Technical aspects of use of ultrasound nazakat2

Department of Textile Engineering
Mehran University of Engineering & Technology, Jamshoro.

M.E RESEARCH PROPOSAL

USE OF ULTRASOUND FOR INTENSIFICATION OF ENZYMATIC BIO-
PROCESSING.

By

Engineer; Nazakat Ali
07 TLE17

Supervised by:
PROFESSOR; RAJA FAHAD ASHRAF
DEPARTMENT OF TEXTILE ENGINEERING
MUET, JAMSHORO.
A research approach for the fulfillment of Masters of Textile
Engineering
Post Graduate Diploma leading to ME Textile Engineering

Mehran University of Engineering & Technology, Jamshoro

TITLE:

OBJECTIVE: USE OF ULTRASOUND FOR INTENSIFICATION OF
ENZYMATIC BIO-PROCESSING.

BACKGROUND:

Use of enzymatic processing in the food, textile, and bio-fuel applications is
becoming increasingly popular, primarily because of rapid introduction of a
new variety of highly efficient enzymes. In general, an enzymatic bio-
processing generates less toxic and readily biodegradable wastewater
effluents. The ever-increasing legislative pressures by governments
worldwide to reduce the quantity/toxicity of industrial wastewaters will ensure
even greater acceptance of enzymatic bio-processing in the future.

1. INTRODUCTION:

Enzymatic processing of cotton textiles, like any wet processing system,
involves transfer of mass (enzyme macromolecules) from the processing
liquid medium (enzyme solution) across the surface of the textile substrate.
Enzymatic reaction involves transport of the enzyme macromolecules and
enzymatic reaction products to and from the fiber surface. Since both stages
are controlled by diffusion, the overall reaction rate of enzymatic hydrolysis is
governed by the diffusion rate of the enzyme macromolecules. In general,
large three-dimensional enzyme macromolecules have low diffusion rates and
tend to react with outlaying cellulose fibers in cotton yarn, which could result
in excessive fiber damage. Sonication of enzyme processing solution
under specific conditions could provide a far more efficient transport
mechanism for “bulky” enzyme macromolecules throughout the
immediate border layer of liquid at the substrate surface.

Generally, an application of ultrasound energy into the liquid medium has two
primary effects: cavitation and heating. Sonication of liquid by low
frequencies dissipates most of the ultrasound energy through cavitation
phenomena, while sonication by high frequencies dissipates a

Nazakat Ali Research Proposal 2010 Page 2 of 13


significant amount of energy through heating (at the expense of
cavitational dissipation). In case of enzymatic bio-processing, the more
important is cavitation - formation, growth, and implosive collapse of
bubbles in a liquid.
The dynamics of cavity growth and collapse are highly dependent on the type
of liquid, presence of dissolved species and gases in the liquid, and
temperature of the liquid. The imploding cavitation bubble causes the nearly
adiabatic compression of the excess vapors inside of the cavity thus raising its
pressures ~ 500 atm. and temperatures ~ 5, 500 oC (plasma conditions). The
powerful ultrasonic irradiation of liquids produces a plethora of high energy
chemical reactions. As the excess vapors in the cavitational bubble are
compressed by the collapse, and its contents reach several thousand Kelvin,
the trapped vapors are largely dissociated.

For water, collapse of the cavitational bubble produces the high-energy
intermediates such as H+ (atomic hydrogen), OH- (hydroxyl), e-(aq)
(solvated electrons), H2O2 (hydrogen peroxide), HO2 (superoxide) and, H2
(molecular hydrogen).
The formation of such highly reactive intermediates by collapsing cavitational
bubbles should significantly affect the long term catalytic stability/activity of
dissolved enzyme macromolecules. The common perception is that these
highly reactive intermediates and powerful shock waves resulting from
collapse of cavitation bubbles could severely damage or at least, inactivate
the very sensitive and intricate structures of enzyme proteins.
Unlike the cavitation bubbles collapse in homogenous systems (liquid-liquid
interface), collapse of a cavitation bubbles in heterogeneous systems (e.g.
enzyme solution – solid substrate) is different because cavitation bubbles that
collapse on or near to a surface are non-symmetrical since the surface
provides resistance to liquid flow. The result is an in-rush of liquid
predominantly from the opposite side of the bubble (remote from the substrate
surface) resulting in a powerful liquid jet (~450 m/sec) being formed and
targeted at the surface. It is also important, that the rapid collapse of the
cavitation bubbles generates significant shear forces in the bulk liquid



