Co-producing Lipopeptides and Poly-y-glutamic Acid by Solid-state Fermentation of Bacillus Subtilis Using Soybean and Sweet Potato Residues and Its Biocontrol and Fertilizer Synergistic Effects

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Co-producing Lipopeptides and Poly-y-glutamic Acid by Solid-state Fermentation of Bacillus Subtilis Using Soybean and Sweet Potato Residues and Its Biocontrol and Fertilizer Synergistic Effects
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  Short Communication Co-producing lipopeptides and poly- c -glutamic acid bysolid-state fermentation of   Bacillus subtilis  using soybean andsweet potato residues and its biocontrol and fertilizer synergistic effects Qijun Wang  a,b , Shouwen Chen  a , Jibin Zhang  a , Ming Sun  a , Ziduo Liu  a , Ziniu Yu  a,* a State Key Laboratory of Agricultural Microbiology, National Engineering Research Centre of Microbial Pesticides,Huazhong Agricultural University, Wuhan 430070, PR China b College of Life Science and Technology, Key Lab of Crop Disease Monitoring and Safety Control in Hubei Province,Xiaogan University, Hubei 432000, PR China Received 15 April 2007; received in revised form 23 May 2007; accepted 23 May 2007Available online 6 August 2007 Abstract A  Bacillus subtilis  strain B6-1, previously isolated from the rhizosphere of vegetable, selectively produced antibiotics or poly-gamma-glutamic acid ( c -PGA) in two kinds of liquid media and their co-productions were obtained when using soybean and sweet potato res-idues in solid-state fermentation. The antibiotics were purified and identified as fengycins. After these residue cultures were introduced,cucumber wilts were effectively suppressed. The introduction also significantly increased the dry weights of roots and shoots of cucumberseedlings, and the roots to shoots ratio, especially at lower nutrition, which indicated the fertilizer synergistic effects. So the product of soybean and sweet potato residues, cultivated with B6-1 co-producing lipopeptides and  c -PGA, can be expected to be used as both bio-control agents and fertilizer synergists.   2007 Elsevier Ltd. All rights reserved. Keywords:  Lipopeptides; Poly- c -glutamic acid ( c -PGA); Biocontrol agent; Fertilizer synergist; Solid-state fermentation 1. Introduction Some of   Bacillus  species could produce antibioticsagainst phytopathogenic fungi, most notably the nonribos-omally synthesized cyclic lipopetides surfactin, iturin andfengycin. These amphiphilic cyclic biosurfactants arealready suggested to play an important role in biologicalactivities. They have many advantages over other pesti-cides: low toxicity, high biodegradability, environmentallyfriendly characteristics (Kim et al., 2004; Maget-Danaand Peypoux, 1994; Stein, 2005; Yoshida et al., 2001; Yuet al., 2002). Several of species of the genus  Bacillus  alsosynthesize poly- c -glutamic acid ( c -PGA), similar to struc-ture of thermal polyaspartate (TPA), polymerized via  c -amide linkage. For its excellent characters,  c -PGA hasattracted a variety of interest for potential application asan environment-friendly fertilizer synergist like TPA andother polyamino acids (Ashiuchi and Misono, 2002; Chenet al., 2005; Shih and Van, 2001).In recent years, there has been an unprecedentedincrease in interest in the more efficient utilization of agro-industrial residues because its application providesan alternative way to reduce the production cost and helpto solve many environmental hazards. Huge quantities of soybean curd and sweet potato residues are discharged asagro-industrial wastes, which usually results in problemsfor disposal and potentially severe pollution. As these res-idues are rich in carbohydrate, protein and many othernutrients, many researchers have investigated the possibil-ity of bioconversion of the residues by submerged andSSF (Adams et al., 2002; Aziz and Mohsen, 2002; Ho¨lkerand Lenz, 2005; O’Toole, 1999; Pandey et al., 2000; Yokoiet al., 2002). 0960-8524/$ - see front matter    2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2007.05.052 * Corresponding author. Tel.: +86 27 87396030; fax: +86 27 87393882. E-mail address:  yz41@mail.hzau.edu.cn (Z. Yu).  Available online at www.sciencedirect.com Bioresource Technology 99 (2008) 3318–3323  To our knowledge, the co-production of lipopeptidesand  c -PGA by microorganisms, especially the use of thesoybean and sweet potato residues cultivated with microor-ganisms as both biocontrol agent and fertilizer synergisthave not yet been reported. The aims of this study wereto purify and determine the antibiotics from  Bacillus sub-tilis  strain B6-1, co-produce the antibiotics and  c -PGA insolid-state fermentation using soybean curd and sweetpotato residues as the main solid substrates, evaluate itsdual function as both biocontrol agent and fertilizersynergist. 