggested that serine hydrolases having perhydrolase activity
followed
a
unique bi-substrate
double
displacement mechanism, also known as the pingpong mechanism, which is applicable to many
enzymes catalyzing partial exchange reactions.44
Tao et al. hypothesized that the same catalytic triad
in the serine hydrolase was responsible for
39
Figure 7. Schematic representation of bi-substrate Ping- perhydrolase activity in BsAXE. Therefore, it is
quite possible that BsAXE will follow the pingpong mechanism74
pong mechanism.
In the scope of the proposed study, it is paramount to establish whether the bi-substrate ping-pong model
can explain the kinetic mechanism of BsAXE. Briefly, in such a mechanism, substrate A binds to the
enzyme E, followed by the release of product P through two binary intermediate equilibrium complexes
EA and FP (Figure 7). The modified enzyme F then binds to the second substrate B, followed by release of
product Q and simultaneous regeneration of enzyme E through two different binary intermediate
complexes. Briggs-Haldane steady state treatment78 at initial rate conditions ([P]=0, [Q]=0) renders the
following initial rate equation:
11
1
𝑣
=
1
𝑉𝑚𝑎𝑥
+
𝐾𝑎
𝐾𝑏
+
𝑉𝑚𝑎𝑥 [𝐴]
𝑉𝑚𝑎𝑥 [𝐵]
(9)
where Vmax=k3k7[ET]/(k3+k7), Ka=k7(k2+k3)/{k1(k3+k7)}, and Kb=k3(k6+k7)/{k5(k3+k7)}; ET is the total
enzyme concentration, Ka and Kb are apparent Michaelis constants. Due to the absence of a [A][B] term,
ping-pong rate law is distinct from ordered or random bi-bi mechanisms.77
Kinetic experiments will be performed by
holding individual substrates, triacetin (A)
or H2O2 (B) constant, while the other is
varied. HPLC will be used as the analytical
method for substrate and product analysis
for precise estimation of all components, as
described by Tao et al.39 Alternatively, the
spectrophotometric method can also be
used for PAA concentration measurements
at very low substrate concentration.7979 Both
methods require derivatization of PAA. For
initial rate measurements, the assays will be
run for <30 seconds if the reaction rate
remains linear within this range. Whether
ping-pong mechanism will be applicable for
BsAXE can be verified by LineweaverBurke plots at different H2O2 levels, while
varying triacetin and vice versa. Substrate
Figure 8. Predicted product inhibition patterns for the Ping Pong
inhibition, if exists, will appear as a UBi Bi kinetic mechanism.74
shaped non-linearity in the plots at high
concentration of the substrates.80
To verify product inhibitions, initial rate experiments will be conducted as described above with either
product (peracetic acid or glycerol-diacetate) set to zero in the initial reaction mixture. If the ping-pong
mechanism is proved valid, higher concentration of the first product will generate a competitive inhibition
pattern in the lineweaver-Burk plot (Figure 8) of the second substrate variation (converging 1/v vs 1/
[second substrate]) and vice versa according to the following equations:
1
𝑣
𝑎
𝑏
= 𝑉 + 𝑉 [𝐴]
when [P]=0
[1 + 𝐾 ] + 𝑉 [𝐵]
1
1
𝑣
𝑎
𝑏
= 𝑉 + 𝑉 [𝐴]
+ 𝑉 [𝐵]
[1 + 𝐾 ] when [Q]=0
1
1
1
𝐾
[𝑄]
1
𝑖𝑞
𝐾
𝐾
1
1
𝐾
(10)
1
[𝑃]
(11)
𝑖𝑝
V1 is the maximum forward reaction rate (refer to Figure 7) and Kiq and Kip are the product inhibition
constants of the two products considered here (glycerol diacetate and peracetic acid). Similar inhibition
patterns will also be verified for the BsAXE@GA/PA110 by performing initial rate experiments. Kiq and
Kip will be calculated by evaluating the secondary plots for both BsAXE and BsAXE@GA/PA110. It is
expected that the immobilized BsAXE@GA/PA110 will exhibit reduced substrate/product inhibition which
will be indicated by lower magnitude of Kiq and Kip compared to that of free BsAXE.
1.3.3.
Objective 3: Continuous PAA production using immobilized BsAXE in packed bed reactor
The most important objective of the proposed research is to demonstrate continuous production of PAA
using BsAXE@GA/PA110. For this purpose, a lab-scale packed bed reactor will be constructed using the
immobilized enzyme as the packing. Borosilicate glass columns of different lengths and diameters with
fritted discs will be obtained from commercial vendors. A flow-through system will be set up where an
aqueous feed at constant flow rates and known concentrations of triacetin and H2O2 will be flowed through
the reactor and the outlet concentrations will be analyzed using HPLC as discussed in section 1.3.2.2.
Response surface methodology will be used for optimization with central composite design as the method
12
for DoE, as discussed in detail in section 1.3.1.4. To evaluate the hypothesis presented in section 1.2 (b),
PAA concentration in the effluent will be used as the response variable. Five factors will be considered
within a reasonable operating range- length of catalyst (BsAXE@GA/PA110) bed, feed flow rate, inlet
concentration of triacetin, inlet concentration of H2O2 and the internal diameter of the reactor. Model fitting
will be done followed by ANOVA in Minitiab® software package. For numerical optimization, feed low
rate will be set to maximum, inlet concentrations of triacetin and H2O2 will be set to minimum, internal
diameter of the reactor will be set as range based on standard tube diameters available commercially, and
the response variable value (PAA concentration) will be set to an arbitrary number using a reasonable
approximation. Concentration profile of reactants and products will be obtained by keeping all other
operating variables at constant value while varying the length of packed bed.
