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Production of antifungal materials by bioconversion of shellfish chitin wastes fermented by Pseudomonas fluorescens K-188
Enzyme and Microbial Technology 36 (2005) 49–56
Production of antifungal materials by bioconversion of shellfish chitin wastes fermented by Pseudomonas fluorescens K-188 San-Lang Wanga,b,∗ , Yue-Horng Yenc , Guo-Chuan Tzengc , Chienyan Hsiehc a
Graduate Institute of Life Sciences, Tamkang University, Tamsui 251, Taiwan Life Science Development Center, Tamkang University, Tamsui 251, Taiwan Department of Bioindustry Technology, Da-Yeh University, Chang-Hwa 515, Taiwan b
c
Received 8 September 2003; accepted 17 March 2004
Abstract Pseudomonas fluorescens K-188 produced antifungal materials that inhibited fungal phytopathogens Fusarium oxysporum, Fusarium solani, Trichoderma harzianum, and Pythium ultimum. In the concentration of antifungal materials, ethanol precipitation was better than ammonium sulfate precipitation. The antifungal materials retained almost 70% of the original antifungal activity even after being heated at 100 ◦ C for 10 min. Microscopic examination showed, when the antifungal materials were added, the hyphae of F. oxysporum became thinner and the germination of the spores was inhibited. The antifungal activity of P. fluorescens K-188 primarily should be correlated with cells themselves and an antifungal protein with molecular weight of 11 kDa. © 2004 Elsevier Inc. All rights reserved. Keywords: Pseudomonas fluorescens; Antifungal; Chitin; Fusarium oxysporum; Shrimp and crab shell
1. Introduction Chemical fungicides continue to play the most important role in current farming practices for the protection of crops against diseases. However, their utilization is under increased scrutiny and examen in recent years because many chemical fungicides are very toxic, and cause contamination of the environment, leave fungicide residues in food products, and induce pathogen resistance. Because of these limitations of chemical fungicides, it seems necessary to search for an alternative control strategy. Biological control or the use of microorganisms or their secretions to prevent plant diseases, offers an attractive harmless alternative or supplement for the control of plant diseases. Therefore, biological control tactics become an important approach to facilitate sustainable agriculture [1–4]. Chitin and its derivatives are of interest because they have various biological activities and applied in such as ∗
Corresponding author. Tel.: +886 2 2626 9425; fax: +886 2 8631 1015. E-mail address: [email protected] (S.-L. Wang).
0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2004.03.024
immunoadjuvant and a flocculant of wastewater sludge, and agrochemical uses [5]. Shrimp and crab shell contain chitin, protein and inorganic compounds mainly composed of calcium carbonate [6–8]. Traditional methods for the preparation of chitin include demineralization and deproteinization of the waste material with strong acids and bases (e.g., HCl and NaOH) [8–10]. The production of chitin and its hydrolyzed derivatives, such as acetylglucosamine and chitooligosaccharide, from waste of the shellfish industry has been limited due to the high cost of chitinase and the shrimp and crab shell pretreatment process [5,8]. Chitinases, a group of enzymes capable of directly degrading chitin to low molecular weight products, have been found to be produced by a number of microorganisms. The production of inexpensive chitinolytic enzymes is an important element in the utilization of shellfish wastes. The discovery of inexpensive chitinolytic enzymes not only solves environmental problems but also promotes the economic value of the marine products [11]. To further enhance the utilization of chitin-containing waste, we recently investigated
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the bioconversion of shrimp and crab shell powder (SCSP) for the production of chitinase and antifungal compounds [1–5,8,11–18]. In this research, we found that the strain Pseudomonas fluorescens K-188 exhibited antifungal properties in the presence of shellfish chitin wastes. The optimal culture conditions and some properties of the antifungal materials from culture broth are described. The effects of the antifungal materials on the germination of spores of pathogenic F. oxysporum were investigated.
2. Matreials and methods 2.1. Materials Chitin flake and chitosan powder from crab shell were purchased from Biotech Co. (Kau-Shyuon, Taiwan). Powdered chitin was purchased from Sigma Chemical Co., St. Louis, MO. Metarhzium anisopliae was kindly supplied by Dr. JenYuen Shieh, Department of Bioindustry Technology, Da-Yeh University, Chanhwa, Taiwan. 2.2. Shrimp and crab processing waste Five preparations of the same types of shrimp and crab processing wastes were used in this study, namely: (i) Untreated shrimp and crab shell powder (SCSP): The SCSP used in these experiments was prepared as described earlier [1]. In the preparation of the SCSP, the shrimp and crab shells collected from a marine foodprocessing factory were washed thoroughly with tap water followed by steaming. The solid material obtained was dried, milled, and sieved to powder with diameters <0.053 mm. (ii) SCSP treated with HCl (HCl–SCSP): In this process, the SCSP was treated with 2N HCl at room temperature for 2 days. The ratio of SCSP to solvent was 1:8 (w:v). This is identical to the demineralization method for the preparation of crustacean chitin [19]. The demineralized material was recovered by filtration followed by thoroughly rinsed with deionized water, and dried at 65 ◦ C. This product is referred to as HCl–SCSP. (iii) The filtrate from SCSP treated with HCl (HCl-extract): This extract was the filtrate obtained from the HCl–SCSP preparing process as described above. The filtrate was adjusted to pH 7 by NaOH solution. This product is referred to as HCl-extract. (iv) SCSP treated with NaOH (NaOH–SCSP): In this process, SCSP was treated with 2N NaOH at 100 ◦ C for 30 min. The ratio of SCSP to solvent, 3:40 (w:v), was referred from the deproteinization method for the preparation of crustacean chitin [20]. The deproteinized material was recovered by means of filtration, thoroughly rinsed with deionized water, and dried at 65 ◦ C.
