Bioresource Technology 101 (2010) 9264–9271

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Bioresource Technology

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Evaluation of metal ions (Zn2+, Fe3+ and Mg2+) effect on the production offusaricidin-type antifungal compounds by Paenibacillus polymyxa SQR-21

Waseem Raza, Xingming Yang, Hongsheng Wu, Qiwei Huang, Yangchun Xu, Qirong Shen *

Jiangsu Provincial Key Lab for Organic Slid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 12 April 2010Received in revised form 13 July 2010Accepted 14 July 2010Available online 17 July 2010

Keywords:FusaricidinsIronMagnesiumPaenibacillus polymyxa SQR-21Zinc

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.07.052

* Corresponding author. Address: Jiangsu ProvincWaste Utilization, Nanjing Agriculture University, N210095, Jiangsu Province, PR China. Tel.: +86 1390158

E-mail address: shenqirong@njau.edu.cn (Q. Shen)

Effect of metal ions (Mg2+, Zn2+ and Fe3+) on the production of fusaricidin-type antifungal compounds byPaenibacillus polymyxa SQR-21 was studied in liquid culture. First, one-factor; three-level experimentswere conducted to find out optimal concentrations of each metal ion for maximum production of fusa-ricidins. Later, three-factor; five-level experiments were performed and a quadratic predictive modelwas developed using response surface methodology (RSM). The results indicated that Fe3+ and Mg2+ pos-itively affected the growth of P. polymyxa as determined by measuring the OD600 of the liquid culture. Theproduction of fusaricidin-type antifungal compounds was significantly inhibited by Zn2+ (P = 0.0114) andincreased by Mg2+ (P = 0.0051) but the effect of Fe3+ (P = 0.2157) was non-significant. However, a syner-gistic positive effect of Mg2+ and Fe3+ on the production of antifungal compounds was observed. Thisstudy sheds lights on the pertinent effects of the individual and combined metal ions on the productionof fusaricidins in P. polymyxa. It provides the key information for optimization of the metal ions in thefermentation media to achieve the maximum antibiotic production in this strain.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Metals are an integral part of all ecosystems. Some of them arevital components of living systems and known as essential metalions (Doelman, 1978). Secondary metabolisms are affected by thepresence or absence of these essential metal ions, as they may beresponsible for activation of some of the biosynthetic pathways(Behal, 1986). This regulatory effect of metal ions on microbial sec-ondary metabolism has been recorded for a variety of species(Weinberg, 1977), but little work has been reported for Paenibacil-lus species. Among Paenibacillus spp., P. polymyxa strains are capa-ble of suppressing plant diseases caused by soil-borne pathogensand promoting plant growth (Ryu et al., 2006). P. polymyxa strainsare known to produce two types of peptide antibiotics. One groupcomprises the antibiotics active against bacteria, including the pol-ymyxins, etc. (Shoji et al., 1977). The other group is made up of thepeptide antibiotics active against fungi and Gram-positive bacteriaand includes fusaricidins A, B, C, and D (Beatty and Jensen, 2002;Raza et al., 2009). In addition, there are many reports where thenature of the inhibitory agent is undefined (Liang et al., 1996). Inthis experiment, we used P. polymyxa strain SQR-21 (P. polymyxa)that was checked for all possible antibiotic compounds and was

ll rights reserved.

ial Key Lab for Organic Slido. 1, Weigang Road, Nanjing6468; fax: +86 2584432420.

.

found to produce only fusaricidin type of antifungal compounds,fusaricidin A, B, C and D, composed of a group of cyclic depsipep-tides that have molecular masses of 883, 897, 948, and 961 Da,respectively, with an unusual 15-guanidino-3-hydroxypentadeca-noic acid moiety bound to a free amino group (Raza et al., 2009).The fusaricidin biosynthetic gene cluster of P. polymyxa PKB1 wascloned and sequenced. It spans 32.4 kb, including an open readingframe, coding for a six-module non-ribosomal peptide synthetase(Li and Jensen, 2008).

Previously, we have studied the effect of four metal ions likeCa2+, Ni2+, Mn2+ and Cu2+ on fusaricidin production by P. polymyxa(Raza et al., 2010), however, still there is no information about theeffect of metal ions like Mg2+, Zn2+ and Fe3+ on fusaricidin produc-tion by P. polymyxa. The previous reports have indicated that thesemetals ions can significantly affect the production of antifungalcompounds. For example, Mg2+ up to 1.25 mM, increased bulbifor-min production and growth of Bacillus subtilis (Vasudeva et al.,2008). In Streptomyces sp., Fe3+ is required for production of theantibiotics actinomycin, neomycin, streptomycin and chloram-phenicol (Weinberg, 1970). The extracellular antifungal antibioticproduction by Streptomyces galbus was promoted by the additionof 200 lM Zn2+ (Paul and Banerjee, 1983).