Although initial rate equation will be determined as an outcome of objective 2, it is also important to
consider and evaluate mass transfer limitations in immobilized reactor systems.81,82 Especially because most
published studies used the simplified single-substrate Michaelis-Menten model for the rate equation.83,84 In
the proposed study, it is already hypothesized that BsAXE will follow a bi-substrate ping-pong model, the
initial rate model of which is distinct from that of Michaelis-Menten. In simplified terms, the apparent
reaction rate constant to appear lower in magnitude than that of the calculate value of the rate constant due
to mass transfer limitations. For a packed bed reactor, at steady state, the mass balance equation can be
written as84:Although initial rate equation will be determined as an outcome of objective 2, it is important
also to consider and evaluate mass transfer limitations in immobilized reactor systems.81,82 Especially
because most published studies used the simplified single-substrate Michaelis-Menten model for the rate
equation. In the proposed study, it is already hypothesized that BsAXE will follow a bi-substrate ping-pong
model, the initial rate model of which is distinct from that of Michaelis-Menten. In simplified terms, the
apparent reaction rate constant to appear lower in magnitude than that of the calculate value of the rate
constant due to mass transfer limitations. For a packed bed reactor, at steady state, the mass balance equation
can be written as84:
𝜕[𝐴]
(1−𝜀)𝜌
𝑉max⁡ [𝐴][𝐵]
𝑢 𝜕𝑥 + 𝜀 𝑃 𝐾 [𝐵]+𝐾
=0
(12)
[𝐴]+[𝐴][𝐵]
𝑎
𝑏
where, u=velocity of feed (cm/s), x= differential length of the packed bed (cm), [A]=triacetin concentration
(mM), [B]=H2O2 concentration (mM), ε=void fraction, ρP= weight of catalyst/bed volume (g/cm3), and
Vmax, Ka and Kb are kinetic parameters from equation (9). Tao et al. had showed in their work that the
required H2O2 concentration was much larger than triacetin in BsAXE catalyzed reaction.39 In the proposed
study, where the target product (PAA) concentration is very low (<1 mM), the change in concentration of
H2O2 is expected to be negligible. Also, since [B]>>[A], equation (12) can be re-written as:
𝜕𝐴
+ 𝑘′𝑟 [𝐴] = 0
(13)
𝜕𝑥
1 (1−𝜀)𝜌𝑃
𝑉max⁡ [𝐵]
𝜀
𝐾𝑎 [𝐵]+𝐾𝑏 [𝐴]+[𝐴][𝐵]
where 𝑘′𝑟 = 𝑢
is the 1st order reaction rate constant. This 𝑘′𝑟 is the theoretical rate
constant and can be compared to the experimentally determined rate constant (𝑘′𝑟,𝑒𝑥𝑝 ) from the
concentration profile. If 𝑘𝑚 is the mass transfer coefficient and the catalyst particle is assumed to be
spherical, then,
1
1
1
= 𝑘′ + 𝑘 𝑎
(14)
𝑘′
𝑟,𝑒𝑥𝑝
𝑟
where 𝑎𝑚 =
𝑚 𝑚
6
,
𝜌𝑃 𝑑𝑃
[dp=average diameter of a resin particle]. Effect of flow rate on mass transfer coefficient
will be experimentally verified. It is expected that at higher flow rates, mass transfer limitations will be
reduced. Locally resourced municipal wastewater secondary effluent will be used for the preparation of
feed solution, and it is expected that the optimized operating parameters for continuous PAA production at
real wastewater conditions can be obtained upon completion of this objective.
1.3.4.
Objective 4: Disinfection studies, optimization of target PAA concentration and contact time,
and implementation in pilot scale
13
The final objective of the proposed work is to evaluate the effectiveness of this in situ continuous PAA
synthesis system towards disinfection. To do that, disinfection studies need to be performed. Secondary
effluents from local wastewater treatment plants will be collected, and E.coli will be quantified after
membrane filtration using cytochrome detection test strips from Merck as described elsewhere.32 Different
doses of PAA will be exogenously added to study the E.coli reduction at different contact times. PAA will
be quenched with sodium thiosulfate in adequate amounts. The experimental data will be fitted to the Hom
model8585 to evaluate the concentration and contact time for maximum E. coli removal, given by the
following equation:
𝑁
ln 𝑁𝑡 = −𝑘𝐶 𝑛 𝑡 𝑚
(15)
0
where, Nt and N0 are the number of E. coli at time t and time zero, C is the PAA dosage and n, m, and k are
constants. Contact time data is not required to be optimized because it will not have any effect on the reactor
configuration. The optimum PAA dosage data from this set of experiments will be used in the reactor
optimization model, where the PAA concentration will be used as the response variable (see section 1.3.3).
With the revised optimized reactor operational conditions, coliform reduction studies will then be
performed using feed solutions to the reactor prepared with this municipal secondary effluent mentioned
above. Upon successful coliform reduction studies, pilot scale testing will be done at local facilities like the
Kansas River Wastewater Treatment Plant (KRWWTP). It is expected that using the optimized flow reactor
system, a similar level of coliform reduction will be achieved.
1.4. Project Timeline
The activities described in section 1.3 will be completed within a 3-year period. The hypotheses stated
in section 1.2 will be tested progressively through these activities. A comprehensive distribution of these
activities will be undertaken throughout these three years as proposed in Table 3. The KU team is optimistic
that the overall goal of developing the continuous enzymatic PAA synthesis system for wastewater
disinfection will be achieved at the end of this timeline.
Table 3. Timeline for Objectives 1-4 for synthesis of BsAXE@GA/PA110, and its utilization in continuous PAA
production for wastewater disinfection
Year 1
Q1
Q2
Objective 1
Objective 2
BsAXE
purification,
activity
assays, and
kinetic
studies
PA110 chracterization: gas
adsorption, TGA, FTIR, SEM, CO2
chemisorption
Q3
Year 2
Q1
Q3
Year 3
Q4
Preparation of GA/PA110 and
BsAXE@GA/PA110; activity studies
of BsAXE@GA/PA110;
optimization of immobilization
parameters
BsAXE
purification
and activity
assay and
when
necessary
Thermodynamic and kinetic stability
studies of BsAXE@GA/PA110
Repeat optimization of
immobilization parameters (based on
outcome of Objective 3 in Q1 and Q2
Q1
Q2
Q3
Q4
Objective 4
Disinfection studies for
optimization of PAA dosage and
contact time using PAA from
commercial source
Chracterization of GA/PA110 and
BsAXE@GA/PA110: FTIR, FESEM
Q4
Q2
Objective 3
Continuous PAA synthesis
studies using packed bed
reactor: optimization of reactor
for production of target PAA
concentration
Mass transfer limitation studies
BsAXE@GA/PA110 additional
characterization: CO2 chemisorption,
gas adsorption
Q1, Q2, Q3 and Q4 represents quarters 1, 2, 3 and 4 respectively in each year
14
In situ disinfection studies for
optimization using the
continuous packed bed reactor;
pilot scale testing
1.5. Broader Impacts
With continuously depleting water resources globally, adequate water recycling and reuse is paramount.
