Liu Yueying, Fu Jinkun^#, Zhou Zhaohui^#, Lin Zhongyyu^#, Li Renzhong
(Department of Biology, ^#Department of Chemistry, Xiamen University, Xiamen, 361005,China)

Received Dec. 21, 1999; Supported by the National Natural Science Foundation of China. (No.29743001 and 29876026)

Abstract Highly dispersive supported palladium catalyst was prepared by the reduction of Bacillus licheniformis R08 biomass. TEM analysis indicated that R08 biomass was able to adsorb and reduce Pd²⁺ to Pd⁰ particle. IR spectrum study showed that -COO^- and -HPO₄²- groups on the cell wall may involve in the process of adsorbing Pd²⁺. XPS detection showed that  Pd²⁺ ions on g-Al₂O₃ were reduced to Pd⁰ by R08 biomass. The resulted Pd⁰/g-Al₂O₃ catalyst after  heating treatment possesses much high efficiency for catalyzing the oxidation of carbon monoxide, comparing with the catalyst prepared by the common impregnated method_. Moreover, TEM micrograph indicated that the Pd⁰ particles on the supporter g-Al₂O₃ were highly dispersive with mean size of 5nm after heat treatment.
Keywords Palladium catalyst, biosorption, bioreduction, Bacillus licheniformis.

1. INTRODUCTION
The catalytic properties of supported metal catalysts are closely related to the dispersive degree of metal ^[1,2]. Therefore, the preparations of highly dispersed supported metal catalysts have attracted a great deal of attention ^[3]. In our previous paper, we reported D01 biomass to reduce Au³⁺ for the preparation of highly dispersed supported gold catalyst Au/a -Fe₂O₃ ^[4]. As a  part of our research work on the preparations of highly dispersive precious metal catalysts by microbial method, here we further report the preparation of palladium catalyst, a much more common used catalyst for hydrogenation and oxidation. In the preparation, the biosorption of Pd²⁺ by R08 biomass (a dead biomass) was characterized with TEM and IR techniques. The Pd/ g-Al₂O₃ catalyst was prepared from the bioreduction of Pd²⁺ impregnated on g-Al₂O₃ and heat treatment, then characterized by TEM and XPS techniques. The catalyst showed much high efficiency for the catalytic oxidation of carbon monoxide compared with the common impregnated catalyst.
2. EXPERIMENTAL
2.1 Bacterial cultivation and biomass preparation
The bacterial strain R08 used in this study was isolated from pit water and identified as Bacillus licheniformis R08. The strain R08 was cultivated as previously reported ^[4]. The cultures were harvested by centrifuging (3500 rpm, 15 min) and the cell pellets were subsequently washed three times with deionized water. The resulted biomass was dried at 60^oC and ground with a mortar and pestle. The dry, dead biomass was stored in a dryer for use.
2.2 Measurement of IR spectra
The blank R08 biomass and the biomass were contacted with PdCl₂ solution for 1 h and then dried at 60^oC respectively. Their IR spectra were measured on 740sx FT-IR spectrometer.
2.3 Preparation of Pd⁰/g-Al₂O₃ catalyst by impregnated method
Stoichiometric amounts of PdCl₂ aqueous solution was impregnated on g-Al₂O₃ (30-60 meshes, BET surface area 240m²/g) for the preparation of catalyst precursor Pd²⁺/g-Al₂O₃. The loading weight of Pd²⁺ was 2 %. Pd²⁺/g-Al₂O₃ was pre-dried under vacuum at 80^oC for 4 h and heated to 600^oC at a rate of 5^oC/min in a reaction tube. The resulted catalyst was kept at 600^oC for 1.5 h and reduced with hydrogen at 250^oC for 1h for the preparation of Pd⁰/g-Al₂O₃ catalyst.
2.4 Preparation of supported Pd⁰/g-Al₂O₃ catalyst with microbial reduction 
The above precursor Pd²⁺ /g-Al₂O₃, R08 biomass and deionized water were mixed with weight ratio of 1:0.3:5. The mixture was incubated at 30^oC and then was kept at pH 3.5 for the preparation of Pd⁰/g-Al₂O₃. The sample was periodically removed and detected with a XPS (ESCALRB MKI X-ray photoelectron spectrometer) for the determination of the reduced degree of the sample until Pd²⁺ ions supported on g-Al₂O₃ were completely reduced to Pd⁰. The resulted Pd⁰/g-Al₂O₃ was heated in a oven at 600^oC for 1.5 h to destroy the biomass adsorbed on the Pd⁰/g-Al₂O₃, and further used for the preparation of TEM micrograph and the test of the catalytic reaction.
2.5 Test of the catalytic activity
The above 2 % Pd⁰/g-Al₂O₃ catalysts (0.5 g) were filled in a fixed bed microreactor (0.6cm i.d.) and heated at 150^oC. CO (2%)-air mixture was flowed with GHSV 20000 ml· g^-1·h^-1 (bioreduced method) or GHSV 1000 ml · g^-1·h^-1 (conventional method) for the test of catalytic activity. The activities of Pd⁰/g-Al₂O₃ catalyst prepared by two different methods were compared. The conversion of 2% CO balanced with air was analyzed according to CO percent content in the concentration of initial and finial gas by an online ST03 model gas chromatograph with a thermal conductivity detector and 5Å molecular sieve column.
2.6 Preparation of TEM micrographs
The sample was taken out by the copper grid. The sample on the copper grid was dried at room temperature and then recorded with a TEM (JEM 100 CXII transmission electron microscope) at an accelerating voltage of 100 kV.
3. RESULTS AND DISCUSSION
3.1 TEM characterization of adsorption of Pd²⁺ by R08 biomass
Fig.1-a and 1-b were the transmission electron micrographs of R08 biomass contacted without and with PdCl₂ solution respectively. It can be found that Pd²⁺ ions were adsorbed and reduced to metallic palladium by R08 biomass. The obvious reduction of Pd²⁺ ions by R08 biomass indicated that R08 biomass could be used as Pd²⁺ reductant for the preparation of supported palladium catalyst.