Studies involving the effect of individual metal ions on theproduction of metabolic products by microorganisms can beinformative, but in natural environment, metals are presenttogether at various concentrations. Thus, there is a need to evalu-ate their interactive behavior so that we can develop optimal

W. Raza et al. / Bioresource Technology 101 (2010) 9264–9271 9265

recipe concentrations to produce maximum amounts of metabolicproducts. The traditional ‘‘three-factor; three-level” techniqueused for optimizing a multi-variable system with all potential com-binations, not only require many experiments but also may resultin wrong conclusions. Under these circumstances, RSM is an attrac-tive alternative that can be used to study the effect of several fac-tors that influence the dependent responses by varying the factorssimultaneously and carrying out a limited number of experiments(Li et al., 2001). RSM has been successfully applied in many areas ofbiotechnology such as optimization of culture conditions forhydrogen production by Ethanoligenens harbinense B49 (Guoet al., 2009) and Nattokinase production by B. subtilis (Deepaket al., 2008). However, it has not previously been used to studyand evaluate the effect of metal ions (Mg2+, Zn2+ and Fe3+) on fusa-ricidin-type antifungal compounds production. Thus, experimentswere planned to investigate the effect of three metal ions (Mg2+,Zn2+, and Fe3+) on growth and fusaricidins production by P. poly-myxa and to develop a quadratic predictive model. In addition,the effect of most effective metal ions combination on overall met-abolic activity of P. polymyxa was also determined by measuringintra and extracellular proteins and carbohydrates, intracellular li-pid and total RNA contents.

2. Methods

2.1. Bacterial and fungal cultures

A fusaricidin producing strain, P. polymyxa SQR-21 was isolatedfrom the rhizosphere soil of healthy watermelon plant from aheavily wilt diseased field (GenBank Accession No. FJ600406; Chi-na General Microbiology Culture Collection Center (CGMCC),Accession No. 1544). A tested pathogenic strain Fusarium oxyspo-rum f. sp. cucumerinum (F. oxysporum) were provided by the soil–microbe interaction laboratory, Nanjing Agriculture University,Nanjing, China. The P. polymyxa SQR-21 bacterial culture wasmaintained on Luria Bertani (LB) agar plates and was stored at�80 �C in tryptic soya broth (TSB) containing 20% glycerol for fur-ther use. The fungal pathogenic strain, F. oxysporum, was main-tained by cultivation on potato dextrose agar (PDA) plates for3 days at 28 �C and then the plates were sealed with parafilmand stored at 4 �C. The pathogen was subcultured onto a freshPDA plate after every month.

2.2. Preparation of metal ion media

Liquid-culture experiments were performed in 500 ml of tryp-tone broth (tryptone, 10; NaCl, 5 and sucrose, 10 g L�1; pH 7.5). Ini-tial Mg2+, Zn2+ and Fe3+ contents in tryptone broth, determined bySpectra AA, 220 FS atomic absorbance spectrometer, were 100, 4,and 10 lM, respectively. For the estimation of optimal concentra-tion of each metal ions needed to produce maximum fusaricidin-type antifungal compounds by P. polymyxa, a series of one-factor;three-level experiments were conducted. The levels considered,in the final one-factor, three-level experiments, for Mg2+ were0.65, 1.3 and 2.6 mM and for Zn2+ and Fe3+ these levels were 25,50 and 100 lM each. Each experiment had three replicates includ-ing control cultures without supplemented metal ions. The finalone-factor; three-level experiment was conducted twice for eachmetal ion.

For the isolation of antifungal compounds, tryptone broth wasinoculated with overnight culture of SQR-21 in TSB and incubatedin an incubator shaker (170 rpm, 30 �C). After 4 days, OD600 wasmeasured and for the quantification of fusaricidins, liquid culturewas centrifuged at 12,000g for 10 min and antifungal compoundswere extracted with an equal volume of n-butanol twice, concen-

trated by rotary evaporator and dissolved in methanol (1 ml).These extracts were used to determine fusaricidins production byagar diffusion assay against F. oxysporum (150 ll in each well).The diameter of inhibition zone was measured after 48 h and fusa-ricidins concentration was determined by comparing with stan-dards. The standards were developed by using 5–25 lg ml�1 ofpurified fusaricidins in methanol against F. oxysporum. The treat-ment differences were assessed with one-way ANOVA. Duncan’smultiple-range test was applied when one-way ANOVA revealedsignificant differences (P 6 0.05). All statistical analysis was per-formed with SPSS BAS Ever.11.5 statistical software (SPSS, Chicago,IL).

