Simulation of onsite vacuum heap aeration and soil surface enlargement by a closed agitated soil bioreactor

Microbiol. Res. (1996) 151, 29 - 35

Microbiological Research © Gustav Fischer Verlag Jena

Simulation of onsite vacuum heap aeration and soil surface enlargement by a closed agitated soil bioreactor M. Saner, R. Bachofen, K. Schneider Institute of Plant-Biology, Department of Microbiology, University of Zurich, Zollikerstr. 107, 8008 Zurich, Switzerland. Accepted: September 26, 1995

Abstract Onsite bioremediation techniques are widely established due to their ease of monitoring and the possibility of optimising the degrading conditions. The aim of this work was to simulate the vacuum heap principle with a closed system and to show the efficiency of soil surface enlargement on the degrading activity of soil populations. The containment allows the aeration of up to 100 litres of soil under realistic conditions. The aerobic activity of the soil organisms is determined online by the measurement of O2 and CO 2 by mass spectroscopy. The introduction of a mixing principle to enlarge the surface of the soil, leads to a drastic increase of biological activity in the soil. As a PAR model substance, phenanthrene was used as C-source within 2 days without addition of adapted strains. Key words: soil - simulation - solid phase - bioremediation - PAR

Introduction Most commonly sanitation of contaminated soil areas are carried out on site. On- or off-site techniques offer a more or less closed system in terms of hindering contaminants from a transfer vertically to the subsurface as well as horizontally to clear areas. Large volumes of contaminated soil are mechanically mixed, homogenized and irrigated with the appropriate nutrient solution. The heap is then set up in layers and in between those, a perforated plastic piping is installed, which is connected with manifolds to a vacuum blower system (D. Eiermann, 1992). The Corresponding author.' M. Saner

efficiency of such on-site technology allows to degrade hydrocarbons to a final concentration of 50 -100 mg' kg- 1 dry soil within a reasonable time scale. (M. Biehler, 1994). Degradation time and residual polluant concentration are largely related to the class of chemicals to be degraded. The degradation of poly aromatic hydrocarbons (P AH), relates strongly to the soil type and degrading strategies of the microorganisms. It has been shown that naphtlalene must desorb from the soil matrix before degradation starts (Bashir et al., 1990). In contrast, other studies have shown that biodegradation of pentachlorophenol and toluene was not retarded by interactions with the soil matrix (Bellin et al., 1990). The understanding of the strategies of soil organisms to desorb and degrade hydrocarbons is somewhat contradictory. One major problem in the degradation of hydrocarbons in soil is their bioavaiability to the metabolically active population (Volkering et al., 1992). The present paper supports the hypothesis that enlarging the surface of the soil will increase the bioavaiability of hydrocarbons to degrading populations.

Theory Biomass formation of the degrading culture is a function of the yield and substrate concentration (Tempest et al., 1970): dX dS -=-y.dt dt

(1),

where X is the biomass concentration (kg· m - 3) and S the total concentration of substrate (kg· m - 3). Y is the yield constant (kg biomass/kg substrate). The substrate concentration is a function of the sorption Microbiol. Res. 151 (1996) 1

29

process

dQ

Tt and

. the total substrate concentratIon C1

(kg· m - 3) in solution: dC! -dS = -1 . -dQ +-

~

dt

V

dt

(2)

dt

The desorption process is dependent on the surface of the soil particles A (m2), the desorption constant k (m . h -1), the substrate equilibrium concentration Ceq (kg· m - 3) and the total substrate concentration in solution C1 (kg· m - 3). The process can be written as a first order reaction dQ

-Tt = k . A . (Ceq -

(3)

Ct )

(Perry et al., 1963). If Ct ~ 0 becomes true at low cell densities, the culture is able to grow exponentially. At high cell densities however, C1 is negligible compared to Ceq and considered to be constant, it follows dC! =0 dt

As a result the maximal mass transfer is dQt -=k·A·C dt eq

(4)

dX

Y dQt

y. k . A . Ceq V

(5)

(Volkering et al., 1992). Thus, when the soil surface area (A) is enlarged, the formation of biomass will increase. Furthermore, the desorption constant (k) is also dependent of the soil surface. The desorbing process will only take place if process water and surfactants (produced by degrading cultures or added by experimentators), have access to the adsorbed hydrocarbons at the soil surface. The yield (Y) of the populations is a biological constant and is strongly specific to different substrates and organisms and can not be changed by the experimenter (Egli et al., 1982).