Fig.1 Transmission electron micrographs of B. licheniformis R08 biomass
(a) R08 biomass (b) R08 biomass contacted with PdCl₂ solution

3.2 IR spectra characterization of R08 biomass contacted with Pd²⁺
The IR spectra of the biomass and the biomass contacted with Pd²⁺solution for 1 h were measured. The bands at 1547 cm^-1 and 1402 cm^-1 are corresponded to asymmetric and symmetric vibration of C=O bond respectively. Moreover, two bands at 1230 cm^-1 and at 1286 cm^-1 may associate with the vibration of n (C-O ) and n (P-O) ^[5]. The bands of R08 biomass contacted with Pd²⁺ shifts obviously to lower frequency from 1547 cm^-1 to 1539 cm^-1, which suggest the existence of interaction between COO^-group of the biomsss and Pd²⁺ ions. In addition, the spectral band of n (P-O) shifts from 1286 cm^-1 to 1281cm^-1, which may result from the interaction between palladium and HPO₄²-group of the biomass ^[5]. The results are in agreement with some reports on biosorption of metal ions ^[6,7].
3.3 XPS spectral characterization of reduction of Pd²⁺/g-Al₂ O₃ by R08 biomass
The reduced degree of Pd²⁺ supported on g-Al₂ O₃ may be monitored according to the ratio of the total area of spectral peaks for the bonding energy of Pd²⁺ to Pd⁰. The bioreduction of Pd²⁺/g-Al₂O₃ by R08 biomass was examined by XPS. Fig.2-a was the XPS spectrum of catalytic precursor Pd²⁺/g-Al₂O₃ contacted with R08 biomass for 12 h. It may be defined that about 20% of Pd²⁺ ions were reduced to Pd⁰. Fig.2-b shows the XPS spectrum of the sample of Pd²⁺/g-Al₂O₃ reduced by R08 biomass for 48 h. The results indicated that the Pd²⁺supported on g-Al₂O₃ were completely reduced to Pd⁰.
    The above results further supported that R08 biomass could be used as a reductant for preparation of Pd⁰/g-Al₂O₃ and the mechanism of Pd²⁺ biosorption involved a reduction of Pd²⁺ ions. In the bioreduction, the enzymatically mediated bioreduction of metals has been well documented ^[8]. However, non-enzymatically mediated bioreduction of metals including palladium has only received little or no attention. Brierley et al ^[9] have indicated that the biosorption of platinum group metal ions by MAR (a metal removal agent prepared by spent dead biomass from fermentation processes) followed by an apparent reduction of the metal ions. Recently, Lloryed et al ^[8] reported that the resting cells of Desulfovibrio desulfurcans could reduce enzymatically soluble Pd²⁺ ions to Pd⁰, using pyruvate, formate, or H₂ as the electron donor without biochemical cofactors. The process of Pd²⁺ reduction was O₂ insensitive. Our results showed that the biosorption and bioreduction of Pd²⁺ by R08 dead biomass was a non-enzymatic reaction, in which metabolic process is not involved. The reduction of Pd²⁺ by R08 dead biomass may relate with -HPO₄²- and -COO^- groups on the biomass surface.