The metal concentrations that showed maximum fusaricidinsproduction were used as the highest level (+1 level) for interactivestudy (three-factor; five-level experiments). For the interactivestudy experiment, the tryptone medium was sterilized and the cul-tures were supplemented with MgSO4, ZnSO4 and FeCl3, for Mg2+,Zn2+ and Fe3+, respectively. Each experiment was repeated twicewith three replicates.

2.3. Extra and intracellular chemical composition

The effect of metal ions on the extra and intracellular chemicalcomposition of P. polymyxa was determined by measuring intra (IP)and extracellular protein (EP), intra (IC) and extracellular polysac-charides (EPS) and intracellular lipid (IL) contents. The P. polymyxaliquid culture (tryptone broth) samples (2 ml) were centrifuged(12,000g) for 10 min. The pellets were suspended in 2 ml of deion-ized water for washing and centrifuged three times. These pelletswere used for the determination of total intracellular protein, car-bohydrate and lipid contents. For total protein contents, thewashed cells were resuspended in deionized water and incubatedin 1 N NaOH at 90 �C for 10 min to solubilize cellular protein. Pro-teins were measured according to Bradford (1976) with bovine ser-um albumin standards ranging from 10 to 100 lg ml�1. Totalcarbohydrate was estimated by the phenol–sulfuric acid method(Dubois et al., 1956). The lipid contents of bacterial cells were mea-sured using the phosphoric acid–vanillin reagent method of Izardand Limberger (2003) with Triolein standards ranging from 10 to100 lg. The cell free liquid culture was used for the estimation ofextracellular protein and polysaccharide (EPS) contents by theabove-described methods. Before assaying protein, the resultingEPS solution was dialyzed using a membrane of 1000 Da molecularweigh cut-off against ultra pure water for 2 days at 4 �C to removethe small molecules and entrained media residues.

2.4. RNA extraction and primers design

To check the expression of fusaricidin synthetase gene (fusA),total RNA was isolated using Trizol reagent (Invitrogen, Shanghai)according to manufacturer’s instructions. To remove DNA contam-ination, 10 U DNase1 (Takara, Dalian) along with 20 U RNase inhib-itor (Takara, Dalian) (37 �C, 40 min) were used in the reactionmixture of 50 ll containing 20–50 lg RNA. RNA was estimatedby measuring the absorbance at 260 nm. Specific primers for fusA(111 bp) and 16S rRNA gene (16s) (210 bp) were designed by usingprimer premier 5 software (PREMIER biosoft international). Thedesigned primers were as follows, fusA1, 50-GCAGAGGATGA-TAGTGTTGGTC-30, fasA2, 50-CAGCACATCATGCGTTCC-30, 16s1, 50-CATTCATCGTTTACGGCGT-30 and 16s2, 50-TGTTAATCCCGAGGCTC-ACT-30.

2.5. Reverse transcription and real time PCR assay

For the synthesis of first strand cDNA, 3 lg of RNA, 200 U ofRevertAid M-MuLV reverse transcriptase (Fermantas), 20 U RNase

9266 W. Raza et al. / Bioresource Technology 101 (2010) 9264–9271

inhibitor (TaKaRa, Dalian), 0.2 lg of random hexamer primer and1 mM dNTP in the total volume of 20 ll were used. RT was per-formed by denaturation at 65 �C for 5 min, incubation at 42 �Cfor 60 min and inactivation at 95 �C for 5 min. Target genes fromcDNA were amplified separately using 3 ll aliquots of RT productas template and 30 pmol of each primer pair (fusA1 and fusA2,16s1 and 16s2). Reaction mixtures for PCR contained 2.5 U Taqpolymerase, 20 nmol of dNTP and 100 nmol Mg2+. The PCR proto-col included incubation at 95 �C for 5 min, followed by 30 cycles,each including 94 �C for 30 s, 58 �C for 30 s, 72 �C for 1 min andthen at 72 �C for 2 min. Amplified products were separated, stainedand viewed to check the band intensity and cDNA quality. Single-plex relative Real Time PCR was performed using an iCycler MyiQsingle color Real Time PCR detection system (BioRad). Reactionsmixtures (20 ll) contained 1 mM primers, 3 ll cDNA and 10 ll ofSYBR� Premix Ex Taq (Perfect Real Time) (TaKaRa, Dalian) includingTaKaRa Ex Taq HS and SYBR� Green I, dNTP and buffer. The PCRprotocol included a denaturation at 95 �C for 10 min followed by40 cycles with 95 �C for 30 s, 58 �C for 30 s and 72 �C for 1 min.Detection of the fluorescent product was carried out at the endof the 72 �C extension period (2 min). After the PCR, these sampleswere heated from 58 to 95 �C. When the temperature reaches theTm of each fragment, there was a steep decrease in fluorescenceof the product. The 2 ll cDNA of each treatment were mixed to-gether to prepare relative standards. The whole experiment wasrepeated twice.