2. Methods  2.1. Microorganisms and culture conditions The  B. subtilis  strain B6-1 was isolated from the rhizo-sphere of vegetable, and identified using biochemical, phys-iological and 16S rDNA sequence analysis methods(GeneBank accession number EF420247). B6-1 was main-tained at 28   C on slant tube of nutrient agar (NA: 5 g/lbeef extract, 10 g/l peptone, 5 g/l sodium chloride, 18 g/lagar and pH 7.2–7.4).  Fusarium oxysporum  f. sp.  cucumer-inum , the pathogen of cucumber fusarium wilts, was main-tained on slant tube of potato dextrose agar (PDA) at28   C.  2.2. Characterization of antagonistic compound(s) of culture filtrates Hemolytic activity was tested according to Pabel et al.(2003). The cell-free supernatant was subjected to acid pre-cipitation according to Kim et al. (2004). Oil displacementactivity was tested according to Morikawa et al. (2000).The aggregational behavior of antifungal compounds wasperformed according to Lin and Jiang (1997). The ultrafil-tration cartridge with molecular weight cutoff (MWCO) of 6–8 kDa was used. The retentates in first time were dilutedto 10 times with methanol. The stability tests of the anti-fungal compound(s) against heat, pH and light were per-formed according to Yu et al. (2002).  2.3. Isolation and purification of antifungal compounds After cultivating the cells in NB at 30   C for 96 h, thecells and debris were removed by centrifugation at10,000  g   for 15 min. The cell-free supernatant was passedthrough an ultrafiltration cartridge with MWCO of 6– 8 kDa. The retentates were precipitated by adding 3 NHCl to final pH of 2.0 and stored at 4   C overnight. Theprecipitates were collected by centrifugation at 10,000  g  for 20 min at 4   C, and extracted with a chloroform/meth-anol (2:1, v/v) solvent. The solution was dried using arotary vacuum evaporator. The resulting residue wasextracted with methanol and filtered through a 0.45  l mnylon membrane. The filtrate was applied to the AccuBondODS-C18 solid phase extraction (SPE) cartridge (500 mg,3 ml) (Agilent Technologies, USA), and then eluted succes-sively with 0%, 20%, 40%, 50%, 70%, 80%, and 100% aque-ous methanol. The most active fractions, the eluates of 70%, 80%, and 100% aqueous methanol, were pooled andconcentrated by evaporation at 40   C. The solution wasanalyzed by HPLC (515 pump with 996 photodiode arraydetector; Waters) equipped with an analytical (ODS-C18column, 250  ·  4.6 mm internal diameter; Hanbon, China)grade of reversed phase column at a flow rate of 1 ml/min. The mobile phase components were acetonitrile/water/trifluoroacetic acid (TFA) (50:50:0.1, v/v/v). Sam-ples eluted were collected at every 1-min interval. Anti-fungal activity of the collected samples was determined.Various concentration methanol solutions (only solvent)were as controls. Quattro Micro (Waters, USA) LC–MSsystem was chosen to measure the mass of the antifungalcompounds, equipped with SunFire TM ODS-C18 column(150  ·  2.1 mm). The mobile phase was the same, flow rate0.2 ml/min.  2.4. High-yield production of   c -PGA in submerged  fermentation In some media B6-1 also produces extracellular viscousmaterials that were identified as  c -PGA. For high-yieldproduction of   c -PGA, B6-1 was grown in 80 ml of ouroptimized medium (in 500 ml flask) composed of 80 g/l glu-cose, 30 g/l monosodium glutamate, 18 g/l tri-sodium cit-rate, 10 g/l NH 4 SO 4 , 1 g/l K 2 HPO 4 , 0.2 g/l CaCl 2  Æ  2H 2 O,0.12 g/l ZnSO 4  Æ  7H 2 O, 0.06 g/l MgSO 4  Æ  7H 2 O, pH 7.2– 7.4. They were incubated at 37   C at 200 rpm for 36 h.  2.5. Extraction and quantification of   c -PGA The extraction and quantification of   c -PGA were doneaccording to Chen et al. (2005) without filtration. At thesame time the antifungal activity of   c -PGA fermentationbroth was detected. And the quantitative analysis of   c -PGA in NB was also carried out.  2.6. Co-producing the lipopeptides and   c -PGA in solid-state fermentation Twenty grams (dry weight) medium that consisted of 10 g perlite, 5 g sweet potato residue and 5 g soybean curdresidue were placed in 250 ml conical flask. The final con-centrations of mineral salts were set to be 0.5 g/kgMgSO 4  Æ  7H 2 O, 0.15 g/kg CaCl 2  Æ  2H 2 O, 0.04 g/kgFeCl 3  Æ  6H 2 O, 0.104 g/kg MnSO 4  Æ  H 2 O. Initial pH andmoisture content of the substrates were adjusted to 7.2and 60%, respectively. The substrates were mixed thor-oughly to achieve uniformity, sealed and then autoclavedat 121   C for 20 min. A loopful of cells from the freshNA plate were transferred into 250 ml flask containing30 ml NB and incubated for 8–10 h on a rotary shaker at200 rpm at 37   C. The autoclaved substrates