(v) The filtrate from SCSP treated with NaOH (NaOHextract): This extract was the filtrate obtained from the NaOH–SCSP preparation process described above. This preparation is referred to as NaOH-extract. The concentrations of the four preparations other than the raw SCSP are expressed as the weight percentage of raw SCSP used before treatment. For example, X g of raw SCSP, treated with acid or alkali, produced X wt.% of SCSP in the acidic/alkaline solution (w/v). 2.3. Isolation and screening of antifungal materials producing strains Microorganisms isolated from soils collected at different locations in northern Taiwan were screened on agar plates containing 0.2% colloidal chitin, 0.1% K2 HPO4 and 0.05% MgSO4 ·7H2 O, 0.1% NaNO3 , and 2% agar (pH 7). The plates were incubated at 30 ◦ C for 2 days. Colonies that grew well or showed a clear zone around the colonies under such conditions were isolated and retained for subsequent screening. Those organisms obtained from the first screening were subcultured in liquid media (containing 1% SCSP, 0.1% K2 HPO4 and 0.05% MgSO4 ·7H2 O) in shaken flasks at 30 ◦ C and shaken at 180 rpm for 2 days. After centrifugation (8000 × g, 4 ◦ C, for 20 min, Beckman J2-21 M/E), the supernatants were collected for measurement of antifungal activity using the procedure described below. The strain K-188, which showed the highest antifungal activity, was isolated, kept on nutrient agar, and used for the study. 2.4. Identification of strain K-188 After study of the morphological, cultural, biochemical and physiological characteristics of the organism, identification was made in accordance with the method described in Bergey’s Mannual of Systematic Bacteriology [21]. Strain K-188 was identified as P. fluorescens (identified by Culture Collection and Research Center, Shin-Chu, Taiwan). 2.5. Effect of culture conditions In the investigation of culture conditions, growth was carried out in a basal medium containing 0.1% K2 HPO4 and 0.05% MgSO4 ·7H2 O (pH 7), and gradually supplemented with the various ingredients to be investigated. The major ingredients investigated including SCSP, carbon sources, nitrogen sources, and inorganic salts were added and investigated separately. One hundred milliliters of the resultant medium in a 250-mL Erlenmeyer flask was aerobically incubated at 30 ◦ C for 48 h on a rotary shaker (180 rpm). After centrifugation (8000 × g, 4 ◦ C, for 20 min, Beckman J221 M/E), the supernatant was used for bioassay. Usually an effective experimental prior condition was used as the basis
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for the later experiment until the best culture condition was obtained. With the use of this culture composition, the effects of the initial pH, temperature, culture volume and cultivation time on the production of antifungal activity was investigated in the same way until the optimum culture condition was found. 2.6. Dual culture The laboratory screened antifungal compounds producing strains, P. fluorescens K-188, Pseudomonas aeruginosa K187 [5], Bacillus amyloliquefaciens V656 [1], Bacillus subtilis W113 [3], and B. subtilis W118 [3], were dual cultured and mix cultured with M. anisopliae in potato dextrose agar (PDA) and potato dextrose broth (PDB), respectively, to observe the growth condition and the change of spore production. 2.7. Measurement of protease activity For measuring protease activity, a diluted enzyme solution (0.2 mL) was mixed with 2.5 mL of 1% casein in pH 7 phosphate buffer and incubated at 37 ◦ C for 10 min. The reaction was terminated by adding 5 mL of 0.19 M trichloroacetic acid (TCA). The reaction mixture was centrifuged and the soluble peptide in the supernatant fraction was measured by the method of Todd with tyrosine as the reference compound [22]. 2.8. Preparation of the antimicrobial materials For the production of antifungal materials, P. fluorescens K-188 was grown aerobically at 37 ◦ C for 4 days in an Erlenmeyer flask (250 mL) containing 200 mL of liquid medium consisting of 1% SCSP, 0.05% MgSO4 ·7H2 O, and 0.1% K2 HPO4 (pH 7). The culture broth was centrifuged for 20 min at 8000 × g, and the supernatant was used for the concentration of the antifungal materials. The supernatant was subjected to ammonium sulfate precipitation and ethanol precipitation as described below. (a) Ammonium sulfate precipitation. Ammonium sulfate (608 g/mL) was added to the supernatant (800 mL). The resultant mixture was kept at 4 ◦ C overnight, and the precipitate formed was collected by centrifugation at 4 ◦ C for 20 min at 12,000 × g. The precipitate was dissolved in a small amount of 50 mM sodium phosphate buffer (pH 7) and dialyzed against the buffer. The resultant dialysate (45 mL) was referred as concentrated solution A. (b) Ethanol precipitation. 95% ethanol (1:9, v/v) was added to the supernatant. The resultant mixture was kept at 4 ◦ C over night, and the precipitate formed was collected by centrifugation at 4 ◦ C for 20 min at 12,000 × g. The ethanol-free precipitate was dissolved in a small amount of 50 mM sodium phosphate buffer (pH 7) and referred as concentrated solution B.
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2.9. Antifungal activity of the materials The antifungal activity for the materials was estimated using a growth inhibition assay described earlier [1]. Fungal spores of F. oxysporum were grown on Petri plates filled with potato dextrose agar (PDA). After 10 days of incubation at 25 ◦ C, the fungal colonies were recovered with sterile water containing 0.1% (v/v) Tween 80. The resulting suspension was filtered aseptically through sterilized gauges. The filtrate was adjusted with sterile water to a concentration of 1 × 106 spores/mL, and stored at 4 ◦ C. To test the antifungal effect of the material produced by P. fluorescens K-188, Petri plates were filled with molten PDA precooled to 45 ◦ C, and divided into two groups, namely experimental group (E) and control group (C) (triplicate for each). To each plate in the experimental group (E), an appropriate amount of the antifungal materials was added. To those of the control group (C), an equal amount of sterile buffer was added. After the plates were cooled, the fungal inoculum was then placed onto the agar surface. Both groups were incubated at 25 ◦ C for 72 h. The diameters of the largest and smallest fungal colonies were recorded and the averages were calculated. The inhibition ratios were calculated with the following formula. If the inhibitory ratio was greater than 20%, the test strain would be considered of being inhibited and the minimal inhibitory concentration (MIC) for that strain was then determined. Inhibition ratio (%) = (C − E)/C × 100% where C is the average diameter of the largest and smallest colonies of the control groups; E is the average diameter of the largest and smallest colonies of the experimental groups. 2.10. pH and thermal stability of the antifungal materials The pH stability of the antifungal materials was determined by measuring the residual inhibitory activity at pH 7 as described above after dialyzing the samples against a 50 mM buffer solution of various pHs (pH 3–11) in seamless cellulose tubing (Sankyo, Japan). The buffer systems used were glycine–HCl (50 mM, pH 3), acetate (50 mM, pH 4.5), phosphate (50 mM, pH 6–8), Na2 CO3 –NaHCO3 (50 mM, pH 9–11). The thermal stability of the antifungal materials was studied by heating the samples at 100 ◦ C for various time periods. The residual inhibitory activity was measured as described above (using F. oxysporum as the target). 2.11. Spore germination affected by the antifungal material Half milliliter of fungal spores (106 /mL) of F. oxysporum, 0.5 mL of potato dextrose broth, and 0.5 mL of antifungal material were added into the eppendorf tube (1.5 mL). The resultant mixture was incubated at 25 ◦ C, and the spores were observed by using a phase-contrast microscope at various intervals [14].