immediately surrounding the bubbles and, as a result, produces a strong
stirring mechanical effect.
These effects can significantly increase mass and heat transfer to the
surface of the substrate by disrupting the interfacial boundary layers and
also activate the catalytic performance of the enzyme macromolecules
adsorbed onto the surface of substrate. It is well known that the diffusion
transport of enzyme macromolecules toward the surface of a solid substrate
could be enhanced to a certain degree by simple mechanical agitation of the
processing solution, but it is also recognized that mechanical agitation is not a
very effective stirring mechanism for the immediate border layer of liquid at a
solid - liquid interface where the enzymatic reaction actually occurs. The first,
immediate layer of liquid at solid - liquid interface is motionless, and then
velocities of following layers quickly increase to the maximum constant value
defined by the power of agitation of the bulk solution. Since the immediate
adjusted layer of liquid at a solid – liquid interface is practically immobile the
only available transport mechanism for enzyme macromolecules to reach
substrate surface is diffusion.
When microscopic cavitation bubbles collapse in the immediate vicinity of a
substrate surface, they generate powerful shock waves that cause effective
stirring/mixing of this adjusted layer of liquid. These shock waves generated
by cavitation bubbles collapsing on and near the surface of substrate (e.g.
cellulosic fibers) are an ideal stirring mechanism for the immediate layer of
liquid at the solid-liquid interface where enzyme reactions take place. The
forceful stirring/mixing of this normally immobile layer of liquid greatly
improves the supply of enzyme macromolecules to the surface of a substrate.
The following specific features of cavitation phenomena are very important for
practical bio-processing application:
a) Effect of cavitation is several hundred times greater in
heterogeneous (e.g. all textile wet processes) than in homogeneous
systems and



b) In water, maximum effects of cavitation occur at ~500 C, which is the
nearly optimum temperature for many enzymatic bio-processing
applications.

However, when ultrasound specifically is used to inactivate enzymes and
terminate enzymatic activity its actual efficiency is quite low. For
example, the combined effects of heat, ultrasonic waves, and pressure may
be used for inactivation of certain thermostable enzymes. The comprehensive
overview of the combined effects of heat, pressure and ultrasound on
microorganisms and enzymes enfold that the resistance of most
microorganisms and enzymes to ultrasound is so high that the required
intensity of ultrasound treatment would be impractical.



2. SCOPE OF PROJECT: In summary, we can formulate the
necessary requirements to maximize the benefits of ultrasound energy for
enzymatic bio-processing as follows:
· Ultrasound Frequency: it appears that optimum sonication frequency
should be in range of 20—100 kHz. These low sonication frequencies are
more beneficial because:
a) Most of the introduced ultrasound energy is dissipated through the
cavitation mechanism rather than through heating.
b) The lower sonication frequencies produce larger cavitation bubbles and
therefore, more powerful “jets”, thus providing more vigorous stirring/mixing of
the border layer of liquid at solid - liquid interface.
c) Hardware for generation of low frequencies usually is more cost efficient.

It is also important that in case of heterogeneous systems most of cavitation
bubbles are generated close to the surface of the substrate thus providing an
important additional benefit of the “opening up” of the surface of solid
substrates as result of mechanical impacts produced by powerful “jets” of
collapsing cavitational bubbles. Another imperative consideration is that,
despite their close-packed and generally well-ordered structures, enzyme
macromolecules are usually not entirely rigid and have some conformational
flexibility in solution that helps them to properly position the “active” domain
relative to the substrate.

Therefore, vigorous stirring/agitation of the normally immobile border layer of
liquid at the liquid-solid interface caused by sonication should help enzyme
macromolecules more easily position themselves “fittingly” onto the substrate.

And lastly, another valuable benefit of the intensive stirring/agitation of this
border layer by collapsing cavitation bubbles is enhanced removal of the
products of hydrolysis reaction from the reaction zone that should contribute
to an overall increase in the reaction rate.