2.6. Methodology and design of experiments

The most common experimental design in RSM is the centralcomposite design (CCD) (Bas� and Boyac, 2007). In our study, theCCD allowed us to develop a quadratic predictive model that hada minimal number of experimental runs. The CCD used was gener-ated by ‘‘Design-Expert” software (Trial version 7.1.6; Stat-EaseInc., Minneapolis, MN, USA). According to this design, 20 experi-ments containing six replications were conducted at the centerpoint for estimating the purely experimental uncertainty variancein triplicates. The parameters for three metal ions (Mg2+, Zn2+ andFe3+) were chosen as main variables and designated as x1, x2 and x3,respectively. The low, middle and high levels of each variable weredesignated as �1.68, �1, 0, +1 and +1.68.The behavior of the sys-tem was explained by the following second-degree polynomialequation:

Table 1Effect of the different Mg2+, Zn2+ and Fe3+ concentrations on OD600, production of fusextracellular protein (EP) and polysaccharide contents (EPS), RNA contents and relative efour days incubation. Values having same letters are less significant with each other at P =

Levels OD600 Fus (lg ml�1) IP (lg mg�1) IL (lg mg�1) IC (

Mg2+ (mM)0 0.692 11.3 c 455.6 d 69.6 b 1070.65 0.691 13.7 b 485.3 c 88.1 a 1621.3 0.736 15.8 a 504.9 a 66.1 c 1942.6 0.754 10.3 c 498.6 b 55.1 d 193LSD – 1.07 0.85 2.22 11.2

Zn2+ (lM)0 0.750 c 10.0 c 490.6 a 62.9 a 11625 0.830 b 12.7 b 449.1 b 53.8 b 10250 0.910 a 13.7 ab 420.2 c 38.5 c 100100 0.930 a 15.4 a 421.4 c 36.3 c 97.4LSD 0.07 1.96 6.44 2.37 11.7

Fe3+ (lM)0 0.736 c 11.5 c 445.2 68.5 b 11425 0.755 b 13.6 b 447.0 75.8 a 11550 0.766 ab 15.6 a 449.3 69.6 b 120100 0.773 a 12.6 bc 450.8 65.7 b 125LSD 0.09 1.80 – 5.67 7.01

Y ¼ B0 þXn

i¼1

Bixi þXn

i<j

Bijxixj þXn

j¼1

Bjjx2j ð1Þ

where Y is response; B0 is a constant, Bi is the linear coefficient, Bij isthe second-order interaction, and Bjj is the quadratic coefficients.The variable, xi, is the non-coded independent variables. It mustbe noted that in the present study, three variables are involvedand hence n takes the value 3. Thus, by substituting the value 3for n, Eq. (1) becomes:

Y ¼ B0 þ B1x1 þ B2x2 þ B3x3 þ B12x1x2 þ B13x1x3 þ B23x2x3

þ B11x21 þ B22x2

2 þ B33x23 ð2Þ

where Y is the predicted response, and x1, x2 and x3 are input vari-ables. B0 is a constant and B1, B2 and B3 are linear coefficients. B12,B13 and B23 are cross-product coefficients and B11, B22 and B33 arequadratic coefficients.

3. Results and discussion

3.1. One-factor; three-level experiments

For the estimation of optimum concentration of each metal ionfor maximum production of fusaricidins, the influence of one-fac-tor (one metal ion) was determined with three concentration levelexperiments (Table 1). The results revealed that Zn2+ and Fe3+ sig-nificantly increased the growth, as measured by optical density(OD600) of P. polymyxa while the effect of Mg2+ on growth of P. poly-myxa was non-significant. The increase in the concentrations ofZn2+, Fe3+ and Mg2+ in the liquid culture increased the productionof fusaricidins and the relative expression of fusA gene. However,at the highest levels of Fe3+ (100 lM) and Mg2+ (2.6 mM), decreasein the production of fusaricidins and in the relative expression offusA gene was determined. The expression of positive control gene16S rRNA was remained nearly constant among all treatments.Four kinds of fusaricidins were produced by P. polymyxa that wereeluted in two peaks (Raza et al., 2010) but their production ratiowas not affected by the metal ions so the quantity of fusaricidinspresented in the results, represents total amount of fusaricidins.To determine the effect of metal ions alone on the overall meta-bolic activity of P. polymyxa SQR-21, intracellular protein (IP), lipid(IL) and carbohydrate (IC) contents, extracellular protein (EP) andpolysaccharide (EPS) contents and RNA contents were determined.

aricidins (Fus), intracellular protein (IP), carbohydrate (IC) and lipid contents (IL),xpression of fusA gene (RE) of P. polymyxa SQR-21 against Fusarium oxysporum after

0.05, LSD = least significant differences.