0

~

l.

The maximal substrate uptake rate cannot exceed the desorption rate of the substrate. The maximal growing rate is then:

dt = -V . at =

The soil can be aerated through different aeration tubes. A sprinkling device is mounted on the top of the reactor to provide nutrients and to control the water content of the soil. At the bottom 3 drains are provided to lead offliquids in case of excess the water holding capacity of the soil. In Figure 1, a schematic overview is given. Aeration of the reactor is controlled by an mass flow controller (Brooks mass flow 5800, Veenendaal, NL). The reactor is air tight up to pressures of 2 bar. This ensures that anaerobic experiments can be carried out with a gentle stream of N 2' Surface enlargement of the soil was achieved by the introduction of rotating axles fitted with various small "soil-cutters". These soil cutters fractionate the soil to small particles. The axles are periodically guided through the soil. Nearly the whole volume unit becomes mixed within one mixing period of one revolution of the central axis. Due to the small cutting elements (4.5 cm; 1.5 cm; 0.2 cm) which move through the soil, the soil-matrix is intensively mixed rather then transported. The problems of pellet formation often observed with spiral mixers or paddles do not occur.

~ 3.

.. /,S>f-HH"r

I10-

2.

6.

o -

0 L...I

L...J

7.

L...J

4. S' .

Fig. 1. Reactor scheme. 1. Gear disc with 15 couplings for mixing axles, 2. Containment with bull eye and 2 sample ports, 3. Mixing axle, 4. second gear disc, 5. closings, 6. central axis, 7. drainage. Material: Steel V2A. Gear discs: Aluminium coated with Teflon. Length: 82 cm; Diameter: 50 em; Volume: 100 litres. Aeration of the soil provided through all mixing axles. Gear-axle unit powered by a DC Motor 110 W, max. rph 15/h. Mixing axles powered by a DC Motor 600 W, max. rpm 100/ min (outer axles).

- - - ',

..

Material and methods As a closed containment supplied with different aeration tubes, a planetoid mixing reactor was constructed. Up to 100 litres of soil can be placed into the containment. The reactor is made of V2A steel.

30

Microbiol. Res. 151 (1996) 1

f

Fig. 2. Part of one mixing axle. These axles can easily be coupled with the coupling devices at the two gear discs. The aeration through the axles provides an excellent diffusion of O2 in the soil matrix. 3 axles are placed in the reactor for the simulation of the vacuum heap method. a) exhaust, b) sprinkler device, c) central axis, d) soil, e) mixing/aeration axles, f) drainage

Soil contamination. To obtain a good dispersion of phenanthrene, used as model contaminant, was dissolved in acetone and thoroughly mixed with the soil. 8 g of phenanthrene were dissolved in 400 ml of acetone. The solution was mixed with 20 litres of soil (A-horizon), 23% water content. The contaminated soil was placed in a dark room for 72 h to removemost of the solvent content (acetone) and then filled into the reactor. Process control system. The soil reactor and additional pumps, submerse reactor and analytical instruments are connected to a process control system: (DDC, PCS Wetzikon, CH, K. Schneider, 1987). The software was developed at the institute. The software package has proved to be reliable and easy to handle in different commercial fermentation plants. The system runs on a VME OS-9 computer which offers real-time, multi-tasking, multi-user operation. Input and output of process data and controls via Phoenix Interbus S Modules offer a very high reliability and allow easy digital data transfer from the computer to the process plant and vice versa. The frequency of the online data acquisition can be set between 4 milliseconds and a few hours. In the experiments periods were set between 5 min., to 10 min. Process outputs/inputs are transformed from analog to digital signals via 12 Bit AD/DA converters in the Phoenix Interfaces near the bioreactor. This ensures that no high frequency distortion will be captured throughout the signal path. All analog signal sections are galvanically disconnected via Phoenix DC/DC converters. Analysis and determination ofsoil activity. To measure the activity of aerobic soil organisms CO 2 production and O 2 uptake during the fermentation process is measured online by mass spectroscopy (PGA 100, Leybold K51n, BRD). Gas samples from the bioreactor are continously cooled and dried by an PeltierCooler (Antec, Wettigen, CH). They are transferred to the mass spectrometer through Nylon tubes (12 m, id 4 mm). To reduce the pressure, the sample is transferred into the mass spectrometer by a capillary of 2 m length and an id. of 0.3 mm. The electrical output signal is amplified with a DC instrumental amplifier. The detection limit is of 1 ppm CO 2 (G. Jud, 1992). The mass spectrometer is coupled with the process control system byaRS 232 interface. The calibration of the system is software controlled and carried out automatically every 6 h with calibration gases. The mass spectrometer is a poweful tool to determine the activity of microorganisms and offers an unique versatility. Gases which can be quantitifed in

