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박 영 규

경기도 포천읍 선단리 11-1 대진대학교 화학공학과

The Application of High-Pressure Reactor for the Recycling of Waste Pulp

Young G. Park

Department of Chemical Engineering, Univ. of Daejin, Korea

【Address】487-800 Department of Chemical Eng. Univ. of Daejin, Sundan-Ri, Pochun-Eup, Pochun Geun, Kyungki-Do, Korea, Tel)+82-357-539-1994, Fax:+82-357-536-6676, E-Mail: ypark@road.daejin.ac.kr

INTRODUCTION

Waste cellulose like pulp is expected to be valuable renewable resources for energy.  Cellulose conversion to glucose from which most of the petroleum products can be produced has been performed by acid-catalyzed hydrolysis.  So far, similar methods by acid process have been proposed for wastetreatment, there have been few studies for the cellulose conversion using high pressure [Zimmerman, 1970].

Pressure plays an important environmental role in chemical reactions, yet it has seldom been a subject of study for biological wastetreatment experiments.  There have been reported more than several hundreds of enzyme-catalyzed reactions which occur in living cell.  The use of high pressure in the process for cellulose conversion has several important advantages for recycling of wasted materials.  High pressure aids to enhance the permeability of microorganism in the cellulose floc, and it increases the solubility of gases such as oxygen in the water.  It may sometimes aid the hydrolysis of enzyme in the reactor. The purpose of present study is to investigate how the glucose and protein synthesize in the reactor using wasted cellulose and which benefit occurs in the use of the high pressure.

Most studies under high pressure have employed a simple experimental apparatus using anaerobic organism which eliminates the need for the continuous air sparger under high pressure.  In a few cases [Davidson, Torma, Brierley, 1981], organic polymers such as liquid fluorocarbon are employed.  According to our research, no previous studies utilizing a system such as ours has ever been published.  This high pressure system is for aerobic organisms, and is capable of monitoring pH and oxygen under continuous aeration at pressure up to 340 atmospheres.  The operating pressure could be increased if suitable electrodes for pH and oxygen measurement are found.

The microorganism employed in this study is Tricoderma reesei which is widely used for cellulase production.  This microorganism degrades cellulose, the most abundant organic compound, to produce reducing sugars.

EXPERIMENTAL

2.1. High pressure system for cellulose decomposition

A 3.8 liter, 316 stainless steel pressure vessel has been manufactured at our engineering research facility.  The pressure vessel, rated at 340 atm pressure, has a two-stage, magnetic-driven stirrer with a pitched blade.  The vessel's wall has attached baffles of 1 cm wide fixed at an angle of 120o.  Cooling and heating are provided by a coil installed inside the vessel.  A sparger of 28 cm long with 5 holes is installed 2.5cm below the agitator.  A recycle pump continuously withdraws the liquid medium to feed a recycle vessel where a pair of pH electrodes and oxygen probe are installed. A level in the vessel controls the speed of the pump in order to prevent the liquid from over-filling, which are connected to a pH meter/controller installed on the control panel.  Form is controlled by a high pressure pump and by a mechanical foam breaker on the top of the agitation shaft.  Cold water runs continuously in and out of the condenser in the exit gas line and the condensation returns to the fermentor: a gear-driven, pressure-control valve installs in the exit gas line and a manual metering valve installs between the accumulator and the reactor.  The entire system is sterilized by steam.  The gas supply from the accumulator is sterilized before entering the reactor.  Samples are collected by means of a sampling device.

2.2. Materials

The microorganism used in this study is Tricoderma reesei (ATCC 26921).  Stock cultures are stored at 4oC on potato-dextro-agar (PDA) slant.  The composition of medium for inoculum is developed by Mandel and Reesei [1957].  The PDA plates cultured at 30oC for seven days are used to harvest the spores, using 10 ml of distilled water per plate.  Seed inoculum is prepared by suspending the spores harvest in a one-liter flask, and adjusting the final spore concentration within 80,000 spores/ml.

Wasted cellulose has been used as a hammer milled sulfite pulpe and uninked nespaper.  This cellulose has been selected a well-treated production in the paper-mill manufacture.

2.3. Analytical methods

The dinitrosalicylic acid (DNS) method for reducing sugars [Sigma method] and the enzyme-glucose oxidase method is used for the determination of sugars liberated from cellulose and the measurements of enzyme activities.  Estimates of soluble proteins per ml of culture are expressed as units which are equivalent to glucose produced (mg)/min ml.