lg mg�1) EP (lg ml�1) EPS (lg ml�1) RNA (lg mg�1) RE

.4 c 307.7 c 1232 b 27.1 a 0.85 c

.0 b 364.5 a 1271 a 26.3 a 1.20 b

.7 a 323.7 b 1257 ab 23.8 a 1.26 a

.4 a 289.6 d 1186 c 19.8 b 0.54 d4 0.50 25.49 3.46 0.03

.9 a 355.5 b 1410 a 34.3 a 0.64 c

.3 b 364.5 b 1284 ab 33.6 a 0.83 ab

.9 b 480.7 a 1203 b 29.8 a 0.97 abb 257.7 b 775 c 19.0 b 1.12 a

3 116.4 126.7 4.48 0.27

.5 b 324.6 b 1354 c 27.8 ab 0.74 c

.7 b 328.0 b 1444 b 28.7 ab 1.04 b

.0 ab 331.6 b 1699 a 31.4 a 1.27 a

.8 a 345.7 a 1717 a 26.4 b 0.63 c7.08 39.19 4.37 0.22

Table 3Analysis of variance for predictive equation for fusaricidins production by P. polymyxa

W. Raza et al. / Bioresource Technology 101 (2010) 9264–9271 9267

The results showed that the increase in the concentration of Mg2+

in the liquid culture increased the IP, IC and EPS contents (except atthe highest level, 2.6 mM Mg2+), however, maximum IL and EP con-tents were determined at 0.65 mM Mg2+ concentration. The in-crease in the concentration of Fe3+ in the liquid culture increasedthe IC, EP and EPS contents while the effect on IP contents wasnon-significant. The maximum IL contents were determined at25 and maximum RNA contents were determined at 50 lM Fe3+

concentration. The increase in the concentration of Zn2+ in the li-quid culture decreased the IP, IL, IC, EPS and RNA contents. Themaximum EP contents were determined at 50 lM Zn2+ concentra-tion, while the EP contents at all other concentration levels of Zn2+

were non-significant with each other. The effect of these metal ionson antibiotic production by different microbes has been reportedearlier. MgSO4 at 2 mM concentration increased iturin A produc-tion by Bacillus amyloliquefaciens B128 (Lin et al., 2007). Additionof Zn2+ has a stimulatory effect on the activity of fatty acid synthe-tase and on aflatoxin B1 production in Aspergillus parasiticus (Re-ding and Harrison, 1994). Concentrations of Fe3+ up to 1.8 M hadstimulatory effects on the production of antitubercular antibioticrifamycin by Nocardia mediterranea A TCC 13685 (Mukhtiar, 2000).

3.2. Three-factor; five-level experiments

The values of the responses (fusaricidins type antifungal com-pounds and OD600) obtained under the different experimental con-ditions are summarized in Table 2.

By applying multiple regression analysis to the experimentaldata of fusaricidin-type antifungal compounds, the response andtest variables were found to be related by the following second-or-der polynomial equation:

YFusaricidins ¼ 11:57þ 1:06x1 � 0:92x2 þ 0:39x3 � 0:99x1x2

� 1:56x1x3 � 0:14x2x3 þ 1:88x21 þ 0:44x2

2

þ 1:58x23 ð3Þ

By applying multiple regression analysis to the experimentaldata of OD600, the response and test variables were found to be re-lated by the following second-order polynomial equation:

Table 2Central composite design arrangement and responses.

Trialno.

Variables YFusaricidins ðlg ml�1Þ YOD600

x1 Mg2+

(mM)x2 Zn2+

(lM)x3 Fe3+

(lM)Observed Predicted Observed Predicted

1 0.65 50 25 12.60 12.24 0.86 0.792 1.30 50 25 20.60 19.47 0.82 0.773 0.65 100 25 13.20 12.65 0.65 0.664 1.30 100 25 15.10 15.93 0.70 0.705 0.65 50 50 18.00 16.43 0.89 0.886 1.30 50 50 17.60 17.41 0.95 0.947 0.65 100 50 15.90 16.29 0.87 0.918 1.30 100 50 13.70 13.31 0.97 1.039 0.43 75 37.5 14.20 15.09 0.85 0.8610 1.52 75 37.5 18.50 18.67 0.94 0.9411 0.98 25 37.5 12.80 14.38 0.82 0.9012 0.98 125 37.5 11.80 11.28 0.94 0.8713 0.98 75 16.5 15.00 15.36 0.50 0.5614 0.98 75 58.5 16.00 16.69 0.96 0.9115 0.98 75 37.5 10.90 11.57 0.90 0.8916 0.98 75 37.5 13.00 11.57 0.86 0.8917 0.98 75 37.5 11.10 11.57 0.89 0.8918 0.98 75 37.5 11.40 11.57 0.86 0.8919 0.98 75 37.5 11.70 11.57 0.92 0.8920 0.98 75 37.5 11.50 11.57 0.90 0.89