The proposed research seeks to provide a safer, greener alternative for in situ disinfection of treated
wastewater with peracetic acid which is considered to have the least environmental impact. Fundamental
yet unexplored aspects of perhydrolase enzyme kinetics and product inhibition will be studied in this body
of work. Ingenious approaches like optimization-based immobilization of perhydrolase enzyme on
functionalized polymeric resin will be implemented in this study. Stabilization effects which are recognized
as concurrent upon enzyme immobilization will be studied from thermodynamic and kinetic standpoints.
Investigation on biocatalytic method of continuous organic synthesis will promote the bridging of
biotechnology and chemical engineering horizons. Additionally, utilization of renewable feedstocks like
glycerol derivative (triacetin) is particularly appealing from a sustainability perspective. At KU,
sustainability has been the basic theme of prolific research centers such as the Center for Environmentally
Beneficial Catalysis (CEBC), Institute for Bioengineering Research (IBER), and the Institute for
Sustainable Engineering (ISE). Collaboration with Fort Lewis College will bring crucial expertise in
biotechnology required for enzyme synthesis and purification.
1.5.1.
Outreach and Dissemination
It is the vision of the KU team to collaborate with local partners to justify the broader purpose of the
research. Therefore, upon successful completion of the overall objective, the continuous reactor module
will be tested in pilot scale at a local municipal wastewater treatment facility like the Kansas River
Wastewater Treatment Plant (KRWWTP). Education modules on wastewater disinfection appropriate for
high school STEM curriculum will be generated and disseminated to local school districts.
Dissemination will primarily occur through publication of scientific articles in esteemed peer-reviewed
journals. Graduate and undergraduate students will also participate in national as well as international
scientific conferences to present the research results in the form of posters and oral presentations. Student
exchange programs with other public research universities will also be undertaken for further dissemination
of knowledge gathered from the proposed research activities.
1.5.2.
Personnel Development
A sizable number of undergraduate students at the public R1 University of Kansas are enrolled in
engineering majors. The PI will assist students at various educational levels in doing worthwhile research
with immobilized enzymes and continuous reactors. Opportunities will be provided to students to engage
in experimental and modeling activities as an addition to the undergraduate chemical engineering program.
Through the initiatives Chemical Engineering Experience in Research (ChEER) and Research Experience
for Teachers, the KU team will also support the engagement of educators and high school students in
meaningful research (RET). Exposure to scientific research may improve students' interest in STEM
(Science, Technology, Engineering, and Math) careers and provide instructors with inspiration for sciencerelated lessons.
1.5.3.
Diversity, Equity, Inclusion, and Belonging
Diversity in research environments is crucial because it contributes original thoughts and passions to
scholarly conversations. To encourage a more welcoming and diverse workplace for underrepresented
groups, KU provides a variety of initiatives. The IHAWKe (Indigenous, Hispanic, African American,
Women, KU Engineering) program supports faculty in fostering a friendly academic environment by
encouraging students to engage in professional and leadership development. By encouraging students from
minority backgrounds to participate in research and by supporting their growth both academically and
personally, the P.I. will contribute to the promotion of diversity, equity, inclusion and belonging.
15
2. References
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
Moumouni, D. A.; Andrianisa, H. A.; Konaté, Y.; Ndiaye, A.; Maïga, A. H. Inactivation of
Escherichia Coli in a Baffled Pond with Attached Growth: Treating Anaerobic Effluent under the
Sahelian
Climate.
Environ.
Technol.
2016,
37
(9),
1054–1064.
https://doi.org/10.1080/09593330.2015.1098732.
Sharafi, K.; Moradi, M.; Karami, A.; Khosravi, T. Comparison of the Efficiency of Extended
Aeration Activated Sludge System and Stabilization Ponds in Real Scale in the Removal of
Protozoan Cysts and Parasite Ova from Domestic Wastewater Using Bailenger Method: A Case
Study, Kermanshah, Iran. Desalination Water Treat. 2015, 55 (5), 1135–1141.
https://doi.org/10.1080/19443994.2014.923333.
Martin. Water and Sanitation. United Nations Sustainable Development.
Sperling, M. von. Wastewater Characteristics, Treatment and Disposal; Biological wastewater
treatment series; IWA Publ. [u.a.]: London, 2007.
Aghalari, Z.; Dahms, H.-U.; Sillanpää, M.; Sosa-Hernandez, J. E.; Parra-Saldívar, R. Effectiveness
of Wastewater Treatment Systems in Removing Microbial Agents: A Systematic Review. Glob.
Health 2020, 16, 13. https://doi.org/10.1186/s12992-020-0546-y.
Naidoo, S.; Olaniran, A. O. Treated Wastewater Effluent as a Source of Microbial Pollution of
Surface Water Resources. Int. J. Environ. Res. Public. Health 2014, 11 (1), 249–270.
https://doi.org/10.3390/ijerph110100249.
Hawrylik, E. Methods Used in Disinfections of Wastewater and Sewage Sludge – Short Review.
Archit. Civ. Eng. Environ. 2020, 13 (2), 57–63. https://doi.org/10.21307/acee-2020-017.
Wu, C.; Zhou, Y.; Sun, X.; Fu, L. The Recent Development of Advanced Wastewater Treatment by
Ozone and Biological Aerated Filter. Environ. Sci. Pollut. Res. 2018, 25 (9), 8315–8329.
https://doi.org/10.1007/s11356-018-1393-8.
Combined Sewer Overflow Technology Fact Sheet: Chlorine Disinfection. 10.
Amin, M. M.; Hashemi, H.; Bovini, A. M.; Hung, Y. T. A Review on Wastewater Disinfection. Int.
J. Environ. Health Eng. 2013, 2 (1), 22. https://doi.org/10.4103/2277-9183.113209.
Foroughi, M.; Khiadani, M.; Kakhki, S.; Kholghi, V.; Naderi, K.; Yektay, S. Effect of OzonationBased Disinfection Methods on the Removal of Antibiotic Resistant Bacteria and Resistance Genes
(ARB/ARGs) in Water and Wastewater Treatment: A Systematic Review. Sci. Total Environ. 2022,
811, 151404. https://doi.org/10.1016/j.scitotenv.2021.151404.