our system are: O 2 , CO 2, N 2 , Ar, H 2 , CH 4. In addition the relative change of the soil temperature proofed to be an excellent parameter to qualitatively indicate general biological activity in the soil in general. To measure the hydrocarbon concentration in soil, 3 g of soil were extracted with 3 ml of ethylacetate for 10 min., followed by 3 min. of ultrasonic treatment and shaked for 24 h and finally again extracted in the ultrasonic bath for 3 min. (modified after Eschenbach et al., 1994). The suspension was then centrifuged at 3 500 rpm for 10 min. 1 III was injected in the DANI GC 86.10 HT. Column type DB 5. Conditions:

Injector 250 °C 1.0 ml/min Helium FID 275°C 35 °C 1 min hold 35 -200°C 10 °C/min 200-250 °C 5°C/min Split 1: 50

The nutrient solution (without C-source) contained: 3.3 g·l-l Na2HP04; 1.6 g .1- 1 KH 2 P0 4; 0.1 g .1- 1 MgS0 4 · 7H 2 0; 0.02 g .1- 1 CaCI 2 · 2H 2 0; 1.0 g .1- 1 NH 4Cl. Additionaly, vitamins and trace elements were added to the medium as described by D. H. Eickelborn (1975) and Pfennig et al. (1966).

Results 30 litres of soil out of the top 15 cm of an uncontaminated area near the Institute were sieved to exclude particle greater than 4 mm. The soil was placed carefully on the 3 aeration tubes which had been placed before into the containment. The aeration rate was set to 2.51· min -1. Figure 3 shows that CO 2 concentration in the off gas is dependent on the aeration rate. The change in soil temperature when aeration is started indicates clearly its positive effect on the activity of the soil organisms. However, after 5 -10 h the concentration of CO 2 as well as the temperature decrease to nearly the original level between the two maxima. The fast increase of CO 2 concentration in the off gas at the beginning of the aeration process results from the purging of CO 2 formed before the aeration period. Later the concentration of CO 2 shows a continuous slow decrease. It may be assumed, that static aeration of soil heaps results in the formation of ducts and drains inside the soil heap, resulting in an inhomogeneous dispersion of O 2 and nutrients in the soil. Due to the fact that every kind of matter has the tendency to select the way of least resistance, the formation of ducts are even forced within the Microbiol. Res. 151 (1996) 1

31

duration of such aeration processes. However, the same experiment carried out with the same and Diesel contaminated soil (6 g' kg- 1 dry soil) shows no decrease in the CO 2 concentration during the same time period. In Figure 4 at the time of 45 h the soil was aerated by the aeration pipes inside the soil heap. At the time of 80 h the mixing starts. At the time of 140 h the addition of 0.5 I nutrient solution showed no influence because of the carbon limitation of the system. The influence of enlarging the soil surface is demonstrated in Figure 5, using similar starting condi26

30

r----r---r---""T"'-~r__-..,

2.5

...... .----...·-·r------.. -----

2.0

,I

O8Iof1a\

0.13

II II I

15

24 0.011

C 10

E

==. ~

~

j

8v

0.()3

22

.gC 05 ~

Q)

'"

0.0

10

0

nme(h)