were inocu-lated with a 5% inoculum level, and then incubated in a Q. Wang et al. / Bioresource Technology 99 (2008) 3318–3323  3319  chamber with relative humidity above 80% at 37   C for72 h.The extraction and quantification of   c -PGA were testedaccording to Chen et al. (2005). For lipopeptides, the fer-mented substrates were mixed thoroughly with distilledwater (1:10, w/v) and filtered through two-layer muslincloth. The obtained extracts were autoclaved at 121   Cfor 15 min. The isolation of lipopeptides was performedas described above. To compare the production in SSFand submerged fermentation, a series of dilutions of cul-ture were measured to determine the maximum dilutionthat can be detected by the antifungal activity assays.During solid-state fermentation, samples were takenevery 6 h to determine pH, viable cell number, yield of   c -PGA and maximum dilution that can be detected by anti-fungal activity assays.  2.7. Biological control tests Cucumber seeds sterilization, germination and prepara-tion of microconidia suspensions were carried out accord-ing to Soriano-Martı´n et al. (2006). The perlites wereautoclaved at 121   C for 1 h two times at 12-h intervals.The pots were introduced with mixtures of perlites and res-idue cultures of B6-1 (4:1, v/v). The germinated seeds wereplanted in plastic pots, one to each pot. The seedlings werefertilized daily with a Yamasaki’s cucumber nutrient solu-tion. The pathogen used was  F. oxysporum  f. sp.  cucumer-inum . One millilitre of fresh microconidial suspension(5  ·  10 6 microconidia/ml) was dropped along the stem of each seedling with two true leaves. They were placed in agrowth chamber at 30   C with 90% relative humidity under16-h photoperiod (about 8000 lx intensity). The controlsincluded: (a) pathogen infection and residue mixtures with-out inoculum of B6-1, (b) pathogen infection but no resi-due cultures, and (c) residue cultures but no infection.After 3 weeks, the percentage of diseased seedlings wasdetermined.  2.8. Fertilizer synergist tests To investigate the fertilizer synergistic effects of residuecultures, the pots were introduced with mixtures of perlitesand residue cultures of B6-1 (9:1, v/v). After the planting of seedlings, the pots were fertilized daily with the nutrientsolution. Treatments included: fertilized daily with a full,1/2, 1/3 nutrient solution, respectively. In respective con-trol the mixtures were not inoculated with B6-1. The dryweight of roots and shoots were measured after 5 weeks. 3. Results and discussion 3.1. Isolation and determination of antifungal compounds from culture filtrate of strain B6-1 The antifungal compounds could be precipitated withHCl and remain its activities against heat, UV for30 min, at pH 3–12 for 24 h. The culture broth showedhemolytic and oil displacement activity. In the first ultrafil-tration, the antifungal compounds were in the retentates.But in the second, the compounds were in the permeates.The results suggested the antifungal compounds can formaggregates, but the aggregates can be ruptured to monomermolecular in solvent. So from the surfactant characteristicsof the antibiotics, it could be deduced the antifungal com-pounds belong to the lipopeptides (iturin, surfactin orfengycin) because it is generally accepted that a combina-tion of fungicidal and surfactant activity in  Bacillus  is avalid indicator for the presence of lipopeptides from iturin,surfactin or fengycin class (Maget-Dana and Peypoux,1994; Pabel et al., 2003).In our further studies, the cell-free supernatant was con-centrated by an ultrafiltration cartridge with a MWCO of 6–8 kDa. The retentates showed strong antifungal activity.After acid precipitation and two times of extractions, thesupernatant showed stronger antifungal activity. It waspassed through the C18 SPE cartridge, and eluted succes-sively with aqueous methanol. Only the fraction elutedwith concentration >50% showed antifungal activity (vari-ous concentration methanol solutions did not affect thefungal growth). The most active fractions (the eluates of 70%, 80%, 100% methanol) were pooled, subjected toHPLC and separated further. The major antifungal com-pounds showed same UV spectra in methanol and ahomologous [M+H] + ion peak at  m/z  1450, 1464, 1478,1506, respectively (data not shown). These mass data corre-sponded well to those lipopeptides determined by otherauthors, indicating the presence of C15, C16, C17, C19 fen-gycins (Pabel et al., 2003; Williams et al., 2002), corrobo-rating the deduction above.The present purification methods of lipopeptide typi-cally include acid precipitation, SPE, ultrafiltration or thecombination of them. Acid precipitation is the most suc-cessfully used (Lin and Jiang, 1997). In our studies, afteracid