Li, G.; Ben, W.; Ye, H.; Zhang, D.; Qiang, Z. Performance of Ozonation and Biological Activated
Carbon in Eliminating Sulfonamides and Sulfonamide-Resistant Bacteria: A Pilot-Scale Study.
Chem. Eng. J. 2018, 341, 327–334. https://doi.org/10.1016/j.cej.2018.02.035.
Collivignarelli, C.; Bertanza, G.; Pedrazzani, R. A Comparison Among Different Wastewater
Disinfection Systems: Experimental Results. Environ. Technol. 2000, 21 (1), 1–16.
https://doi.org/10.1080/09593332108618137.
Gibson, J.; Drake, J.; Karney, B. UV Disinfection of Wastewater and Combined Sewer Overflows.
In Ultraviolet Light in Human Health, Diseases and Environment; Ahmad, S. I., Ed.; Advances in
Experimental Medicine and Biology; Springer International Publishing: Cham, 2017; pp 267–275.
https://doi.org/10.1007/978-3-319-56017-5_22.
Wagner, M.; Brumelis, D.; Gehr, R. Disinfection of Wastewater by Hydrogen Peroxide or Peracetic
Acid: Development of Procedures for Measurement of Residual Disinfectant and Application to a
Physicochemically Treated Municipal Effluent. Water Environ. Res. 2002, 74 (1), 33–50.
https://doi.org/10.2175/106143002X139730.
Baldry, M. g. c. The Bactericidal, Fungicidal and Sporicidal Properties of Hydrogen Peroxide and
Peracetic Acid. J. Appl. Bacteriol. 1983, 54 (3), 417–423. https://doi.org/10.1111/j.13652672.1983.tb02637.x.
Kitis, M. Disinfection of Wastewater with Peracetic Acid: A Review. Environ. Int. 2004, 30 (1), 47–
55. https://doi.org/10.1016/S0160-4120(03)00147-8.
16
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
Kim, J.; Huang, C.-H. Reactivity of Peracetic Acid with Organic Compounds: A Critical Review.
ACS EST Water 2021, 1 (1), 15–33. https://doi.org/10.1021/acsestwater.0c00029.
Malchesky, P. S. Peracetic Acid and Its Application to Medical Instrument Sterilization. Artif.
Organs 1993, 17 (3), 147–152. https://doi.org/10.1111/j.1525-1594.1993.tb00423.x.
Carletto, D.; Furtado, F.; Zhang, J.; Asimakopoulos, A. G.; Eggen, M.; Verstege, G. C.; Faggio, C.;
Mota, V. C.; Lazado, C. C. Mode of Application of Peracetic Acid-Based Disinfectants Has a
Minimal Influence on the Antioxidant Defences and Mucosal Structures of Atlantic Salmon (Salmo
Salar) Parr. Front. Physiol. 2022, 13.
Liberti, L.; Notarnicola, M. Advanced Treatment and Disinfection for Municipal Wastewater Reuse
in Agriculture. Water Sci. Technol. 1999, 40 (4), 235–245. https://doi.org/10.1016/S02731223(99)00505-3.
Koivunen, J.; Heinonen-Tanski, H. Peracetic Acid (PAA) Disinfection of Primary, Secondary and
Tertiary Treated Municipal Wastewaters. Water Res. 2005, 39 (18), 4445–4453.
https://doi.org/10.1016/j.watres.2005.08.016.
Flores, M. J.; Lescano, M. R.; Brandi, R. J.; Cassano, A. E.; Labas, M. D. A Novel Approach to
Explain the Inactivation Mechanism of Escherichia Coli Employing a Commercially Available
Peracetic Acid. Water Sci. Technol. 2013, 69 (2), 358–363. https://doi.org/10.2166/wst.2013.721.
Usepa, O.; Nrmrl, L.; Materials Management Division. Wastewater Disinfection with Peracetic Acid
(PAA) and UV Combination: A Pilot Study at Muddy Creek Plant. 19.
Eckert, K. Disinfection Performance of Peracetic Acid in Florida Wastewater Reuse Applications.
UNF Grad. Theses Diss. 2013.
Luukkonen, T.; Prokkola, H.; Pehkonen, S. O. Peracetic Acid for Conditioning of Municipal
Wastewater Sludge: Hygienization, Odor Control, and Fertilizing Properties. Waste Manag. 2020,
102, 371–379. https://doi.org/10.1016/j.wasman.2019.11.004.
Poffé, R.; de Burggrave, A.; Houtmeyers, J.; Verachtert, H. Disinfection of Effluents from Municipal
Sewage Treatment Plants with Peroxy Acids. Zentralbl. Bakteriol. [B] 1978, 167 (4), 337–346.
Lefevre, F.; Audic, J. M.; Ferrand, F. Peracetic Acid Disinfection of Secondary Effluents Discharged
off
Coastal
Seawater.
Water
Sci.
Technol.
1992,
25
(12),
155–164.
https://doi.org/10.2166/wst.1992.0347.
Baldry, M. G. C.; French, M. S. Disinfection of Sewage Effluent with Peracetic Acid. Water Sci.
Technol. 1989, 21 (3), 203–206. https://doi.org/10.2166/wst.1989.0100.
Luukkonen, T.; Heyninck, T.; Rämö, J.; Lassi, U. Comparison of Organic Peracids in Wastewater
Treatment: Disinfection, Oxidation and Corrosion. Water Res. 2015, 85, 275–285.
https://doi.org/10.1016/j.watres.2015.08.037.
Pironti, C.; Dell’Annunziata, F.; Giugliano, R.; Folliero, V.; Galdiero, M.; Ricciardi, M.; Motta, O.;
Proto, A.; Franci, G. Comparative Analysis of Peracetic Acid (PAA) and Permaleic Acid (PMA) in
Disinfection
Processes.
Sci.
Total
Environ.
2021,
797,
149206.
https://doi.org/10.1016/j.scitotenv.2021.149206.
Luukkonen, T.; Pehkonen, S. O. Peracids in Water Treatment: A Critical Review. Crit. Rev. Environ.
Sci. Technol. 2017, 47 (1), 1–39. https://doi.org/10.1080/10643389.2016.1272343.