20

15

25

Fig. 3. The CO 2 concentration in dependence of the aeration and the influence of the aeration to the soil temperature indicate clearly the positive effect to the activity of the soil organisms. However the concentration of CO 2 as well as the temperature are rapidly decreasing to a level between the two maximas. 0.4

tions. The aeration rate was again set to 2.5 1· min -1. During the first 17 h the mixing system was switched off. The concentration of CO 2 was similar to the previous experiment. After 17 h the mixing was started, the axles having a rotation speed of 60 min -1. The concentration of CO 2 in the off gas and the soil temperature increased significantly. When the axles were switched off, the CO 2 concentration as well as the temperature decreased to the original level, indicating that growth of the soil organisms is limited. The correlation between the two signals is very high. Control experiments without soil clearly proved, that the temperature is not dependent probably to the heat from the reactor. The soil temperature is not only in a bioreactor but also under field conditions an easy to measure and reliable parameter in de terming the soil activity qualitatively. Figure 6 shows the degradation activity of 20 litres of soil (A-horizon) with a water content of 23% which had been contaminated with 220 mg . kg -1 (dry weight) of phenanthrene. The reactor was aerated with 2 I· min - 1. The mixing axles were periodically rotated through the soil, for a period of 15 min., followed by a pause of 30 min. The lag-phase until the process starts had a length of 50 h. No nutrients were added to the system at the beginning of the experiment. After this lag, the CO 2 concentration in the exhaust gas increased compared to the background CO 2 concentration of 0.1 %. This CO 2 production is of microbial origin, as the profile of O2 and CO 2 in the head space correlate. The amount of the formed and used O2 fraction in the head space were integrated for the second peak within 50 hours from 70 h to 120 h resulting in 4.8 g

6 r----,----""T"~--_,.---____,

5

0.3 4

0.2

3

2

0.1

,Y'"

~ 1

Ii .....

~

0 U

:;;

•

.

'ii

M

0.0

0

,••

0

50

100

time [hI

32

Microbiol. Res. 151 (1996) 1

150

200

Fig. 4. During the first 45 h the soil was aerated from the top of the containment. From 45 h to 80 h the soil heap was aerated through the aeration pipes inside the soil heap. The CO 2 concentration is increased compared to the beginning of the experiment and shows no decrease. The mixing of the soil and therefore the decreased mass transfer limitations results in an even higher CO 2 production rate. The fast decrease of the diesel concentration at the beginning is not only due to microbial dedradation, but also from the blowing out of volatile fractions.

O 2 used and 4.65 g C(02) formed during this period. 600 data points were taken into account for this calculation.

CPR

OUR

= RQ = 0.97

The respiratory quotient yields to nearly 1.0, indicating that the culture growths under fully oxidative conditions. 28

0.3

The mlmma seen after 70 h is the effect of a limitation of nutrients. After the addition (pulse) of 0.51 of nutrient media at time of 70 h the CO 2 concentration increases again. After about 115 h the soil culture becomes carbon limited. The addition of 0.5 I of media after 170 h therefore shows no influence. The rate of degradation of phenanthrene correlates with the CO 2 production rate of the system.

10

Fig. 5. The CO 2 production is drastically increased due to the soil surface enlargement. After the mixing is switched off the CO 2 concentration in the exhaust gas decreases to the original level. 21.25

0.7

21.00

0.6

20.75

0.5

20.50

0.4

20,25

0.3

20.00

0.2

250 r - - - - - - - , - - - - - - , - - - - - - - - - ,

150

100 C02

0-

g a>

~50

19,75

~ 0,1

~

N

0

u

0

N

19,50

£ 6 c

a Q)

0,0

O~--------_P~---a----~-------~ 50

100

time (h)

150

200

Fig. 6. The formation of CO 2 is based on the mineralisation of phenanthrene. The background level of CO 2 in the exhaust gas from the natural soil, is 0.1 % . The mineralisation of phenanthrene in the soil is very fast. After 100 h, only traces of phenanthrene < 1 mg . 1- 1 could be measured. The speed of mineralisation is dependent on the CO 2 production rate. The pointers mark the addition of 0.5 I nutrient solution. Microbiol. Res. 151 (1996) 1

33

Thus, a higher CO 2 emission rate indicates a faster phenanthrene degradation rate. For a further control the emission of CO 2 during the mineralisation of phenanthrene was balanced from the start until 125 h. C-source which was added: 8 g of phenanthrene which represents 7.54 g of carbon. For the formation of energy and biomass out of hydrocarbons, the general equation can be formulated typified as benzene (Mitchell et al., 1992), (biomass)