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2.12. Molecular weight determination The molecular weight of sample was determined by GPC using a size exclusion column (TSK-G3000SWXL ). The 0.2 M phosphate buffer (pH 6.8) was used as a mobile phase at a flow rate of 0.5 mL/min, and the sample loop loaded was 20 L. The effluent was monitored by UV detecter at 254 nm (JASCO UV-1570) and the data processed with data manager (VISCOTEK DM400). The standards polymer used for the calibration were bovine serum albumin (67,000 MW), (-chymotrysinogen (25,700 MW), myoglobin (17,600 MW), and Aprotinin (6500 MW).
3. Results and discussion 3.1. Effect of P. fluorescens K-188 on the growth of M. anisopliae It was observed in the dual culture test that both B. amyloliquefaciens V656 and P. fluorescens K-188 grew better with M. anisopliae. This means both grew at same rate and did not compete for nutrition sources (data not shown). The mixture of M. anisopliae and P. fluorescens K-188 was also inoculated on liquid medium (PDB) to observe the growth condition of the colonies. It was also found that mixture culture of P. fluorescens K-188 and B. amyloliquefaciens V656 showed least interference on M. anisopliae colonies (data not shown). Concluded from above results, among the laboratory strains, P. fluorescens K-188 and B. amyloliquefaciens V656 had the least influence on the growth of M. anisopliae. Although both species of Pseudomonas and Bacillus were reported to be used as bio-pesticides, Bacillus has narrower range of utilization than Pseudomonas, which can be utilized as both pesticides as well as germicides. Therefore, P. fluorescens K-188 was chosen for further study in this research. The spore production was observed since the current application of M. anisopliae focused on its spore products. The amount of spore produced by M. anisopliae was not changed when it was mix-cultured with P. fluorescens K-188 (data not shown), i.e., P. fluorescens K-188 had no effect on the spore production of M. anisopliae. Therefore, it is possible that P. fluorescens K-188 can be utilized together and cooperate with M. anisopliae. 3.2. Effect of culture conditions To study the effect of carbon sources on the production of antifungal materials, growth was carried out in basal medium as described above. Both the media with and without the addition SCSP/chitin were used to investigate the effect of SCSP/chitin on the production of antifungal materials. It was found that the antifungal activity of K-188 was enhanced by the addition of SCSP into the medium. The optimum antifungal activity (0.58 U/mL) was achieved when medium
Fig. 1. Antifungal materials production in the presence of various concentration of the preparations after two days of cultivation. (), SCSP; (), HCl–SCSP; (), HCl-extract; (X), NaOH–SCSP; ( ), NaOH-extract.
contained 1% SCSP and decreased with medium contained higher concentration of SCSP (data not shown). In our previous study [8], we found that SCSP was a more suitable carbon source than powdered chitin for production of antimicrobial compounds by P. aeruginosa K-187. Besides the investigation of the effect of SCSP on the production of antifungal materials, other chitin-related materials from the chitin preparation sources were also used in this study to investigate their effect on the production of antifungal materials. A series of experiments on the production of antifungal activity was also carried out to compare the inducing effect with the other four related preparations (HCl–SCSP, HClextract, NaOH–SCSP, NaOH-extract) described in Section 2. The production of antifungal activity with strain K-188 under these conditions is shown in Fig. 1. It was shown that the other four preparations were all less effective as carbon sources than SCSP in enhancing antifungal activity production. SCSP (1%) was chosen as carbon source and added into the cultures in the following experiments. The effect of time on the production of antifungal activity was investigated. K-188 strain was grown aerobically in 100 mL of optimal media at 25, 30, and 37 ◦ C. During the period of incubation, the antifungal activity was monitored in 24 h intervals for up to 6 days. As shown in Fig. 2, P. fluorescens K-188 incubated at 37 ◦ C for 4 days showed the highest antifungal activity (0.97 U/mL).
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3.3. Antifungal mechanism of P. fluorescens K-188
Fig. 2. Time-course of cultivation.
To study the effect of initial pH on the production of antifungal activity, growth was carried out in the optimal medium (1% SCSP and basal medium) at various initial pHs at 37 ◦ C for 4 days. The result showed that P. fluorescens K188 produced higher antifungal activity when the initial pH was 7. The optimal culture condition for P. fluorescens K-188 was grown aerobically in an orbital shaking incubator at 37 ◦ C for 4 days in an Erlenmeyer flask (250 mL) containing 100 mL of liquid medium consisting of 1% shrimp and crab shell powder, 0.1% K2 HPO4 , and 0.05% MgSO4 ·7H2 O, pH 7.
The antifungal mechanism of P. fluorescens K-188 is investigated herein. Take the concentration of the original culture supernatant as 100%. The final culture supernatant was adjusted to 33% (v/v) of its original concentration and incubated at the same condition. After the incubation, it was examined with phase-contrast microscope. The result was shown in Fig. 3. For control group shown in Fig. 3A, after being incubated at 25 ◦ C for 72 h, mycelium of F. oxysporum was uniform in thickness and showed intact appearance. For experiment group (medium, spore suspension, and antifungal materials was mixed in a volume ratio of 1:1:1) shown in Fig. 3B, the spores of F. oxysporum almost did not germinate at all. The germination rate of F. oxysporum decreased significantly. Comparing with Fig. 3C, even germination occurred, germ tubes were thinner than normal ones and no spore formation phenomenon was found. However, neither hyphae cytolysis nor tail-end expansion was observed. This inhibitory phenomenon was different from those by chitinase enzymes [1,2,5,23–25] and by other non-enzymatic compounds [14,15,26,27]. From above observations, it was inferred that the antifungal activity of P. fluorescens K-188 was not originated from fungal cell wall hydrolase such as chitinase. The inhibitory mechanism of P. fluorescens K-188 should be attributed to nutrition competition and/or germination inhibition. 3.4. Concentration of antifungal materials Many publications reported that the antifungal activities of microorganisms were resulted from the hydrolytic enzymes
Fig. 3. Effect of the antifungal materials from P. fluorescens K-188 on morphology of F. oxysporum. (A), normal mycelia of F. oxysporum; (B) (C), F. oxysporum hyphae in the presence of P. fluorescens K-188 antifungal materials (33%, v/v).