Uhl, A.; Bitzer, M.; Wolf, H.; Hermann, D.; Gutewort, S.; Völkl, M.; Nagl, I. Peroxy Compounds,
Organic. In Ullmann’s Encyclopedia of Industrial Chemistry; John Wiley & Sons, Ltd, 2018; pp 1–
45. https://doi.org/10.1002/14356007.a19_199.pub2.
Bloomfield, S.; Druitte, M.; Vandenberg, D. Process for Producing Peracetic Acid. EP1004576A1,
May 31, 2000.
Block, S. S. Disinfection, Sterilization, and Preservation; Lea & Febiger: Philadelphia, 1991.
Kim, J.; Du, P.; Liu, W.; Luo, C.; Zhao, H.; Huang, C.-H. Cobalt/Peracetic Acid: Advanced
Oxidation of Aromatic Organic Compounds by Acetylperoxyl Radicals. Environ. Sci. Technol.
2020, 54 (8), 5268–5278. https://doi.org/10.1021/acs.est.0c00356.
17
(37)
(38)
(39)
(40)
(41)
(42)
(43)
(44)
(45)
(46)
(47)
(48)
(49)
(50)
(51)
(52)
(53)
(54)
(55)
Kim, J.; Zhang, T.; Liu, W.; Du, P.; Dobson, J. T.; Huang, C.-H. Advanced Oxidation Process with
Peracetic Acid and Fe(II) for Contaminant Degradation. Environ. Sci. Technol. 2019, 53 (22),
13312–13322. https://doi.org/10.1021/acs.est.9b02991.
DiCosimo, R.; Ford, C.; Gavagan, J. E.; Scott, M. (54) PERHYDROLASE FOR ENZYMATIC
PERACID PRODUCTION. 66.
Tao, W.; Xu, Q.; Huang, H.; Li, S. Efficient Production of Peracetic Acid in Aqueous Solution with
Cephalosporin-Deacetylating Acetyl Xylan Esterase from Bacillus Subtilis. Process Biochem. 2015,
50 (12), 2121–2127. https://doi.org/10.1016/j.procbio.2015.10.009.
M. Abdelraheem, E. M.; Busch, H.; Hanefeld, U.; Tonin, F. Biocatalysis Explained: From
Pharmaceutical to Bulk Chemical Production. React. Chem. Eng. 2019, 4 (11), 1878–1894.
https://doi.org/10.1039/C9RE00301K.
Choi, J.-M.; Han, S.-S.; Kim, H.-S. Industrial Applications of Enzyme Biocatalysis: Current Status
and
Future
Aspects.
Biotechnol.
Adv.
2015,
33
(7),
1443–1454.
https://doi.org/10.1016/j.biotechadv.2015.02.014.
DiCosimo, R.; Gavagan, J. E.; Payne, M. S.; III, F. B. C. Production Of Peracids Using An Enzyme
Having Perhydrolysis Activity. US20090005590A1, January 1, 2009.
Park, S.-M. Acetyl Xylan Esterase of Aspergillus Ficcum Catalyzed the Synthesis of Peracetic Acid
from Ethyl Acetate and Hydrogen Peroxide. J. Biosci. Bioeng. 2011, 112 (5), 473–475.
https://doi.org/10.1016/j.jbiosc.2011.07.014.
Bernhardt, P.; Hult, K.; Kazlauskas, R. J. Molecular Basis of Perhydrolase Activity in Serine
Hydrolases.
Angew.
Chem.
Int.
Ed.
2005,
44
(18),
2742–2746.
https://doi.org/10.1002/anie.200463006.
Zhang, Y.-H. P. Production of Biocommodities and Bioelectricity by Cell-Free Synthetic Enzymatic
Pathway Biotransformations: Challenges and Opportunities. Biotechnol. Bioeng. 2010, 105 (4),
663–677. https://doi.org/10.1002/bit.22630.
Hutchison, J. M.; Mayer, B. K.; Vega, M.; Chacha, W. E.; Zilles, J. L. Making Waves: Biocatalysis
and Biosorption: Opportunities and Challenges Associated with a New Protein-Based Toolbox for
Water
and
Wastewater
Treatment.
Water
Res.
X
2021,
12,
100112.
https://doi.org/10.1016/j.wroa.2021.100112.
Iyer, P. V.; Ananthanarayan, L. Enzyme Stability and Stabilization—Aqueous and Non-Aqueous
Environment.
Process
Biochem.
2008,
43
(10),
1019–1032.
https://doi.org/10.1016/j.procbio.2008.06.004.
Polizzi, K. M.; Bommarius, A. S.; Broering, J. M.; Chaparro-Riggers, J. F. Stability of Biocatalysts.
Curr. Opin. Chem. Biol. 2007, 11 (2), 220–225. https://doi.org/10.1016/j.cbpa.2007.01.685.
Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R.
Improvement of Enzyme Activity, Stability and Selectivity via Immobilization Techniques. Enzyme
Microb. Technol. 2007, 40 (6), 1451–1463. https://doi.org/10.1016/j.enzmictec.2007.01.018.
Khan, M. R. Immobilized Enzymes: A Comprehensive Review. Bull. Natl. Res. Cent. 2021, 45 (1),
207. https://doi.org/10.1186/s42269-021-00649-0.
Derewenda, U.; Brzozowski, A. M.; Lawson, D. M.; Derewenda, Z. S. Catalysis at the Interface:
The Anatomy of a Conformational Change in a Triglyceride Lipase. Biochemistry 1992, 31 (5),
1532–1541. https://doi.org/10.1021/bi00120a034.
Nguyen, H. H.; Kim, M. An Overview of Techniques in Enzyme Immobilization. Appl. Sci.
Converg. Technol. 2017, 26 (6), 157–163. https://doi.org/10.5757/ASCT.2017.26.6.157.
Homaei, A. A.; Sariri, R.; Vianello, F.; Stevanato, R. Enzyme Immobilization: An Update. J. Chem.
Biol. 2013, 6 (4), 185–205. https://doi.org/10.1007/s12154-013-0102-9.
Saravanakumar, T.; Palvannan, T.; Kim, D.; Park, S. Optimized Immobilization of Peracetic Acid
Producing Recombinant Acetyl Xylan Esterase on Chitosan Coated-Fe3O4 Magnetic Nanoparticles.