3. AIMS AND OBJECTIVES:
Ultrasound Energy: it appears that optimum sonication power should be in
range of 2-10 W/cm3.
The low energy sonication of the enzyme solution improves the transport of
enzyme macromolecules but does not generate an excessive amount of
highly reactive intermediates.
It is critically important to introduce ultrasound energy into the processing bath
in the most uniform way. That criterion insures the uniform generation of
cavitation bubbles throughout the bath and, as a result, uniform enhancement
of transport of enzyme macromolecules toward substrate.
On the whole, despite the apparent attractiveness of introduction of
ultrasound energy for intensification of enzymatic bio-processing of natural
fibers, it was unclear how ultrasound affects the complex structures of
enzyme macromolecules and how significant the benefits of introduction of
ultrasound energy could be.

The objectives of our experiments were to study the influence of low level,
uniform ultrasound field on the enzymatic bio-scouring and bio-finishing of
cotton textiles.



4. APPLICATIONS OF THE PROJECT:
Application of Ultrasound in Heterogeneous vs. Homogeneous Systems,
since the effects of cavitation are several hundred items greater in
heterogeneous than in homogeneous systems, it is appears that introduction
of ultrasound could be economically justified only for solid-liquid systems. In
homogeneous systems, the much less expensive mechanical agitation would
probably suffice.

It come into sight that sonication of the enzyme processing solution does not
reduce the specific activity of enzyme macromolecules in any significant way,
on a laboratory scale, introduction of ultrasonic energy in the reaction
chamber during enzymatic bio-preparation/bio-finishing of cotton resulting in a
significant improvement in enzyme efficiency.

One possible explanation of the apparent inefficiency of ultrasound to
inactivate enzyme macromolecules could be just their extremely low ratio to
the huge quantity of solvent molecules (e.g. water) at the typically used
enzyme processing concentrations of 4-5 g/liter.
The combination of enzymatic bio-preparation/bio-finishing with sonication
could significantly advance this new “green chemistry” process and made it
more suitable for widespread industrial implementation, and this could greatly
reduce the amount/toxicity of wastewater, energy consumption and
processing costs. Further work can be made on different areas of textile wet
processing such as bio-finishing, scouring, bleaching e.t.c.



5. DATA SOURCE AND RESEARCH METHODOLOGY.

Materials: Desized, scoured, bleached, and mercerized clean cotton cloth will
be used for all bio-finishing tests and original greige cotton cloth will be used
in all bio-scouring tests. The weight loss and wettability for all samples treated
under various conditions with enzyme and/or ultrasound will be determined
and compared with samples of the original untreated cotton fabric.

Ultrasonic Reactor: The enzymatic bio-processing of all textile samples shall
be conducted in a parallel-plate Near-Field Acoustical Processor. This dual
frequency ultrasonic reactor with thermal control capabilities will be equipped
with two variable power generators that provided 0 – 3 W/cm2 of ultrasonic
energy inside the reaction chamber.

Pectinase bio-scouring of greige cotton: Raw unscoured (greige) cotton
contains ~ 90% cellulose and various non-cellulosics such as waxes, pectins,
proteins, fats, and coloring matter. To remove these hydrophobic non-
cellulosics and produce a highly absorbent fiber that can be dyed and finished
uniformly, the greige cotton is traditionally processed with boiling sodium
hydroxide solution in the presence of wetting and sequestering agents. This
industrial process requires large quantities of water and energy, and
generates a highly alkaline wastewater effluent. It is suggested that alkaline
pectinases might be a valuable alternative to harsh alkaline solutions in the
preparation of cotton.

The primary objective of test is to determine how enzymatic bio-scouring of
cotton cloth with various concentrations of pectinase enzyme (0.1-1.0 g/L) are
affected by sonication or not. A treatment time (30 min) will be selected
primarily to emphasize the possible effect of sonication on the enzymatic bio-
scouring rather than to achieve maximum absorbency of the sample. The
average wettabilities of original samples and samples treated with various
concentrations of BioPrep L pectinase with/without ultrasound will also be
analyzed.