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Table 1 Some properties of the materials concentrated from culture supernatant of P. fluorescens K-188
Antifungal activity (U) Protease activity (U) Chitinase activity (U) Volume/dry weight
Culture supernatant
Concentrated solution A
B
970 ND ND 1000 mL
ND ND ND 30 mg
33,100 131 ND 1655 mg
such as chitinase produced by the microorganisms [1–4]. Consequently, it was further investigated that whether hydrolytic enzymes such as chitinase, protease, cellulase, xylanase were present in the fermentation broth of P. fluorescens K-188. To avoid the interfering of the enzyme activity analysis by reducing sugars, the culture supernatant was first concentrated with ammonium sulfate precipitation or ethanol precipitation to remove the above impurities. The recovered amount, antifungal activity, and enzyme activity of the resultant concentrated solution (concentrated solution A and concentrated solution B, respectively) were shown in Table 1. Concentrated solution A, purified by ammonium precipitation, possessed neither antifungal activity nor enzymatic activities of protease and chitinase. Concentrated solution B, purified by ethanol precipitation, possessed both antifungal activity and protease activity. Since the chitinase activity was not found, it was inferred that the antifungal activity of P. fluorescens K-188 should not be related to chitinase. This is different from other antimicrobial strains of Pseudomonas species. The antimicrobial compounds produced by P. aeruginosa K-187 and P. fluorescens were a bifunctional chitinases/lysozyme [5,14,15] and a phenazine antibiotic [26,27], respectively. Besides, protease activity was not found in the original culture supernatant but was detected in the concentrated solution purified by ethanol precipitation (concentrated solution B). Based on this result, it is inferred that protease and protease inhibitor probably existed together in the culture broth. Consequently, no protease activity was observed in the supernatant of P. fluorescens K-188. After the protease inhibitor was removed or inactivated by ethanol during the ethanol precipitation, the protease activity exhibited. Similar phenomenon of simultaneous existing of enzymes and enzyme inhibitors in fermentation broth has been reported previously. For example, lysozyme and lysozyme inhibitor existing together was found in culture broths of B. subtilis I-139 [28], P. aeruginosa M-1001 [29], and Staphylococcus aureus M18 [30]. 3.5. Effect of pH and temperature on antifungal activity and protease activity To investigate if there is any relationship between antifungal activity of P. fluorescens K-188 and protease, the effect of pH and temperature on protease activity and antifungal
activity of concentrated solution from ethanol precipitation (concentrated solution B) was analyzed. After being heated at 100 ◦ C for 10 min, close to 70% of residual antifungal activity was retained. After heated at 100 ◦ C for 2 min, near 90% of residual protease activity was retained. The protease activity completely lost after heated at 100 ◦ C for 4 min. As for optimum pH, it was pH 7–9 for both antifungal activity and protease activity (data not shown). Schokker et al. [31] reported that the extracellular protease purified from P. fluorescens 22F was thermal unstable. Judged from above, the thermal stable protease from P. fluorescens K-188 should be different from the thermal unstable protease from P. fluorescens 22F. As for the relationship between antifungal activity and protease activity, the thermal stability of antifungal activity was much higher than that of the protease activity although optimum pH was similar for both of them. To investigate the relationship between antifungal activity and protease activity, in this research, the antifungal materials (concentrated solution B) made from ethanol precipitation were further purified by DEAE-Sephadex CL-6B column chromatography. The result showed that antifungal activity was existed in the fractions eluted with 0.2 M NaCl containing phosphate buffer (pH 7) (data not shown). After dialysis with the same buffer, the obtained fraction was then performed by SDS–PAGE and GPC for molecular weight determination. The result from SDS–PAGE showed the antifungal material appeared at molecular weight lower than 14,000 (the smallest standard marker). Consequently, it was inferred that the antifungal material was small protein molecule with molecular weight less than 14,000. This coincides with the 11 kDa determined by GPC (data not shown). The relationship between antifungal activity and protease activity of P. fluorescens K-188 is still waiting to be final judged by the purification and characterization of the protease. According to the further purification and molecular weight determination, it can be inferred that the antifungal activity of P. fluorescens K-188 primarily should be correlated with cells themselves and an antifungal protein with molecular weight of 11 kDa. Furthermore, it was confirmed by the results that antifungal activity of the cells removed antifungal samples, filtered aseptically through 0.45-m pore-size membrane filters, were remarkably decreased (retained near half of its original antifungal activity). As a matter of fact, many reports showed the utilization of Pseudomonas species cells for biocontrol research. The examples included impact of P. fluorescens CHA0 and a derivative with improved biocontrol activity on the cultural resident bacterial community on cucumber [32]; effect of P. putida and a synthetic iron chelator on induction of soil suppressiveness to Fusarium wilt pathogens [33]; P. stutzeri YPL-1 genetic transformation and antifungal mechanism against F. solani, an agent of plant root rot [34]; P. cepacia suppression of sunflower wilt fungus and role of antifungal compounds in controlling the disease [35]; fluorescent Pseudomonas spp. Future study will investigate whether the antifungal activity would be retained after P. fluorescens K-188 cells were immobilized. The re-
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sult would confirm if the antifungal activity of P. fluorescens K-188 was originated from the cells themselves.
4. Conclusion Certain fluorescent Pseudomonas can inhibit important fungal pathogens of crops, and they have been studied for biocontrol of root diseases caused by soil-borne fungi [36]. Often, biocontrol mediated by fluorescent pseudomonas involves the production of secondary metabolites and/or extracellular enzymes that inhibit certain fungal pathogens. Several well-studied biocontrol strains of Pseudomonas e.g., Pf-5 and Q2-87 produce the secondary metabolite 2,4diacetylphloroglucinol (DAPG). The compound is recognized as a key factor in the biocontrol of fungal diseases such as take-all of wheat, black root rot of tobacco, or dampingoff of sugarbeat by biocontrol pseudomonas. Production of DAPG by P. fluorescent CHA0 has been detected in the rhizosphere of plants grown in artificial soil microcosms [37]. Bagnasco et al. [36] reported that P. fluorescens as biocontrol agents against forage legume root pathogenic fungi. We have reported that SCSP of marine wastes is an effective inducer for the production of antimicrobial chitinases by P. aeruginosa K-187 [5], Monascus purpureus CCRC 31499 [2,4], and B. amyloliquefaciens V656 [1]. In this research, SCSP was used as the major carbon source for producing material with antifungal activity. The acid or alkali liquid waste from SCSP treatment in the chitin production process also could be a feedstock for antifungal material production by P. fluorescens K-188. The results showed that P. fluorescens K188 could utilize the acid or alkali liquid waste from the chitin production process to produce antifungal materials. The antifungal activity could be both originated from K-188 cells, an antifungal protein with molecular weight of 11 kDa, and the protease activity.
Acknowledgment This work was supported in part by a grant of the National Science Council, Taiwan (NSC90-2313-B-212-003).