Fig.2 XPS spectra of Pd2+/g-Al2O3 contacted with R08 biomass for different period, (a)12 h, (b) 48h

3.4 Catalytic activity for CO oxidation and TEM characterization of Pd⁰/g-Al₂O₃ catalyst
Pd⁰/g-Al₂O₃ prepared by bioreduced method was used as catalyst for oxidation of CO. The catalyst showed better performance in the conversion of CO to CO₂. The catalytic activity was 100% with higher GHSV (20000ml·g^-1·h^-1) at 150^oC. The catalytic activity was retained for more than 70 h. Although Pd⁰/g-Al₂O₃ catalyst prepared by conventional impregnated method also showed 100% conversion efficiency of CO oxidation, the rate of reaction gas flowed was much low GHSV (1000ml·g^-1·h^-1), and the activity was gradually decreased to 50% after 34 h.
    The supported Pd⁰/g-Al₂O₃ catalyst prepared by bioreduced method were observed by TEM, which showed that Pd⁰ particles on the supporter g-Al₂O₃ were highly dispersive with an average size of 5nm after heat treatment (Fig.3). It seems that the particles do not enlarge obviously, since there were the separation of biomass between Pd⁰ particles. From figure 3, there were many particles adsorbed on supporter g-Al₂O₃. This showed that most of the metallic palladium particles were retained and not oxidized.

Fig.3 The TEM micrograph of Pd0/g-Al2O3 catalyst prepared by bioreduction method

    According to the above results, it is considered that catalyst precursor Pd²⁺/g-Al₂O₃ was completely reduced to Pd⁰/ g -Al₂O₃ by R08 biomass. The biomass on the Pd⁰/g-Al₂O₃ has been destroyed and Pd⁰ on g-Al₂O₃supporter was fully exposed after the heat treatment. Moreover, due to the in-situ reduction of Pd²⁺ ions by R08 biomass supported on g-Al₂O₃ under the normal temperature and the "anchor action" of biomass adsorbed on the Pd⁰/g-Al₂O₃^[10], those effects will reduce the shift and gathering of Pd⁰ particles on g-Al₂O₃ supporter during the heating treatment. Because the Pd particles on the supporter were nano-particles and highly dispersive, the catalyst Pd⁰/g-Al₂O₃ prepared with bioreduced method shows relatively high catalytic activity and long activity life for oxidation reaction of carbon monoxide. The reaction temperature (150^oC) of catalytic reaction was much lower than that of the catalyst Pd⁰/g-Al₂O₃ (the load of Pd²⁺ for 5%) prepared with conventional method reported in literature (230^oC)^[11].
    Under the same Pd²⁺ load weight, the supported catalyst Pd⁰/g-Al₂O₃ prepared with R08 biomass reduction is of much high catalytic activity and long activity life than the catalyst prepared by the conventional method. The differences in the catalytic activity may be resulted from the dispersity and size of Pd⁰ particles on the supporter g-Al₂O₃. Therefore this method is of potential application on the preparation of highly dispersed precious metal catalyst.

REFERENCES
[1] Hao Z P, An L D, Wang H L. J. Mol. Catal. (Fenzi Cuihua), 1996, 10 (3): 235-240.
[2] Minicò S, Scirè S, Visco A M et al. Catal. Lett., 1999, 47: 273-276.
[3] Henry C R. Surface Sci. Reports, 1998, 31 (7-8): 235-325.
[4] Liu Y Y, Fu J K, Hu R Z et al. Acta Microbiol. Sinica (Weishengwu Xuebao), 1999, 39 (3): 260-263.
[5] Wu J G. Modern Fourier-transform infrared spectroscopy and its application (Part II). Beijing: Literature of Science and Technology Press, 1994.
[6] Kuyucak N, Volesky B. Biotechnol. Let., 1988, 10 (2): 137-142.
[7] Chen Y S, Sun Q J, Chen J et al. Adv. Enveron. Sci. (Huanjin Kexue Jingzhan), 1997, 5 (6): 34-43.
[8] Lloyd J R, Yong P, Macaskie L E. Appl. Environ. Microbiol., 1998, 64 (11): 4607-4609.
[9] Brierley J A, Vance D B. Proc. Int. Symp., 1987 (Pub.1988), 477-485.
[10] Fourest E, Roux J C. Appl Microbiol Biotechnol., 1992, 17: 399-403.
[11] Luo M F, Hou Z Y, Yuan X X, Zheng X M. Catal. Lett., 1998, 50: 205-209.

[ Back ] [ Home ] [ Up ] [ Next ] Mirror Site in USA  Europe  China  CSTNet ChinaNet