PROCESS Biochem. 2014, 49 (11), 1920–1928. https://doi.org/10.1016/j.procbio.2014.08.008.
Barbosa, O.; Torres, R.; Ortiz, C.; Berenguer-Murcia, Á.; Rodrigues, R. C.; Fernandez-Lafuente, R.
Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols
18
(56)
(57)
(58)
(59)
(60)
(61)
(62)
(63)
(64)
(65)
(66)
(67)
(68)
(69)
(70)
(71)
(72)
to Opportunities in Tuning Enzyme Properties. Biomacromolecules 2013, 14 (8), 2433–2462.
https://doi.org/10.1021/bm400762h.
Ye, L.; Liu, X.; Shen, G.; Li, S.; Luo, Q.; Wu, H.; Chen, A.; Liu, X.; Li, M.; Pu, B.; Qin, W.; Zhang,
Z. Properties Comparison between Free and Immobilized Wheat Esterase Using Glass Fiber Film.
Int. J. Biol. Macromol. 2019, 125, 87–91. https://doi.org/10.1016/j.ijbiomac.2018.12.055.
Malik, F.; Stefuca, V.; Bales, V. Investigation of Kinetics of Immobilized Liver Esterase by Flow
Calorimetry.
J.
Mol.
Catal.
B-Enzym.
2004,
29
(1–6),
81–87.
https://doi.org/10.1016/j.molcatb.2003.12.016.
Oz, Y.; Surmeli, Y.; Sanli-Mohamed, G. Enhanced Thermostability of the Immobilized
Thermoalkalophilic Esterase onto Magnetic-Cornstarch Nanoparticle. Biotechnol. Appl. Biochem.
https://doi.org/10.1002/bab.2213.
Yoo, H.; Lee, J.; Suh, Y.; Kim, S.; Park, S.; Kim, S. Immobilization of Acetyl Xylan Esterase on
Modified Graphite Oxide and Utilization to Peracetic Acid Production. Biotechnol. BIOPROCESS
Eng. 2014, 19 (6), 1042–1047. https://doi.org/10.1007/s12257-014-0298-8.
Johnson, K. A.; Goody, R. S. The Original Michaelis Constant: Translation of the 1913 Michaelis–
Menten Paper. Biochemistry 2011, 50 (39), 8264–8269. https://doi.org/10.1021/bi201284u.
Holtze, C.; Boehling, R. Batch or Flow Chemistry? – A Current Industrial Opinion on Process
Selection. Curr. Opin. Chem. Eng. 2022, 36, 100798. https://doi.org/10.1016/j.coche.2022.100798.
Anand, A.; Weatherley, L. R. The Performance of Microbial Lipase Immobilized onto Polyolefin
Supports for Hydrolysis of High Oleate Sunflower Oil. Process Biochem. 2018, 68, 100–107.
https://doi.org/10.1016/j.procbio.2018.01.027.
Tian, Q.; Song, P.; Jiang, L.; Li, S.; Huang, H. A Novel Cephalosporin Deacetylating Acetyl Xylan
Esterase from Bacillus Subtilis with High Activity toward Cephalosporin C and 7Aminocephalosporanic Acid. Appl. Microbiol. Biotechnol. 2014, 98 (5), 2081–2089.
https://doi.org/10.1007/s00253-013-5056-x.
China Center of Industrial Culture Collection. http://english.china-cicc.org/goods.php?id=124751
(accessed 2022-08-22).
Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of
Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72 (1), 248–254.
https://doi.org/10.1016/0003-2697(76)90527-3.
Pizarro, C.; Fernández-Torroba, M. A.; Benito, C.; González-Sáiz, J. M. Optimization by
Experimental Design of Polyacrylamide Gel Composition as Support for Enzyme Immobilization
by Entrapment. Biotechnol. Bioeng. 1997, 53 (5), 497–506. https://doi.org/10.1002/(SICI)10970290(19970305)53:5<497::AID-BIT7>3.0.CO;2-C.
Asadi, N.; Zilouei, H. Optimization of Organosolv Pretreatment of Rice Straw for Enhanced
Biohydrogen Production Using Enterobacter Aerogenes. Bioresour. Technol. 2017, 227, 335–344.
https://doi.org/10.1016/j.biortech.2016.12.073.
Thommes, M.; Kaneko, K.; Neimark, A. V.; Olivier, J. P.; Rodriguez-Reinoso, F.; Rouquerol, J.;
Sing, K. S. W. Physisorption of Gases, with Special Reference to the Evaluation of Surface Area
and Pore Size Distribution (IUPAC Technical Report). Pure Appl. Chem. 2015, 87 (9–10).
https://doi.org/10.1515/pac-2014-1117.
Brunauer, S.; Emmett, P. H.; Teller, E. Adsorption of Gases in Multimolecular Layers. J. Am. Chem.
Soc. 1938, 60 (2), 309–319. https://doi.org/10.1021/ja01269a023.
Barrett, E. P.; Joyner, L. G.; Halenda, P. P. The Determination of Pore Volume and Area
Distributions in Porous Substances. I. Computations from Nitrogen Isotherms. J. Am. Chem. Soc.
1951, 73 (1), 373–380. https://doi.org/10.1021/ja01145a126.
Alesi, W. R.; Kitchin, J. R. Evaluation of a Primary Amine-Functionalized Ion-Exchange Resin for
CO2 Capture. Ind. Eng. Chem. Res. 2012, 51 (19), 6907–6915. https://doi.org/10.1021/ie300452c.
Harada, T.; Moriyama, H. Solid-State Circular Dichroism Spectroscopy. In Encyclopedia of
Polymer
Science
and
Technology;
John
Wiley
&
Sons,
Ltd,
2013.
https://doi.org/10.1002/0471440264.pst587.
19
(73)
(74)
(75)
(76)
(77)
(78)
(79)
(80)
(81)
(82)
(83)
(84)
(85)
Miles, A. J.; Ramalli, S. G.; Wallace, B. A. DichroWeb, a Website for Calculating Protein Secondary
Structure from Circular Dichroism Spectroscopic Data. Protein Sci. 2022, 31 (1), 37–46.
https://doi.org/10.1002/pro.4153.