C6 0 6 + 1.5 HC0 3 -

+ 1.5NH4 -t 1.5C SH 70 2N + 1.5H 20

(energy) C60 6

+ 7.50 2 -t 6C0 2 + 3H2 0

The yield of biomass formation out of hydrocarbons without the energy used for maintenance is therefore 1.0, which correlates with figures out of literature which vary from 0.7 to 1.0, (Schlegel, 1987; Rosenberg, 1993). To deliver energy from hydrocarbons, the oxidation of intermediates and formation of reduction equivalents ends with the oxidation of intermediates, i.e. formiate to carbondioxide, decreasing the yield coefficient for the biomass formation. Since we deal with mixed populations the yield coefficient of the soil organisms is determined by calculation of the CO 2 mass flow. CPR = carbon production rate, VAFR = volume air flow rate (2.01 . min -1) CPR

PH 20)' 44·273.15 22.4· (273.15 + T) . 760

100

_

0.033) 100

(6)

In 2 = 0.029, the growth rate of the mixed population tS/2

can be determined according to, dS 1 dX1 --=-·W X =_·dt Y x /s dt YXjS =

0.0173 h - 1 .

As a control 1 litre of twice autoc1aved and contaminated soil was placed beside the reactor in the same

34

Microbiol. Res. 151 (1996) 1

The complexity of the soil and therefore the difficulty to interpret laboratory scale experiments hinder to find solutions for the problems commercial bioremediations deal with. The simulation of the vacuum heap technology by means of aeration of an soil heap with aeration pipes under conditions close to field applications and the benefit of powerful process control systems and analytical methods can help to understand the integral functions of biodegradation processes in soil and to uncover short-comings of certain techniques. The fast decrease of CO 2 concentration and soil temperature when the mixing was switched off indicates obviously that the potential degrading populations suffer from growing restrictions. These results are well in aggreement with the theoretical considerations made. Mass transfer limitations playa major role in growing limitations of soil organisms. The gradient of the potential C-source, equation (2), as demonstrated with phenanthrene, dt

CPR during mineralisation (without the fraction formed out of the uncontaminated soil) is 2.99 g for the period of 125 h. The subtraction of the carbon fraction in the exhaust gas from the carbon fraction in the substrate results in 7.54 g - 2.99 g = 4.55 g, which represents 4.83 g of phenanthrene. The yield coefficient Y x /s is therefore approx. 0.6 for phenanthrene in soil, which is less than expected, but still higher as compared to cultures which grow on carbohydrates (Sonnleitner and Hahnemann, 1994). From the rate of phenanthrene uptake which is

yielding in J-lf}

Discussion

depends on the desorption process - dQ which itselfis

= VAFR· (p -

x (%C02out

room to test for evaporation. The soil was aerated according to the soil in the reactor with 0.11· min -1. After 8 days 93% of the phenanthrene added to the soil were still present. Probably these 7% oflost were stripped together with acetone used as solvent.

a function of the surface (A) of the soil, equation (3). The biomass formation is significantly accelerated due to the substrate which is bioavaiable, equation (1). It must be noticed that the phenanthrene uptake rate found in the soil is identical with the growing rate of mixed cultures J-l = 0.03 found by other researcher in submerged cultures on the same substrate (Volkering et al., 1992). Since we calculated the yield coefficient by head space mass balances which is Y x /s = 0.6 the growing rate of the mixed soil population on phenanthrene decreases to J-ltot = 0.0173. Due to the fact that CO 2 formation during the mineralisation correlates qualitatively as well as quantitatively with the measured substrate concentration, the residuals of phenanthrene and other hydrocarbons are well predictable during an experiment by online calculating of the CPR. Furthermore, by calculating the desorption rate and the max. substrate concentration in solution, which depends on the solubility of the compound(s), based on results out of soil model reactors a mathematical prediction of the time frame within the biodegradation takes place, could be realised. A lot of results out of model reactors and real sanitations are necessary to create such a model.