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[25] Mahadevan B, Crawford DL. Properties of the chitinase of the antifungal biocontrol agent Streptomyces lydicus WYEC108. Enzyme Microb Technol 1997;20:489–93. [26] Thomashow LS, Weller DM. Role of a phenazine anatibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminus var. tritici. J Bacteriol 1988;170:3499–508. [27] Thomashow LS, Weller D, Bonsall RF, Pierson LS. Production of the antibiotic phenazine-1-carboxylic acid by fluorescent Pseudomonas species in the rhizosphere of wheat. Appl Environ Microbiol 1990;56:908–12. [28] Murao S, Kato M, Wang SL, Hoshino M, Arai M. Isolation and characterization of a novel hen egg white lysozyme inhibitor from Bacillus subtilis I-139. Agric Biol Chem 1990;54:1129–36. [29] Wang SL, Shieh ST, Pai CS. Production, purification and characterization of two proteinaceous hen-egg-white lysozyme inhibitor from Pseudomonas aeruginosa M-1001. Proc Natl Sci Counc ROC B 1995;19:166–75. [30] Wadstrom T, Hisarsune K. Bacteriolytic enzymes from Staphylococcus aureus. Purification of an endo-beta-N-acetylglycosamidase. Biochem J 1970;120:725–34. [31] Schokker EP, van Wagengerg ACM, van Boekel MAJS. A note on the use of urea in studying the mechanism of thermal inactivation of
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Production of antifungal materials by bioconversion of shellfish chitin wastes fermented by Pseudomonas fluorescens K-188 San-Lang Wanga,b,∗ , Yue-Horng Yenc , Guo-Chuan Tzengc , Chienyan Hsiehc a
Graduate Institute of Life Sciences, Tamkang University, Tamsui 251, Taiwan Life Science Development Center, Tamkang University, Tamsui 251, Taiwan Department of Bioindustry Technology, Da-Yeh University, Chang-Hwa 515, Taiwan b
c
Received 8 September 2003; accepted 17 March 2004
Abstract Pseudomonas fluorescens K-188 produced antifungal materials that inhibited fungal phytopathogens Fusarium oxysporum, Fusarium solani, Trichoderma harzianum, and Pythium ultimum. In the concentration of antifungal materials, ethanol precipitation was better than ammonium sulfate precipitation. The antifungal materials retained almost 70% of the original antifungal activity even after being heated at 100 ◦ C for 10 min. Microscopic examination showed, when the antifungal materials were added, the hyphae of F. oxysporum became thinner and the germination of the spores was inhibited. The antifungal activity of P. fluorescens K-188 primarily should be correlated with cells themselves and an antifungal protein with molecular weight of 11 kDa. © 2004 Elsevier Inc. All rights reserved. Keywords: Pseudomonas fluorescens; Antifungal; Chitin; Fusarium oxysporum; Shrimp and crab shell
1. Introduction Chemical fungicides continue to play the most important role in current farming practices for the protection of crops against diseases. However, their utilization is under increased scrutiny and examen in recent years because many chemical fungicides are very toxic, and cause contamination of the environment, leave fungicide residues in food products, and induce pathogen resistance. Because of these limitations of chemical fungicides, it seems necessary to search for an alternative control strategy. Biological control or the use of microorganisms or their secretions to prevent plant diseases, offers an attractive harmless alternative or supplement for the control of plant diseases. Therefore, biological control tactics become an important approach to facilitate sustainable agriculture [1–4]. Chitin and its derivatives are of interest because they have various biological activities and applied in such as ∗
Corresponding author. Tel.: +886 2 2626 9425; fax: +886 2 8631 1015. E-mail address: [email protected] (S.-L. Wang).
0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2004.03.024
immunoadjuvant and a flocculant of wastewater sludge, and agrochemical uses [5]. Shrimp and crab shell contain chitin, protein and inorganic compounds mainly composed of calcium carbonate [6–8]. Traditional methods for the preparation of chitin include demineralization and deproteinization of the waste material with strong acids and bases (e.g., HCl and NaOH) [8–10]. The production of chitin and its hydrolyzed derivatives, such as acetylglucosamine and chitooligosaccharide, from waste of the shellfish industry has been limited due to the high cost of chitinase and the shrimp and crab shell pretreatment process [5,8]. Chitinases, a group of enzymes capable of directly degrading chitin to low molecular weight products, have been found to be produced by a number of microorganisms. The production of inexpensive chitinolytic enzymes is an important element in the utilization of shellfish wastes. The discovery of inexpensive chitinolytic enzymes not only solves environmental problems but also promotes the economic value of the marine products [11]. To further enhance the utilization of chitin-containing waste, we recently investigated
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the bioconversion of shrimp and crab shell powder (SCSP) for the production of chitinase and antifungal compounds [1–5,8,11–18]. In this research, we found that the strain Pseudomonas fluorescens K-188 exhibited antifungal properties in the presence of shellfish chitin wastes. The optimal culture conditions and some properties of the antifungal materials from culture broth are described. The effects of the antifungal materials on the germination of spores of pathogenic F. oxysporum were investigated.
2. Matreials and methods 2.1. Materials Chitin flake and chitosan powder from crab shell were purchased from Biotech Co. (Kau-Shyuon, Taiwan). Powdered chitin was purchased from Sigma Chemical Co., St. Louis, MO. Metarhzium anisopliae was kindly supplied by Dr. JenYuen Shieh, Department of Bioindustry Technology, Da-Yeh University, Chanhwa, Taiwan. 2.2. Shrimp and crab processing waste Five preparations of the same types of shrimp and crab processing wastes were used in this study, namely: (i) Untreated shrimp and crab shell powder (SCSP): The SCSP used in these experiments was prepared as described earlier [1]. In the preparation of the SCSP, the shrimp and crab shells collected from a marine foodprocessing factory were washed thoroughly with tap water followed by steaming. The solid material obtained was dried, milled, and sieved to powder with diameters <0.053 mm. (ii) SCSP treated with HCl (HCl–SCSP): In this process, the SCSP was treated with 2N HCl at room temperature for 2 days. The ratio of SCSP to solvent was 1:8 (w:v). This is identical to the demineralization method for the preparation of crustacean chitin [19]. The demineralized material was recovered by filtration followed by thoroughly rinsed with deionized water, and dried at 65 ◦ C. This product is referred to as HCl–SCSP. (iii) The filtrate from SCSP treated with HCl (HCl-extract): This extract was the filtrate obtained from the HCl–SCSP preparing process as described above. The filtrate was adjusted to pH 7 by NaOH solution. This product is referred to as HCl-extract. (iv) SCSP treated with NaOH (NaOH–SCSP): In this process, SCSP was treated with 2N NaOH at 100 ◦ C for 30 min. The ratio of SCSP to solvent, 3:40 (w:v), was referred from the deproteinization method for the preparation of crustacean chitin [20]. The deproteinized material was recovered by means of filtration, thoroughly rinsed with deionized water, and dried at 65 ◦ C.