Gill, P.; Moghadam, T. T.; Ranjbar, B. Differential Scanning Calorimetry Techniques: Applications
in Biology and Nanoscience. 2010, 21 (4), 27.
Rozners, E.; Pilch, D. S.; Egli, M. Calorimetry of Nucleic Acids. Curr. Protoc. Nucleic Acid Chem.
2015, 63 (1). https://doi.org/10.1002/0471142700.nc0704s63.
Breslauer, K. J.; Freire, E.; Straume, M. [28] Calorimetry: A Tool for DNA and Ligand—DNA
Studies. In Methods in Enzymology; DNA Structures Part A: Synthesis and Physical Analysis of
DNA; Academic Press, 1992; Vol. 211, pp 533–567. https://doi.org/10.1016/0076-6879(92)11030M.
Purich, D. L. Enzyme Kinetics: Catalysis & Control a Reference of Theory and Best-Practice
Methods, 1st ed.; Elsevier/Academic Press: Amsterdam, 2010.
Briggs, G. E.; Haldane, J. B. S. A Note on the Kinetics of Enzyme Action. Biochem. J. 1925, 19 (2),
338–339.
Pinkernell, U.; Lüke, H.-J.; Karst, U. Selective Photometric Determination of Peroxycarboxylic
Acids in the Presence of Hydrogen Peroxide. Analyst 1997, 122 (6), 567–571.
https://doi.org/10.1039/A700509A.
Rizzi, M.; Stylos, P.; Riek, A.; Reuss, M. A Kinetic Study of Immobilized Lipase Catalysing the
Synthesis of Isoamyl Acetate by Transesterification in N-Hexane. Enzyme Microb. Technol. 1992,
14 (9), 709–714. https://doi.org/10.1016/0141-0229(92)90110-A.
Miyakawa, H.; Shiraishi, F. A Film Diffusional Effect on the Apparent Kinetic Parameters in
Packed-Bed Immobilized Enzyme Reactors. J. Chem. Technol. Biotechnol. 1997, 69 (4), 456–462.
https://doi.org/10.1002/(SICI)1097-4660(199708)69:4<456::AID-JCTB734>3.0.CO;2-E.
Lilly, M.; Hornby, W.; Crook, E. The Kinetics of Carboxymethylcellulose–Ficin in Packed Beds.
Biochem. J. 1966, 100 (3), 718–723. https://doi.org/10.1042/bj1000718.
Yeo, W. S.; Yuniarto, A. The External Mass Transfer Model for the Hydrolysis of Palm Olein Using
Immobilized Lipase in a Scaled-up Recirculated Packed-Bed Reactor. J. Environ. Chem. Eng. 2019,
7 (4), 103185. https://doi.org/10.1016/j.jece.2019.103185.
Özdural, A. R.; Alkan-Sungur, A.; Boyaci, I. H.; Webb, C. Determination of Immobilized Enzyme
Apparent Kinetic Parameters in Packed-Bed Reactors: Presentation of a New Methodology. Food
Bioprod. Process. 2008, 86 (2), 104–108. https://doi.org/10.1016/j.fbp.2008.02.002.
Hom, L. W. Kinetics of Chlorine Disinfection in an Ecosystem. J. Sanit. Eng. Div. 1972, 98 (1),
183–194. https://doi.org/10.1061/JSEDAI.0001370.
20
3. Facilities, Equipment and Other Resources
Graduate and undergraduate students involved in the project will be provided with access to multiple
resources and equipment on campus at KU. Enzyme synthesis and purification will primarily be carried
out by KU’s consultant and collaborator at Fort Lewis College along with differential scanning
fluorimetry tests. Other syntheses and characterization will be conducted on-site with minimum need for
external services.
Department of chemical and petroleum engineering and the Institute for Sustainable Engineering:
Agilent 7890B GC system, HP 1100 HPLC system, Micromeritics ASAP 2020 sorption analyzer,
Micromeritics Autochem 2920 chemisorption analyzer coupled with a HP 5973 mass spectrophotometer,
Carry 300 UV-vis spectrophotometer, TA Instruments SDT-Q600 thermal analyzer
KU Microscopy and Analytical Imaging Research Resource Core Lab (MAI): FEI Versa ThreeDimensional Dual-Beam Field Emission/Low Vacuum Scanning Electron Microscope.
Integrated Science Building (ISB): Shimadzu IRSpirit Fourier transform infrared spectrometer
Institute for bioengineering research: TA Q200 DSC analyzer
Vaccine Analytics and Formulation Center: JASCO J-810 CD Spectropolarimeter
21
4.1. PROPOSED BUDGET
Total other personnel
FRINGE BENEFITS
37% faculty and staff
4,129
15% students (employed 76% or more)
7% students (employed 75% or less)
1,857
Total fringe benefits
Total salaries, wages & fringe benefits
5,986
43,674
EQUIPMENT
1.
2.
3.
Detail or None
-
-
Total equipment
-
TRAVEL
Domestic
Foreign
-
Total travel
PARTICIPANT SUPPORT COSTS
Stipends
-
Travel
-
Subsistence
-
Other
-
OTHER DIRECT COSTS
Research materials & supplies
12,000
Publications (copying and distribution of research results)
-
Consultant Services
4,000
Computer Services
-
22
Subaward #1
-
Subaward #2
-
Subaward #3
Other:
-
-
Tuition
Summer
Fall
Spring
2023
2023
2023
0 Total Number of Participants
Total Participant Support Costs
10,513
1 GRA(s)
1,467
4,621
4,425
Other
10,513
-
Other
-
Communications (long distance, fax, postage)
-
Computer networking and maintenance costs
-
Total "Other"
Total Other Direct Costs
TOTAL DIRECT COSTS
26,513
70,187
BASE
59,674
INDIRECT COSTS - 53% excluding equipment, participant support, tuition and subs in excess of $25k
31,627
$ 101,814
TOTAL PROPOSED COSTS - YEAR 1
23
PROPOSED BUDGET (Continued)
Total other personnel
FRINGE BENEFITS
37% faculty and staff
15% students (employed 76% or more)
7% students (employed 75% or less)
Total fringe benefits
Total salaries, wages & fringe benefits
EQUIPMENT
1. Detail or None
2.
3.