Experimental data: On the wettability of all treated samples will clearly show
that the application of ultrasound energy during pectinase bio-scouring greatly
improves and accelerate the process resulting in much shorter wicking time
(200 vs. 600 sec). Interestingly, that maximum effect provided by sonication
occurs at a concentration of 0.4 g/L, which is lower than recommended by the
manufacturer (0.5 g/L). The overall uniformity of bio-preparation will
substantially better in combined pectinase/ultrasound treatment than in
pectinase only.
Testing/Sampling: The primary objective of testing is to determine how
enzymatic bio-finishing of cotton cloth with various concentrations of cellulase
enzyme (1.0 - 8.0 g/L) would be affected by sonication. Triplicate samples of
desized, scoured, bleached and mercerized cotton cloth (107 g/m2) will be
treated with cellulase solution (Cellusoft L) TM in combination with/without
sonication for 60 min (Liquor Ratio = 200:1).
For all concentration ranges, the average weight loss of samples treated with
a combination of ultrasound and Cellusoft L enzyme will be analyzed and
compared with the samples of treated with enzyme.



5. Results and Discussions:
Results:
However, enzymatic bio-processing has several critical shortcomings that
impede its wide acceptance by industries: expensive processing costs and
slow reaction rates. Our research found that the introduction of a low energy,
uniform ultrasound field into various enzyme processing solutions greatly
improved their effectiveness by significantly increasing their reaction rate. It
has been established that the following specific features of combined enzyme/
ultrasound bio-processing are critically important:
a) The effect of cavitation is several hundred times greater in heterogeneous
systems (solid-liquid) than in homogeneous,
b) In water, maximum effects of cavitation occur at ~500C, which is the
optimum temperature for many enzymes,
c) Cavitation effects caused by ultrasound greatly enhance the transport of
enzyme macromolecules toward substrate surface and,
d) Mechanical impacts, produced by collapse of cavitation bubbles, provide
an important benefit of “opening up” the surface of substrates to the action of
enzymes.
Discussions:
This study has potential for intensification of other technological processes
that involve various types of specific enzymes and matching substrates. We
can assume that practically any solid/liquid system that involves a reaction
between enzyme macromolecules and solid substrate would greatly benefit
from application of ultrasonic energy into the system.



6. Conclusions:
A major incentive for embracing enzymatic bio-processing of natural fibers is
that application of enzymes is much more environmentally benign and the
reactions catalyzed are very specific and ensure more focused performance
as a result. In contrast, traditional chemical processing is much less specific
and often results in undesirable side effects, e.g. significant reduction in the
degree of polymerization of cellulose. Enzymes used in cotton bio-processing
act as catalysts to speed up complex chemical reactions such as the
hydrolysis of cellulose (by cellulases) and pectins (by pectinases). Because
they act as catalysts, relatively small amount of enzymes are required; if
conditions are favorable to the specific enzyme, it will repeat the action
(hydrolysis) many times during the process. Other potential benefits of
enzymatic bio-processing include cost reduction through energy and water
savings and better product quality.

The comprehensive overview of the combined effects of heat, pressure and
ultrasound on microorganisms and enzymes concluded that the resistance of
most microorganisms and enzymes to ultrasound is so high that the required
intensity of ultrasound treatment would be impractical.



7. REFERENCES:

1. W. Aehle: Enzymes in Industry, Wiley-VCH Verlag GmbH & Co. Weinheim,
(2004) 219-231.
2. V. G. Yachmenev, E. J. Blanchard, A. H. Lambert: Use of ultrasonic
energy for intensification of the bio-preparation of greige cotton. Ultrasonics,
42 (2004) 87-91.
3. F. J. Sala, J. Burgos, S. Condon, P. Lopes, L. Raso: Effect of heat and
ultrasound on
microorganizms and enzymes. New Methods of Food Preservation. (1995)
176-204.
4. I. Hua, R. H. Höchemer, M. R. Hoffmann: Sonochemical degradationof p-
nitrophenol in parallel-plate near-field acoustical processor. Environmental
Science & Technology, 29
(1995) 2790-2796.


Technical aspects of use of ultrasound nazakat2

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