Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1997-04-07
2001-12-25
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S163000, C435S170000, C435S072000, C435S099000, C435S173200, C435S173800, C435S277000, C435S278000
Reexamination Certificate
active
06333181
ABSTRACT:
BACKGROUND OF THE INVENTION
Ultrasound can be defined as sound waves above the range of human perception (Price, 1992). Currently, many ultrasonic technologies such as SONAR, medical diagnostics, and surface cleaners are available. SONAR and medical applications typically use low power and high frequency (≧1 MHz). Surface cleaning applications, however, depend on ultrasonic cavitations created by lower frequency (20-50 Khz) and high power ultrasound.
Ultrasonic cavitations result from the rapid compression and expansion of a liquid. In the expansion phase, the liquid is “torn apart”, resulting in the formation of voids or bubbles (Price, 1992; Leeman and Vaughan, 1992). These bubbles gradually increase in size until a critical size is reached, where critical size (usually 100-200 &mgr;m in diameter) is dependent on the frequency of the oscillation and the presence of any nucleating agents, e.g., dissolved gasses, cracks and crevices on a solid surface, or suspended solids (Atchley and Crum, 1988; Price, 1992). Once its critical size is reached, the bubble implodes, at times, generating temperatures approaching 5,500° C. within the bubble (Suslick, 1989, Price, 1992). When collapse of a cavity occurs in a solution free of solid particles, heating is the only consequence. However, if implosion occurs near a solid surface, implosion is asymmetric. As water rushes to fill the void left by the imploding bubble (e.g., at speeds near 400 m/s) shock pressures of 1-5 Kpa can be generated (Suslick, 1988; Suslick, 1989; Price, 1992).
The physical effects of ultrasonic cavitations have been known since the early testing of the first British destroyer, the H.M.S. Daring, in 1894 (Suslick, 1990). The rapid revolution of a ship propeller creates the same, high frequency, compressions and expansions created by ultrasound (Suslick, 1989). Cavitations around the Daring's propeller caused pitting of the metals used. This effect of cavitations on metal surfaces has been confirmed in studies on ultrasonic cavitations (Leeman and Vaughan, 1992; Boudjouk, 1988). High intensity stirring, the dispersal of suspended solids, increased diffusion through cellulose gels, and emulsification of immiscible liquids are other effects attributable to ultrasonic cavitations (Ensminger, 1973).
The high temperatures, pressures and velocities produced by ultrasonic cavitation can also create unusual chemical environments (Suslick, 1989). Compounds in aqueous solution have been shown to form free radicals when subjected to ultrasound. Water, when subjected to ultrasound, creates H. and .OH intermediates, ultimately producing H
2
and H
2
O
2
(Suslick, 1988). Other chemical effects can be caused by high velocity collisions driven by shock waves. The agglomeration of metallic particles in ultrasonic fields has been shown (Suslick, 1989; Suslick, 1990).
Ultrasonic surface cleaners have been available for use since the early 1950's (Shoh, 1988). The mechanism of the cleaning action is reliant on the formation of cavitation bubbles. The contaminant coat can be gradually eroded through cavitational action. Alternatively, the formation of cavitational bubbles between the coat and the surface, effectively peels the coat away from the surface. Other designs of ultrasonic cleaning systems have extremely high efficiency (>95%).
Most biological applications of ultrasonic technology have been directed towards the disruption of cell membranes (Shoh, 1988; Ausubel, 1996). One such device is Fisher Scientific's Model 550 Sonic Dismembrator. Recently, the effects of lower intensities of ultrasound on bacteria have been investigated. It has been shown that nonlethal doses of ultrasound may cause the induction of the SOS response and the transcription of heat shock proteins in
Escherichia coli
(Volmer et al., 1996). Some of the physical damage to
E. coli,
by ultrasonic cavitation, has been illustrated recently (Allison et al., 1996), showing the disruption of the plasma membrane and subsequent leakage of intracellular components.
In the fermentation of milk by
Lactobacillus bulgaricus,
the rate of lactose hydrolysis was increased with the use of discontinuous ultrasound (Wang et al., 1996). Presumably, the cause of the increased rate of hydrolysis was the release of intracellular enzymes into the media. After ultrasonic treatment was stopped,
L. bulgaricus
was able to recover and grow.
Recent interest in ultrasound has been shown by those involved in research in the paper industry investigating its uses as a de-inking device in the recycling of various office paper (Scott and Gerber, 1995; Sell et al., 1995;
Norman
et al., 1994). It was reported that, because of ultrasonic treatment, the structure of the paper was changed such that its water holding capacity increased.
Besides the recycling of paper products, there is an interest in the fermentation of waste paper and other lignocellulosic products into ethanol. The production of ethanol from such products reduces environmental waste problems and reduces reliance on petroleum-based automotive fuels. (Hohmann and Rendleman, 1993; Sheehan, 1993). Accessibility of the substrate to cellulase is a primary factor influencing the efficiency of enzymatic degradation of cellulose (Nazhad et al., 1995).
Cellulase from
T. longibrachiatum
is known to bind to cellulose tightly (Brooks and Ingram, 1995). The binding has also been shown to be dependent on the intensity of agitation (Kaya et al., 1994). Similar effects were seen with an intensive mass transfer reactor, where extremely high rates of hydrolysis were achieved (Gusakov et al., 1996).
SUMMARY OF THE INVENTION
Improved methods for enzymatically converting lignocellulose, for example, to ethanol, are desirable. This invention reports the use of ultrasonic treatment in a Simultaneous Saccharification and Fermentation (SSF) process to enhance the ability of cellulase to hydrolyze mixed office waste paper (MOWP), thereby reducing cellulase requirements by ⅓ to ½. SSF is a process wherein ethanologenic organisms, such as genetically engineered micro-organisms, such as
Escherichia coli
KO11 (Ingram et al., 1991) and
Klebsiella oxytoca
P2 (Ingram et al., 1995), are combined with cellulase enzymes and lignocellulose to produce ethanol. Enzyme cost is a major problem for all SSF processes.
In conducting the invention, enzyme stability is not affected and, surprisingly, continuous ultrasonic treatment results in a decrease in hydrolysis relative to discontinuous treatment. One possible explanation is that the resultant mixing does not allow the cellulase to rebind cellulose long enough for catalysis to occur. Therefore, time to allow catalysis between ultrasonic treatments is desired.
The SSF of waste office paper by
K. oxytoca
may also be “cycle dependent.” Considering the inhibitory effect of ultrasound on the growth of
K. oxytoca
P2, a “recovery period” appears to be desired. With variation in the treatment schedule, such as increasing or decreasing treatment time throughout the course of fermentation, further optimization of the fermentation can be possible.
The use of ultrasound in the conversion of cellulose to ethanol represents a significant improvement in the SSF process. This is particularly true where lignin residues were used to generate the electricity required for the process. Ultrasound can be delivered in other manners as well, with liquid whistle systems, which are able to increase the water holding capacity of recycled paper (Scott and Gerber, 1995). Such a device in a piping loop can produce the desired disruption of the fine structure of cellulose, with a lower energy input.
In one embodiment, the invention comprises a method for the enzymatic degradation of lignocellulose, such as in the production of ethanol from lignocellulosic material, comprising subjecting the material to ultrasound, as in a continuously-operating ultrasonic device, cellulase enzymes, optionally an ethanologenic yeast or an ethanologenic bacterium and/or a fermentable sugar, and maintaining the mixture
Ingram Lonnie O.
Wood Brent E.
Lahive & Cockfield
Lauro, Esq. Peter C.
Prats Francisco
University of Florida Research Foundation Inc.
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