2-4. Operation of high pressure reactor

All the input materials are sterilized in 500ml of distilled water for 30 minutes at 122oC, and cooled to room temperature.  In 48 hours the pH level of the medium drops to between 3.5 and 4.0, and the pH is maintained at 3.5 as a lower limit, using 10 percent NaOH solution. The bioreactor has been pressurized at a rate of 340 KPa (50psi) per minute until it reaches the desired pressure.  Air is aerated at a rate of 0.4vvm(volume of air per unit volume of liquid) to maintain positive dissolved oxygen and good mixing.  The air flow rate is measured at standard conditions and the agitation rate of the bioreactor is 0.1 vvm and 250 rpm, respectively.

RESULTS AND DISCUSSION

Since the maintenance of the system's operating pressure, particularly that of the reactor, is a critical factor during the cultivation of organisms under pressure, it must be tested the system to determine whether it could maintain a stable operating pressure. Several mechanical components of the system may cause unwanted pressure fluctuations which would cause unfavorable culture conditions.  These components are moving parts such as the plungers and diaphragm of the pump and the compressor, the check valves, the control valves, and the sampling system.  Pressure fluctuations of up to 6 atm pressure are observed when the compressor starts to operate.  In order to alleviate this problem the original design is modified to isolate the compressor from the bioreactor by installing two accumulators side by side.  When the first accumulator raises its pressure to a certain level, the system is switched to a second one, while charged gas begins filling the empty accumulator.  This scheme works quite well with minimal pressure fluctuations in the bioreactor.  About 68 kPa of pressure variation is noted during 10 days of reactor operation, except for the period of actual sampling.  When 20 ml of sample is withdrawn through the sampling device up to 200 kPa pressure drop is noticed when the bioreactor is operated at a pressure of 70 atmospheres.

The effect of high pressure on the performance of the pH electrodes and the oxygen probe is enormously changed.  For example, pH is dropped by 50% in comparison with pH state of atmospheric condition. About 250 ml of buffer (pH 7.0) in the recycle vessel is compressed with the probes immersed in it.

Compression of the broth is carried out at a rate of 140 KPa/min until it obtains a set value.   The experimental result shows the time course of the proteins concentration for both reactors at high pressure and atmospheric pressure.  For the high pressure of 6.94MPa, each line represents the cells at different growth phases.  All the pressures and temperatures for the high pressure system are the same 32oC, 6.94MPa, except one batch which run at 44oC, and 12.2 MPa.  Drastic increases in protein production by the microorganisms, at elevated temperature and pressure of 44oC and 12.2 MPa, are noted.  The same cells at the pressure and temperature of 6.94 MPa and 31oC, however, do not improve the protein production as much as the one at the higher temperature and pressure.

Because this is an aerobic condition the rate of protein production should be dependent on the rate of absorption of oxygen into the broth.  The absorption rate is directly proportional to the equilibrium concentration of oxygen in the broth.  Our calculations show that under atmospheric pressure at room temperature the oxygen concentration should be about 5 ppm, that at 69.4 MPa and 31℃the oxygen concentration should be about 260 ppm and that at 12.2 MPa and 44℃, which is the observed result.

The cells at stationary phase (6.94 MPa, 31oC) also demonstrates much better protein secreation (but not better than the batch at an elevated temperature and pressure) than normal atmospheric batch.  Regardless of such a drastic increase in protein content (which is as high as 33% above the atmospheric batch) the glucose production under such an environment does not appear to increase as proportionately as the increase of the protein.  The exact cause of protein "over production" is not presently clear.  The allosteric enzyme's regulation system(which involves the control of amino acid biosynthesis) might have been stimulated by the high pressure, or have changed their molecular conformation of the enzymes involved in the protein synthesis to enhance the protein production and secretion.

CONCLUSION

It is proved that the retardation of protein synthesis under high pressure is counterbalanced by the elevated temperature.  This finding is found to be optimal growth temperature in the high pressure process.  The protein synthesis is obtained by 25% in comparison with the production of protein in the atmospheric pressure.  This result will be on future utilized for the increased production of alcohol with glucose converted by Tricoderma reesei.

REFERENCES

1. Davidson, M.S.,Torma, A.E.,Brierley, J.A. and Brierly, C.L., Biotech.Bioeng. Symposium, 3rd Symposium on Biotech. in Energy Production and Conversion, May (1981).

2. Mandel, M. And Reese, E.T., "Induction of cellulase in Tricoderma virde as influenced by carbon coruces and metals", Journal of Bacteriol., 73, pp269-278 (1957).

3. Sigma method: Sigma Chemical Co.,St. Louis., MO (1986).

4. Zimmerman, A.M., "High Pressure Effects on Cellular Processes", Academic Press (1970).