4,336
1,958
45,986
-
1,660
PARTICIPANT SUPPORT COSTS
Stipends
Travel
-
Subsistence
Other
Total Number of Participants
Total Participant Support Costs
-
OTHER DIRECT COSTS
Research materials & supplies
Publications (copying and distribution of research results)
Consultant Services
Computer Services
Subaward #1
Subaward #2
Subaward #3
Other:
Summer
Tuition
2024
1 GRA(s)
1,532
16,000
4,000
Fall
2024
Spring
2024
4,827
4,621
10,980
-
Other
Other
Communications (long distance, fax, postage)
Computer networking and maintenance costs
24
Total "Other"
Total Other Direct Costs
TOTAL DIRECT COSTS
30,980
78,626
BASE
67,646
INDIRECT COSTS - 53% excluding equipment, participant support, tuition and subs in excess of $25k
TOTAL PROPOSED COSTS - YEAR 2
35,852
$ 114,478
TOTAL PROPOSED COSTS YEAR 1 - YEAR 2
$
25
216,292
PROPOSED BUDGET (Continued)
Year 3: 01/01/25 to 12/31/25
12,304
Total GRA
26,712
Total other personnel Total salaries and wages
29,499
41,803
FRINGE BENEFITS
37% faculty and staff
4,552
15% students (employed 76% or more)
7% students (employed 75% or less)
2,065
Total fringe benefits
Total salaries, wages & fringe benefits
EQUIPMENT
6,617
48,420
1. Detail or None
2.
3.
-
PARTICIPANT SUPPORT COSTS
Stipends
Travel
-
26
Subsistence
Other
Total equipment
TRAVEL
Domestic
Foreign
1,600
-
Total travel
1,600
0 Total Number of Participants
Total Participant Support Costs
OTHER DIRECT COSTS
Research materials & supplies
20,000
Publications (copying and distribution of research results)
-
Consultant Services
4,000
Computer Services
-
Subaward #1
-
Subaward #2
-
Subaward #3
Other:
-
Tuition
1 GRA(s)
Summer
Fall
Spring
2025
2025
2025
1,601
5,043
4,827
Other
11,471
-
Other
-
Communications (long distance, fax, postage)
-
Computer networking and maintenance costs
11,471
Total "Other"
Total Other Direct Costs
TOTAL DIRECT COSTS
35,471
85,491
BASE
74,020
INDIRECT COSTS - 53% excluding equipment, participant support, tuition and subs in excess of $25k
39,231
$
124,722
$
341,014
TOTAL PROPOSED COSTS - YEAR 3
TOTAL PROPOSED COSTS YEAR 1 - YEAR 3
27
PROPOSED BUDGET
Cumulative Total
SALARIES AND WAGES
Senior Personnel
P-Months
Name, PI
acad
summer
-
-
1.80
35,182
-
Name, Co-I
acad
-
-
summer
-
-
acad
-
-
summer
-
-
acad
-
-
summer
-
-
acad
-
-
summer
-
-
name (enter on year 1), Co-I
name (enter on year 1), Co-I
name (enter on year 1), Co-I
Senior Hourly Personnel
calendar
Persons
0.00
-
Total senior personnel
Other Personnel
35,182
Persons
Post Doctoral Associate
calendar
-
-
-
-
Technician(s)
calendar
Graduate Student(s)
acad
3.00
57,024
Total
GRA
summer
3.00
19,008
76,032
Undergraduate Student(s)
calendar
Administrative Assistant
calendar
Persons
3.00
7,969
Persons
-
-
Other personnel
calendar
-
-
Total other personnel
84,001
Total salaries and wages
119,183
FRINGE BENEFITS
37% faculty and staff
13,017
15% students (employed 76% or more)
-
28
7% students (employed 75% or less)
5,880
Total fringe benefits
18,897
Total salaries, wages & fringe benefits
138,080
EQUIPMENT
Total equipment
-
TRAVEL
Total travel
3,260
PARTICIPANT SUPPORT COSTS
0
Stipends
-
Travel
-
Subsistence
-
Other
-
Total Number of Participants
Total Participant Support Costs
-
OTHER DIRECT COSTS
Research materials & supplies
Publications (copying and distribution of research
results)
48,000
Consultant Services
12,000
-
Computer Services
-
Subaward #1
-
Subaward #2
-
Subaward #3
-
Other:
Total Tuition 3 GRA(s)
32,964
Other
-
Other
-
Communications (long distance, fax, postage)
-
Computer networking and maintenance costs
-
Total "Other"
32,964
Total Other Direct Costs
92,964
TOTAL DIRECT COSTS
234,304
BASE
201,340
INDIRECT COSTS - 53% excluding equipment, participant support, tuition and subs in excess of $25k
106,710
$
341,014
TOTAL PROPOSED COSTS
29
4.2. Budget Justification
Principal Investigator: The budget allotted to the principal investigator, Victor Kumar Sharma, will pay
for 3 weeks of compensation each summer. Victor will oversee managing the proposed work's entire
administration, including monitoring costs, scientific direction, and timely development. Throughout the
graduate student's study, he will serve as a mentor and help to their advancement in science.
Graduate Student: This proposal will fund one graduate student at 50% FTE for three years. The
graduate student will primarily be responsible for conducting experiments, collecting, and analyzing
experimental data and review relevant literature. The graduate student will also be responsible to work
with an undergraduate student each year to achieve the requisite goals according to the proposed timeline.
The total requested will also cover the graduate tuition, required by the graduate student to fulfil graduate
course requirements.
Undergraduate Students: One undergraduate student will be working each academic year including the
summer for approximately 158 hours per year. The total requested ($7967) will cover the hourly wage of
the undergraduate student each year.
Fringe Benefits: A total of $18,897 will cover the fringe benefits of the senior personnel and graduate
student, calculated based on their FTE percentages, 37% and 7%, respectively.
Travel: The requested travel budget ($3260) will cover travel expenses for the graduate student in the
second and third year for dissemination in national level scientific conferences.
Other Direct Costs: Funds requested for materials and other supplies result in $92,964 including the
GRA salary and tuition.
Direct Costs: The sum of direct costs involving salaries, fringe benefits, administrative fees, materials,
tuition, and travel expenses is $234,304.
Indirect Costs: Overheard rate of 53% charged to salaries and supplies, in a total of $106,710.
Total Costs: Direct and indirect costs result in a total of $341,014 and equal the funds requested in this
proposal.
30
31
32