YOD600 ¼ 0:89þ 0:025x1 � 0:008x2 þ 0:11x3 þ 0:016x1x2

þ 0:018x1x3 þ 0:040x2x3 þ 0:0049x21 � 0:0021x2

2

� 0:054x23 ð4Þ

The correlation measure for testing the goodness of fit of regres-sion equation is the adjusted determination coefficient R2

Adj. Thevalues of R2

Adj, 0.8440 for Eq. (3) and 0.7547 for Eq. (4), indicate ahigh degree of correlation between the observed and predicted val-ues for the production of fusaricidins and OD600, respectively. Sta-tistical testing of the model was done in the form of analysis ofvariance (ANOVA). The regression model demonstrates that themodel is highly significant, as is evident from the calculated F-val-ues (12.42 for fusaricidins type antifungal compounds and 7.49 forOD600) and a very low probability values (P = 0.0002 for fusarici-dins type antifungal compounds (Table 3) and 0.0021 for OD600

(data not shown). The model also shows statistically non-signifi-cant (P > 0.05) lack of fit for production of fusaricidins and OD600

as is evident from greater computed F-values than the tabulatedF-values (F0.05 (9, 5) = 4.77). The model was, therefore found to beadequate for prediction within the range of variables employed.The coefficient values of Eq. (3), calculated and tested for signifi-cance using the ‘‘Design-Expert” software, indicate that the linearcoefficients (x1, and x2), quadratic term coefficients (x2

1 and x23)

and cross-product coefficient (x1x2 and x1x3) are significant andthe other term coefficients (x3, x2

2 and x2x3) are not significant(Table 4).

The graphical representations of regression Eq. (3), termed asthe 3D plots were obtained using the Design Expert software.Fig. 1A shows the 3D response surface plot at varying Mg2+ andZn2+ concentrations at a fixed Fe3+ concentration of 37.5 lM (0 le-vel). From Fig. 1A, it can be seen that the fusaricidins productiondecreased with an increase in concentration of Zn2+ and increasedwith an increase in concentration of Mg2+. This is an example of aninteractive effect between Mg2+ and Zn2+. Analyzing the Mg2+–Fe3+

plot (Fig. 1B), a significantly (P < 0.05) positive synergistic

SQR-21.

Source Sum ofsquares

df Meansquare

F-value Prob > F

Model 135.72 9 15.08 12.42 0.0002 SignificantResidual 12.14 10 1.21Lack of fit 9.38 5 1.88 3.40 0.1028 Not significantPure error 2.76 5 0.55Cor total 147.86 19

SD = 1.10; R2 = 0.92; R2Adj = 0.84.

Table 4Test of significance for regression coefficients.

Modelterm

Coefficientestimate

df S.E. 95% CIlow

95% CIhigh

F-value Prob > F

Intercept 11.57 1 0.45 10.57 12.57 – –x1 1.06 1 0.30 0.40 1.73 12.74 0.0051**

x2 �0.92 1 0.30 �1.59 �0.26 9.55 0.0114*

x3 0.39 1 0.30 �0.27 1.06 1.75 0.2157x1x2 �0.99 1 0.39 �1.86 �0.12 6.43 0.0296*

x1x3 �1.56 1 0.39 �2.43 �0.69 16.09 0.0025**

x2x3 �0.14 1 0.39 �1.01 0.73 0.12 0.7314x2

11.88 1 0.29 1.23 2.52 41.79 <0.0001**

x22

0.44 1 0.29 �0.20 1.09 2.34 0.1569

x23

1.58 1 0.29 0.93 2.22 29.47 0.0003**

* = <0.05.** = <0.01; CI = confidence limit.

Fig. 1. Response surface 3D plots of the effect of Mg2+ and Zn2+ (A) Mg2+ and Fe3+ (B) and Zn2+ and Fe3+ (C) and their mutual interactions on antifungal compound productionby P. polymyxa SQR-21.

9268 W. Raza et al. / Bioresource Technology 101 (2010) 9264–9271

interactive effect on fusaricidins production was observed at afixed Zn2+concentration of 112.5 lM. The 3D response surface inFig. 1C, which gives the fusaricidins production as a function ofZn2+ and Fe3+ concentrations at a fixed Mg2+ concentration of0.98 mM (0 level), shows that the fusaricidins production de-creased with an increase in the concentration of Zn2+ and increasedwith an increase in concentration of Fe3+.