(v) The filtrate from SCSP treated with NaOH (NaOHextract): This extract was the filtrate obtained from the NaOH–SCSP preparation process described above. This preparation is referred to as NaOH-extract. The concentrations of the four preparations other than the raw SCSP are expressed as the weight percentage of raw SCSP used before treatment. For example, X g of raw SCSP, treated with acid or alkali, produced X wt.% of SCSP in the acidic/alkaline solution (w/v). 2.3. Isolation and screening of antifungal materials producing strains Microorganisms isolated from soils collected at different locations in northern Taiwan were screened on agar plates containing 0.2% colloidal chitin, 0.1% K2 HPO4 and 0.05% MgSO4 ·7H2 O, 0.1% NaNO3 , and 2% agar (pH 7). The plates were incubated at 30 ◦ C for 2 days. Colonies that grew well or showed a clear zone around the colonies under such conditions were isolated and retained for subsequent screening. Those organisms obtained from the first screening were subcultured in liquid media (containing 1% SCSP, 0.1% K2 HPO4 and 0.05% MgSO4 ·7H2 O) in shaken flasks at 30 ◦ C and shaken at 180 rpm for 2 days. After centrifugation (8000 × g, 4 ◦ C, for 20 min, Beckman J2-21 M/E), the supernatants were collected for measurement of antifungal activity using the procedure described below. The strain K-188, which showed the highest antifungal activity, was isolated, kept on nutrient agar, and used for the study. 2.4. Identification of strain K-188 After study of the morphological, cultural, biochemical and physiological characteristics of the organism, identification was made in accordance with the method described in Bergey’s Mannual of Systematic Bacteriology [21]. Strain K-188 was identified as P. fluorescens (identified by Culture Collection and Research Center, Shin-Chu, Taiwan). 2.5. Effect of culture conditions In the investigation of culture conditions, growth was carried out in a basal medium containing 0.1% K2 HPO4 and 0.05% MgSO4 ·7H2 O (pH 7), and gradually supplemented with the various ingredients to be investigated. The major ingredients investigated including SCSP, carbon sources, nitrogen sources, and inorganic salts were added and investigated separately. One hundred milliliters of the resultant medium in a 250-mL Erlenmeyer flask was aerobically incubated at 30 ◦ C for 48 h on a rotary shaker (180 rpm). After centrifugation (8000 × g, 4 ◦ C, for 20 min, Beckman J221 M/E), the supernatant was used for bioassay. Usually an effective experimental prior condition was used as the basis
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for the later experiment until the best culture condition was obtained. With the use of this culture composition, the effects of the initial pH, temperature, culture volume and cultivation time on the production of antifungal activity was investigated in the same way until the optimum culture condition was found. 2.6. Dual culture The laboratory screened antifungal compounds producing strains, P. fluorescens K-188, Pseudomonas aeruginosa K187 [5], Bacillus amyloliquefaciens V656 [1], Bacillus subtilis W113 [3], and B. subtilis W118 [3], were dual cultured and mix cultured with M. anisopliae in potato dextrose agar (PDA) and potato dextrose broth (PDB), respectively, to observe the growth condition and the change of spore production. 2.7. Measurement of protease activity For measuring protease activity, a diluted enzyme solution (0.2 mL) was mixed with 2.5 mL of 1% casein in pH 7 phosphate buffer and incubated at 37 ◦ C for 10 min. The reaction was terminated by adding 5 mL of 0.19 M trichloroacetic acid (TCA). The reaction mixture was centrifuged and the soluble peptide in the supernatant fraction was measured by the method of Todd with tyrosine as the reference compound [22]. 2.8. Preparation of the antimicrobial materials For the production of antifungal materials, P. fluorescens K-188 was grown aerobically at 37 ◦ C for 4 days in an Erlenmeyer flask (250 mL) containing 200 mL of liquid medium consisting of 1% SCSP, 0.05% MgSO4 ·7H2 O, and 0.1% K2 HPO4 (pH 7). The culture broth was centrifuged for 20 min at 8000 × g, and the supernatant was used for the concentration of the antifungal materials. The supernatant was subjected to ammonium sulfate precipitation and ethanol precipitation as described below. (a) Ammonium sulfate precipitation. Ammonium sulfate (608 g/mL) was added to the supernatant (800 mL). The resultant mixture was kept at 4 ◦ C overnight, and the precipitate formed was collected by centrifugation at 4 ◦ C for 20 min at 12,000 × g. The precipitate was dissolved in a small amount of 50 mM sodium phosphate buffer (pH 7) and dialyzed against the buffer. The resultant dialysate (45 mL) was referred as concentrated solution A. (b) Ethanol precipitation. 95% ethanol (1:9, v/v) was added to the supernatant. The resultant mixture was kept at 4 ◦ C over night, and the precipitate formed was collected by centrifugation at 4 ◦ C for 20 min at 12,000 × g. The ethanol-free precipitate was dissolved in a small amount of 50 mM sodium phosphate buffer (pH 7) and referred as concentrated solution B.
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2.9. Antifungal activity of the materials The antifungal activity for the materials was estimated using a growth inhibition assay described earlier [1]. Fungal spores of F. oxysporum were grown on Petri plates filled with potato dextrose agar (PDA). After 10 days of incubation at 25 ◦ C, the fungal colonies were recovered with sterile water containing 0.1% (v/v) Tween 80. The resulting suspension was filtered aseptically through sterilized gauges. The filtrate was adjusted with sterile water to a concentration of 1 × 106 spores/mL, and stored at 4 ◦ C. To test the antifungal effect of the material produced by P. fluorescens K-188, Petri plates were filled with molten PDA precooled to 45 ◦ C, and divided into two groups, namely experimental group (E) and control group (C) (triplicate for each). To each plate in the experimental group (E), an appropriate amount of the antifungal materials was added. To those of the control group (C), an equal amount of sterile buffer was added. After the plates were cooled, the fungal inoculum was then placed onto the agar surface. Both groups were incubated at 25 ◦ C for 72 h. The diameters of the largest and smallest fungal colonies were recorded and the averages were calculated. The inhibition ratios were calculated with the following formula. If the inhibitory ratio was greater than 20%, the test strain would be considered of being inhibited and the minimal inhibitory concentration (MIC) for that strain was then determined. Inhibition ratio (%) = (C − E)/C × 100% where C is the average diameter of the largest and smallest colonies of the control groups; E is the average diameter of the largest and smallest colonies of the experimental groups. 2.10. pH and thermal stability of the antifungal materials The pH stability of the antifungal materials was determined by measuring the residual inhibitory activity at pH 7 as described above after dialyzing the samples against a 50 mM buffer solution of various pHs (pH 3–11) in seamless cellulose tubing (Sankyo, Japan). The buffer systems used were glycine–HCl (50 mM, pH 3), acetate (50 mM, pH 4.5), phosphate (50 mM, pH 6–8), Na2 CO3 –NaHCO3 (50 mM, pH 9–11). The thermal stability of the antifungal materials was studied by heating the samples at 100 ◦ C for various time periods. The residual inhibitory activity was measured as described above (using F. oxysporum as the target). 2.11. Spore germination affected by the antifungal material Half milliliter of fungal spores (106 /mL) of F. oxysporum, 0.5 mL of potato dextrose broth, and 0.5 mL of antifungal material were added into the eppendorf tube (1.5 mL). The resultant mixture was incubated at 25 ◦ C, and the spores were observed by using a phase-contrast microscope at various intervals [14].