The experience with submerged cultures and other applications which need a deeper understanding of fermentation technology can help to develop new bioremediation concepts or to improve already established technologies. Step functions carried out in closed systems and the resulting answers of the biological culture may lead to new approaches in bioremediation processes. From the engineering point of view a variety of improvements may help to speed up degradation processes. Of high priority must be to inhibit the formation of ducts and drains inside the soil heap, causing an uneven dispersion of O 2 and nutrients in the soil. In Figure 4 the depletion of diesel is not only due to microbial activity. The rate of diesel at the beginning of the experiment is obviously to fast, compared to the CO 2 concentration in the exhaust gas. Thus, a significant amount of volatile hydrocarbons had been stripped by the aeration of the system. The quantification of these effects shall be of further interest. Acknowledgements The support of Swiss Priority Programm Environment via the Swiss National Science Foundation for this work is greatly acknowledged. Abbreviations A AD/DA

C,

CO 2 CPR DC

k

O2 PAH

Q

RQ rph rpm

S 15/2

V VAFR

X yx/s JJ.

soil surface area Analog Digital/Digital Analog concentration of substrate in solution carbon dioxide fraction in the exhaust gas carbon production rate direct current desorption constante oxygen fraction in the exhaust gas Poly Aromatic Hydrocarbon amount of solid or adsorbed substrate respiratory quotient rounds per hour rounds per minute total substrate concentration time within half the substrate is taken up volume volume air flow rate biomass concentration yield biomass growth rate

[m 2] [kg· m- 3]

[%] [g·h- 1] [m·h- 1] [%] [kg] [g.g-l] [h -1] [min- 1] [kg·m- 3 j [h -1] [m 3] [1'h- 1] [kg·m- 3] [kg· kg-i) [h -1]

References Bashir, AI, Cseh, T., Leduc, R., Samson, R. (1990): Effect of soil/contaminant interactions on the biodegradation of naphtalene in flooded soils under denitrifying conditions. Appl. Microbiol. Biotechnol. 34, 414-419. Bellin, C. A., O'Conner, G. A., Jin, Y. (1990): Sorption and degradation of pentachlorphenol in sludge amended soils. J. Environ. Qual. 19, 603 - 608. Biehler, M. J., Hagele, S. (1994) : Vergleich von Verfahren zur biologischen Sanierung mineralolkontaminierter BOden. Terra Tech 3, 52-55. Egli, Th., Kappeli, 0., Fiechter, A. (1982): Regulatory flexibility of methylotrophic yeasts in chemostat cultures: simultaneous assimilation of glucose and methanol at a fixed dilution rate. Arch. Microbiol. 131, 1-7. Eiermann, D., Ebiox, R. (1992): Bioremediation of exvacated soil contaminated with mineral oil compounds using vacuum heap technologies, International Symposium, Karlsruhe, BRD. Eschenbach, A., Kastner, M., Bierl, R., Schaefer, G., Mahro, B. (1994): Evaluation of a new, effective method to extract polycyclyc aromatic hydrocarbons from soil samples. Chemosphere 28, 4, 683 - 692. Jud, G. (1992): Untersuchungen zur Wachstumskinetik von Methanobacterium thermoautotrophicum unter besonderer Beriicksichtigung des Massentransfers von Wasserstoff. Ph. D. thesis. University of Zurich. Mitchell, R. (1992): Environmental Microbiology. II Series, Wiley-Liss, QR 100.N48 1992. Perry, R. H., Chilton, CH., Kirkpatrick SD. (1963): Chemical engineers handbook. McGraw-Hill, New York. Pfennig, N., Lippert, K. D. (1966): Uber das Vitamin B-12 Bediirfniss phototropher Schwefelbakterien. Arch. Microbiol. 55: 245 - 256. Rosenberg, E. (1993): Exploiting microbial growth on hydrocarbons. TIBTECH, 11. Schlegel, G.: Allgemeine Mikrobiologie (1992). Thieme Verlag ISBN 3-13-444-607-3 . Schneider, K. (1987): Computeranwendung in der Biotechnologie. Swiss Biotech 5, 2, 13 - 21. Sonnleitner, B., Hahnemann, U . (1994): Dynamics of the respiratory bottleneck of Sachharomyces cerevisiae. J. biotechnol. 38: 63 - 79. Tempest, D. W. (1970) : The continuous cultivation of microorganisms. 1. Theory of a chemostat. Methods Microbiol. 52, 259 - 276. Volkering, F., Breure, A. M., Sterkenburg, A., van Andel, J. G. (1992): Microbial degradation of polycyclic aromatic hydrocarbons: effect of substrate availability on bacterial growth kinetics. Appl. Microbiol. Biotechnol. 36, 548-552. and International Symposium on subsurface Microbiology (ISSM-93) 19-24 Sept. Bath UK.

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