Our previous report has documented the importance of metalions like Ca2+, Mn2+, Ni2+ and Cu2+ for the production of fusaricidinsby P. polymyxa (Raza et al., 2010) but there was a need to explorethe effects of other important metal ions like Mg2+, Zn2+ and Fe3+ asthese metal ions also have been reported to effect the metabolitesproduction significantly by different microorganisms. In thisexperiment, the results showed that Mg2+ was most effective metalion for increasing fusaricidins production by P. polymyxa. The effec-tiveness of Mg2+ to increase the production of fusaricidins is fur-ther supported by Wang and Liu (2008) who reported that Mg2+

was the most effective ion that stimulated the production of poly-myxin E by P. polymyxa Cp-S316. In addition, Mg2+ was an essential

element for the production of PA-5 and PA-7 type antibiotics. Thiswas probably due to its effect on the Mg-requiring acetyl-CoA-car-boxylase activity as shown in Streptomyees strains (Martin andMcdaniel, 1977). The enzyme involved in the synthesis of b-lactamantibiotics, L-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) synthe-tase, also needs Mg2+ for the activation of each amino acid (Rohet al., 1992). The Fe3+ also showed a positive synergistic effect onfusaricidins production with Mg2+ ion. The Fe3+ is most importantmicronutrient used by bacteria and is being required as a cofactorfor a large number of enzymes and iron-containing proteins. AsLubbe et al. (1984) reported that the complete cephamycin path-way benefited from the higher iron concentration. Three of the en-zymes common to the cephalosporin C and cephamycin Cbiosynthetic pathways are known to require iron in their catalyticactivities, namely isopenicillin N synthase (IPNS), deacetoxycepha-losporin C synthase (DAOCS), and deacetoxycephalosporin Chydroxylase (DAOCH).

The data obtained in this experiment clearly depicted that met-als ions alone and in different combinations differently affect

W. Raza et al. / Bioresource Technology 101 (2010) 9264–9271 9269

metabolites production. The optimum concentrations of metalsions required for the maximum fusaricidins production observedin one-factor, three-level experiments were 1.3 mM, 100 lM and50 lM for Mg2+, Zn2+ and Fe3+, respectively. However, when theirinteractive effects were observed then Zn2+ in combination withMg2+ and Fe3+ inhibited the production of fusaricidins. However,the interactive effect of Mg2+ with Fe3+ was positively synergistic.The maximum production of fusaricidin-type antifungal com-pounds (20.6 lg ml�1) was measured at 75 (�1), 25 (�1) lM and1.3 mM (+1) levels of Zn2+, Fe3+ and Mg2+, respectively. Similarkinds of effects of these metal ions have been reported on metab-olites production by other microorganisms. For example, Mg2+ upto 1.25 mM increased bulbiformin production by B. subtilis (Vasud-eva et al., 2008), Zn2+ at a concentration of 2–3 mM nearly stoppedthe pigmentation and antibiotic production of both wild type andstrain NI IS in liquid medium (Bau and Wong, 1979), ferric iron(250–1000 lM) enhanced zwittermicin A accumulation and dis-ease suppression (Milner et al., 1995).

The data regarding the interactive effect of metal ions on bacte-rial growth indicates Mg2+ and Fe3+ together increased the growthof P. polymyxa in liquid culture. However, Zn2+ caused a slight de-crease in the growth of P. polymyxa, as was also depicted from Eq.(4). The maximum growth, as determined by measurement ofOD600 (value of 0.97), was obtained for the combination of Zn2+,Fe3+ and Mg2 at concentrations of 100 (+1), 50 (+1) lM and 1.3(+1) mM, respectively. These results are in agreement with previ-ous reports that the supplementation of the medium with Mg2+

stimulated the growth of Zalerion arboricola (Tkacz et al., 1993).Fe3+ has a marked effect on growth of B. subtilis (Mahmood,

Fig. 2. The effect of optimal fusaricidins producing metal ions concentrations (Zn2+ =intracellular protein (IL), carbohydrate (IC) and lipid (IL) contents, extracellular protein (gene (RE) of P. polymyxa SQR-21. Ck = control; MI = metal ions.

1970) and Zn2+ decreased the growth of Monascus purpureus (Bauand Wong, 1979).

3.3. Effect of optimal metal ion concentrations on overall metabolicactivity of P. polymyxa

The combination of metal ions concentrations that gave maxi-mum fusaricidins production was used to identify their effect onoverall metabolic activity of P. polymyxa. The results showed(Fig. 2) that OD600 (i.e., growth of bacteria), IP, IC and RNA contentsand relative expression of fusA gene were increased by 38%, 5%, 8%,15% and 89%, respectively, while the EP, EPS and IL contents weredecreased by 26%, 2% and 37% over control (without supplementedmetal ions), respectively. These results, in combination with the re-sults of the effect of these metal ions alone on overall metabolicactivity of P. polymyxa where Fe3+ and Mg2+ alone increased mostof tested parameters while the effect of Zn2+ was inhibitory, clearlyshowed that these metal ions (Zn2+, Fe3+ and Mg2+) have a regula-tory role, either directly or indirectly, on bacterial secondarymetabolism, mainly in the production of fusaricidins. The increasein the concentrations of IP, RNA and especially relative expressionof fusA gene indicated that the production of some enzymes, in-volved in fusaricidins synthesis, growth or other activities relatedto different cellular processes might be increased. This resultedin a simultaneous decrease in the IL contents as residual energywas being used for the IP and IC synthesis that promoted theexpression of proteins involved in the fusaricidins production.The EPS that P. polymyxa form, are thought to play a crucial rolein metal biosorption and precipitation as P. polymyxa strains have