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2.12. Molecular weight determination The molecular weight of sample was determined by GPC using a size exclusion column (TSK-G3000SWXL ). The 0.2 M phosphate buffer (pH 6.8) was used as a mobile phase at a flow rate of 0.5 mL/min, and the sample loop loaded was 20 L. The effluent was monitored by UV detecter at 254 nm (JASCO UV-1570) and the data processed with data manager (VISCOTEK DM400). The standards polymer used for the calibration were bovine serum albumin (67,000 MW), (-chymotrysinogen (25,700 MW), myoglobin (17,600 MW), and Aprotinin (6500 MW).
3. Results and discussion 3.1. Effect of P. fluorescens K-188 on the growth of M. anisopliae It was observed in the dual culture test that both B. amyloliquefaciens V656 and P. fluorescens K-188 grew better with M. anisopliae. This means both grew at same rate and did not compete for nutrition sources (data not shown). The mixture of M. anisopliae and P. fluorescens K-188 was also inoculated on liquid medium (PDB) to observe the growth condition of the colonies. It was also found that mixture culture of P. fluorescens K-188 and B. amyloliquefaciens V656 showed least interference on M. anisopliae colonies (data not shown). Concluded from above results, among the laboratory strains, P. fluorescens K-188 and B. amyloliquefaciens V656 had the least influence on the growth of M. anisopliae. Although both species of Pseudomonas and Bacillus were reported to be used as bio-pesticides, Bacillus has narrower range of utilization than Pseudomonas, which can be utilized as both pesticides as well as germicides. Therefore, P. fluorescens K-188 was chosen for further study in this research. The spore production was observed since the current application of M. anisopliae focused on its spore products. The amount of spore produced by M. anisopliae was not changed when it was mix-cultured with P. fluorescens K-188 (data not shown), i.e., P. fluorescens K-188 had no effect on the spore production of M. anisopliae. Therefore, it is possible that P. fluorescens K-188 can be utilized together and cooperate with M. anisopliae. 3.2. Effect of culture conditions To study the effect of carbon sources on the production of antifungal materials, growth was carried out in basal medium as described above. Both the media with and without the addition SCSP/chitin were used to investigate the effect of SCSP/chitin on the production of antifungal materials. It was found that the antifungal activity of K-188 was enhanced by the addition of SCSP into the medium. The optimum antifungal activity (0.58 U/mL) was achieved when medium
Fig. 1. Antifungal materials production in the presence of various concentration of the preparations after two days of cultivation. (), SCSP; (), HCl–SCSP; (), HCl-extract; (X), NaOH–SCSP; ( ), NaOH-extract.
contained 1% SCSP and decreased with medium contained higher concentration of SCSP (data not shown). In our previous study [8], we found that SCSP was a more suitable carbon source than powdered chitin for production of antimicrobial compounds by P. aeruginosa K-187. Besides the investigation of the effect of SCSP on the production of antifungal materials, other chitin-related materials from the chitin preparation sources were also used in this study to investigate their effect on the production of antifungal materials. A series of experiments on the production of antifungal activity was also carried out to compare the inducing effect with the other four related preparations (HCl–SCSP, HClextract, NaOH–SCSP, NaOH-extract) described in Section 2. The production of antifungal activity with strain K-188 under these conditions is shown in Fig. 1. It was shown that the other four preparations were all less effective as carbon sources than SCSP in enhancing antifungal activity production. SCSP (1%) was chosen as carbon source and added into the cultures in the following experiments. The effect of time on the production of antifungal activity was investigated. K-188 strain was grown aerobically in 100 mL of optimal media at 25, 30, and 37 ◦ C. During the period of incubation, the antifungal activity was monitored in 24 h intervals for up to 6 days. As shown in Fig. 2, P. fluorescens K-188 incubated at 37 ◦ C for 4 days showed the highest antifungal activity (0.97 U/mL).
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3.3. Antifungal mechanism of P. fluorescens K-188
Fig. 2. Time-course of cultivation.
To study the effect of initial pH on the production of antifungal activity, growth was carried out in the optimal medium (1% SCSP and basal medium) at various initial pHs at 37 ◦ C for 4 days. The result showed that P. fluorescens K188 produced higher antifungal activity when the initial pH was 7. The optimal culture condition for P. fluorescens K-188 was grown aerobically in an orbital shaking incubator at 37 ◦ C for 4 days in an Erlenmeyer flask (250 mL) containing 100 mL of liquid medium consisting of 1% shrimp and crab shell powder, 0.1% K2 HPO4 , and 0.05% MgSO4 ·7H2 O, pH 7.
The antifungal mechanism of P. fluorescens K-188 is investigated herein. Take the concentration of the original culture supernatant as 100%. The final culture supernatant was adjusted to 33% (v/v) of its original concentration and incubated at the same condition. After the incubation, it was examined with phase-contrast microscope. The result was shown in Fig. 3. For control group shown in Fig. 3A, after being incubated at 25 ◦ C for 72 h, mycelium of F. oxysporum was uniform in thickness and showed intact appearance. For experiment group (medium, spore suspension, and antifungal materials was mixed in a volume ratio of 1:1:1) shown in Fig. 3B, the spores of F. oxysporum almost did not germinate at all. The germination rate of F. oxysporum decreased significantly. Comparing with Fig. 3C, even germination occurred, germ tubes were thinner than normal ones and no spore formation phenomenon was found. However, neither hyphae cytolysis nor tail-end expansion was observed. This inhibitory phenomenon was different from those by chitinase enzymes [1,2,5,23–25] and by other non-enzymatic compounds [14,15,26,27]. From above observations, it was inferred that the antifungal activity of P. fluorescens K-188 was not originated from fungal cell wall hydrolase such as chitinase. The inhibitory mechanism of P. fluorescens K-188 should be attributed to nutrition competition and/or germination inhibition. 3.4. Concentration of antifungal materials Many publications reported that the antifungal activities of microorganisms were resulted from the hydrolytic enzymes
Fig. 3. Effect of the antifungal materials from P. fluorescens K-188 on morphology of F. oxysporum. (A), normal mycelia of F. oxysporum; (B) (C), F. oxysporum hyphae in the presence of P. fluorescens K-188 antifungal materials (33%, v/v).