precipitation and two times of organic extractions,an adequate efficiency of separation was not achievedbecause too many impurities were precipitated andextracted. The validity of SPE is described by otherresearchers (Yoshida et al., 2001; Yu et al., 2002). But herethe concentration of antibiotics was so low that the concen-trated collection from HPLC could not be detected by anti-fungal tests. The ultrafiltration also is used to purifylipopeptides (Lin and Jiang, 1997; Yu et al., 2002). Unfor-tunately, in our researches, although the antibiotics wereall present in the retentates in the first ultrafiltration, inthe second ultrafiltration of 90% methanol of the retentatesthey were almost evenly present in both retentates and per-meates. This result indicates the supramolecular structurescould not be completely ruptured to monomers, even in90% methanol solutions. So a combination of ultrafiltra-tion, acid precipitation and SPE was developed for purifi-cation of the antibiotics. In the first step, ultrafiltrationcartridge was used to concentrate and eliminate smallmolecular impurities. Then acid precipitation and organic 3320  Q. Wang et al. / Bioresource Technology 99 (2008) 3318–3323  extraction were performed to further purification. Afterthat, the SPE on C18 showed high efficacy. Antifungalcompounds were purified from the culture broth of   B. sub-tilis  strain B6-1, and identified as isomers of fengycins. Butthere are no reports about hemolytic activity of fengycins.So other lipopeptides with hemolytic activity may coexist.In generally,  B. subtilis  co-produces various families,homologs or isomers of lipopeptides, causing additionalpurification and quantification problems. Further studiesabout more precise identification must be done in future. 3.2. Co-production of lipopeptides and PGA and time coursein SSF  In the NB, B6-1 produced the lipopeptides but no  c -PGA. And in the optimized medium for production of   c -PGA, the yield of   c -PGA reached 3.47% but the lipopep-tides could not be detected by antifungal activity assays.The average  c -PGA yield in SSF, using perlites, soybeancurd and sweet potato residues as solid substrates, was3.63% in 250 ml flask. Because of the high viscosity andwater-absorbability of   c -PGA, the porosity of SSF becamean especially critical factor. The perlites, an inert and non-nutritive material, were only used as the supporters givingenough room for oxygen, water, nutrition and heat trans-fer. At the same time, the production of lipopeptides wasverified. The maximum dilution in SSF that can be detectedwas 70-folds, remarkably higher compared with 20-folds inNB. The results showed the  B. subtilis  strain B6-1 could co-produce the lipopeptides and  c -PGA and achieve highyields of them when using soybean curd and sweet potatoresidues as the basis of a solid substrate in SSF.The time courses of pH, viable cell number, yields of   c -PGA and lipopeptides are shown in Fig. 1. In the initial12 h pH decreased which could be thought to be mainlydue to acid production by quick consumption of carbonsubstrates. Then pH increased, at last maintained about8.8 mainly by ammonification of degradation of nitroge-nous compounds in soybean residues. The number of viablecells increased rapidly after 6 h, reached the highest12.05  ·  10 9 cfu/g at 36 h, and slowly decreased thereafter.Because the diameter of inhibition zones can not sensitivelyindicate the tiny variation of the lipopeptides, here the max-imum dilution of culture broth that can be detected by theantifungal activity assays was used as the relative amount.The concentration of lipopeptides reached its highest levelat 54 h and remained relatively constant after that time.In the initial about 12 h the production of   c -PGA increasedslowly. After that it increased rapidly, reached the maxi-mum yield (3.63%, w/w) at 42 h and then decreased gradu-ally. The reason is that synthesized PGA would bebiodegraded by depolymerase of itself (Xu et al., 2005). 0123456789101112130 6 12 18 24 30 36 42 48 54 60 66 72Incubation time (h)    N  u  m   b  e  r  o   f  v   i  a   b   l  e  c  e   l   l  s   (   1   0    9    c   f  u   /  g   )  o  r  p   H 012345678    P   G   A  y   i  e   l   d   (   %   )  o  r   t   h  e  m  a  x   i  m  u  m   d   i   l  u   t   i  o  n  o   f   l   i  p  o  p  e  p   t   i   d  e  s   (  ×   1   0   ) Fig. 1. Time courses of   c -PGA yields (  ) which were the percentage of   c -PGA amount to medium amount (dry weight), maximum dilution of lipopeptidesthat can be detected by antifungal activity assays ( j ), pH ( d ), number of viable cells ( m ) in SSF. Q. Wang et al. / Bioresource Technology 99 (2008) 3318–3323  3321
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