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Table 1 Some properties of the materials concentrated from culture supernatant of P. fluorescens K-188
Antifungal activity (U) Protease activity (U) Chitinase activity (U) Volume/dry weight
Culture supernatant
Concentrated solution A
B
970 ND ND 1000 mL
ND ND ND 30 mg
33,100 131 ND 1655 mg
such as chitinase produced by the microorganisms [1–4]. Consequently, it was further investigated that whether hydrolytic enzymes such as chitinase, protease, cellulase, xylanase were present in the fermentation broth of P. fluorescens K-188. To avoid the interfering of the enzyme activity analysis by reducing sugars, the culture supernatant was first concentrated with ammonium sulfate precipitation or ethanol precipitation to remove the above impurities. The recovered amount, antifungal activity, and enzyme activity of the resultant concentrated solution (concentrated solution A and concentrated solution B, respectively) were shown in Table 1. Concentrated solution A, purified by ammonium precipitation, possessed neither antifungal activity nor enzymatic activities of protease and chitinase. Concentrated solution B, purified by ethanol precipitation, possessed both antifungal activity and protease activity. Since the chitinase activity was not found, it was inferred that the antifungal activity of P. fluorescens K-188 should not be related to chitinase. This is different from other antimicrobial strains of Pseudomonas species. The antimicrobial compounds produced by P. aeruginosa K-187 and P. fluorescens were a bifunctional chitinases/lysozyme [5,14,15] and a phenazine antibiotic [26,27], respectively. Besides, protease activity was not found in the original culture supernatant but was detected in the concentrated solution purified by ethanol precipitation (concentrated solution B). Based on this result, it is inferred that protease and protease inhibitor probably existed together in the culture broth. Consequently, no protease activity was observed in the supernatant of P. fluorescens K-188. After the protease inhibitor was removed or inactivated by ethanol during the ethanol precipitation, the protease activity exhibited. Similar phenomenon of simultaneous existing of enzymes and enzyme inhibitors in fermentation broth has been reported previously. For example, lysozyme and lysozyme inhibitor existing together was found in culture broths of B. subtilis I-139 [28], P. aeruginosa M-1001 [29], and Staphylococcus aureus M18 [30]. 3.5. Effect of pH and temperature on antifungal activity and protease activity To investigate if there is any relationship between antifungal activity of P. fluorescens K-188 and protease, the effect of pH and temperature on protease activity and antifungal
activity of concentrated solution from ethanol precipitation (concentrated solution B) was analyzed. After being heated at 100 ◦ C for 10 min, close to 70% of residual antifungal activity was retained. After heated at 100 ◦ C for 2 min, near 90% of residual protease activity was retained. The protease activity completely lost after heated at 100 ◦ C for 4 min. As for optimum pH, it was pH 7–9 for both antifungal activity and protease activity (data not shown). Schokker et al. [31] reported that the extracellular protease purified from P. fluorescens 22F was thermal unstable. Judged from above, the thermal stable protease from P. fluorescens K-188 should be different from the thermal unstable protease from P. fluorescens 22F. As for the relationship between antifungal activity and protease activity, the thermal stability of antifungal activity was much higher than that of the protease activity although optimum pH was similar for both of them. To investigate the relationship between antifungal activity and protease activity, in this research, the antifungal materials (concentrated solution B) made from ethanol precipitation were further purified by DEAE-Sephadex CL-6B column chromatography. The result showed that antifungal activity was existed in the fractions eluted with 0.2 M NaCl containing phosphate buffer (pH 7) (data not shown). After dialysis with the same buffer, the obtained fraction was then performed by SDS–PAGE and GPC for molecular weight determination. The result from SDS–PAGE showed the antifungal material appeared at molecular weight lower than 14,000 (the smallest standard marker). Consequently, it was inferred that the antifungal material was small protein molecule with molecular weight less than 14,000. This coincides with the 11 kDa determined by GPC (data not shown). The relationship between antifungal activity and protease activity of P. fluorescens K-188 is still waiting to be final judged by the purification and characterization of the protease. According to the further purification and molecular weight determination, it can be inferred that the antifungal activity of P. fluorescens K-188 primarily should be correlated with cells themselves and an antifungal protein with molecular weight of 11 kDa. Furthermore, it was confirmed by the results that antifungal activity of the cells removed antifungal samples, filtered aseptically through 0.45-m pore-size membrane filters, were remarkably decreased (retained near half of its original antifungal activity). As a matter of fact, many reports showed the utilization of Pseudomonas species cells for biocontrol research. The examples included impact of P. fluorescens CHA0 and a derivative with improved biocontrol activity on the cultural resident bacterial community on cucumber [32]; effect of P. putida and a synthetic iron chelator on induction of soil suppressiveness to Fusarium wilt pathogens [33]; P. stutzeri YPL-1 genetic transformation and antifungal mechanism against F. solani, an agent of plant root rot [34]; P. cepacia suppression of sunflower wilt fungus and role of antifungal compounds in controlling the disease [35]; fluorescent Pseudomonas spp. Future study will investigate whether the antifungal activity would be retained after P. fluorescens K-188 cells were immobilized. The re-
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sult would confirm if the antifungal activity of P. fluorescens K-188 was originated from the cells themselves.
4. Conclusion Certain fluorescent Pseudomonas can inhibit important fungal pathogens of crops, and they have been studied for biocontrol of root diseases caused by soil-borne fungi [36]. Often, biocontrol mediated by fluorescent pseudomonas involves the production of secondary metabolites and/or extracellular enzymes that inhibit certain fungal pathogens. Several well-studied biocontrol strains of Pseudomonas e.g., Pf-5 and Q2-87 produce the secondary metabolite 2,4diacetylphloroglucinol (DAPG). The compound is recognized as a key factor in the biocontrol of fungal diseases such as take-all of wheat, black root rot of tobacco, or dampingoff of sugarbeat by biocontrol pseudomonas. Production of DAPG by P. fluorescent CHA0 has been detected in the rhizosphere of plants grown in artificial soil microcosms [37]. Bagnasco et al. [36] reported that P. fluorescens as biocontrol agents against forage legume root pathogenic fungi. We have reported that SCSP of marine wastes is an effective inducer for the production of antimicrobial chitinases by P. aeruginosa K-187 [5], Monascus purpureus CCRC 31499 [2,4], and B. amyloliquefaciens V656 [1]. In this research, SCSP was used as the major carbon source for producing material with antifungal activity. The acid or alkali liquid waste from SCSP treatment in the chitin production process also could be a feedstock for antifungal material production by P. fluorescens K-188. The results showed that P. fluorescens K188 could utilize the acid or alkali liquid waste from the chitin production process to produce antifungal materials. The antifungal activity could be both originated from K-188 cells, an antifungal protein with molecular weight of 11 kDa, and the protease activity.
Acknowledgment This work was supported in part by a grant of the National Science Council, Taiwan (NSC90-2313-B-212-003).
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