50 (�1), Fe3+ = 25 (�1) and Mg2+ = 1.3 mM (+1)) on bacterial growth (i.e., OD600),EP) and polysaccharide (EPS) contents, RNA contents and relative expression of fusA

9270 W. Raza et al. / Bioresource Technology 101 (2010) 9264–9271

been used in the biosorption of Cu (Acostal et al., 2005) but thecombination of metal ions that produced maximum fusaricidinsdecreased the EPS production. The biological and chemical charac-teristics of these uptake processes are important as an aid in theunderstanding of the role of metallic ions in basic cellular func-tions. The decrease in EP and EPS concentrations in the presenceof Zn2+ indicated the toxic effect of this metal ion on extracellularenzyme production. This conclusion was further supported by theresults of one-factor with three concentration levels experimentswhere Zn2+ decreased IP, IL, IC, EPS and RNA contents of P. poly-myxa. These toxic effects of Zn2+ are in agreement with previousfindings when Zn2+, at concentrations of 2–3 mM, nearly stoppedthe pigmentation and antibacterial activity of both wild type andstrain NI IS in liquid medium (Bau and Wong, 1979).

During growth, different metal ions as well as their oxide min-erals influence the types and quantity of polysaccharides, proteinsand enzymes secreted by bacteria (Reed, 1987). The mechanisms ofhow metal ions promoted or inhibited the growth and antibioticproduction are obscured and have not yet been elucidated. In ourcase, metal ions might be obstructing with secondary metabolismmore than enzymes or other cellular processes. They may have in-creased or decreased the synthesis of the prepeptide or the activa-tion of the suitable prepeptide maturation enzymes and thetransfer out of the cell. Siezen et al. (1995) predicted the Ca2+ bind-ing sites to be present in NisP peptidase, which cleaves the leaderpeptide from the precursor nisin. Since the precursor is devoid ofantibacterial activity (van der Meer et al., 1993), metal ions areconsidered to be involved in the activation or inactivation of theleader peptidase. The cell wall permeability of P. polymyxa pro-moted by metal ions can increase the concentration of antibioticin the medium. Which has been reported by Petit-Glatron et al.(1993), who studied the capacity of the cell wall to concentrateCa2+ and proposed that the increased concentration of Ca2+ in themicroenvironment of the cell wall could play an important rolein the last step of the secretion. Another possibility is that the me-tal ions might activate such enzymes whose activity change theregulatory functions of the cell in favor of different secondarymetabolites, especially fusaricidins.

Ten verification experiments were also carried out to confirmthe sufficiency of the model equations (i.e., Eqs. (3) and (4)), underdifferent combinations of metal ions and the results are shown inTable 5. The validation data were analyzed using SPSS software(Version 16.0, SPSS Inc.). The correlation coefficients (R) betweenthe experimental and predicted values of the size of inhibitionzone and growth (i.e., OD600) were 0.864 and 0.929, respectively.The experimental values were in accord with the predicted valuesand sustained the conclusion that the models of Eqs. (3) and (4)presented adequate and precise results. This clearly showed themarked advantage of using RSM that allowed us to obtain a regres-sion equation predicting the growth and fusaricidins production by

Table 5Verified results of the model equation for growth and antifungal production by P.polymyxa SQR-21.

Trail no. Variables YInhibition zone (mm) YOD600

x1 x2 x3 Observed Predicted Observed Predicted

1 0.98 37.5 25 13.3 13.6 0.675 0.7602 1.02 39.5 26 13.7 13.6 0.776 0.7703 1.06 41.5 27 14 13.7 0.78 0.7814 1.10 43.5 28 14.4 13.8 0.797 0.7925 1.14 45.5 29 14.5 13.9 0.808 0.8056 1.18 47.5 30 15.1 14.1 0.813 0.8187 1.22 49.5 31 15.3 14.2 0.855 0.8338 1.26 51.5 32 15.5 16.6 0.873 0.8599 1.28 53.5 33 16 16.6 0.883 0.87010 1.30 55.5 34 16.6 16.7 0.9 0.881

P. polymyxa and could identify which of the metal ions were mostimportant in this process.

4. Conclusions

This regression equation model is the first application of RSM todescribe the effect of metal ions like Mg2+, Zn2+ and Fe3+ on theproduction of fusaricidin-type compounds by P. polymyxa. Thefusaricidins have great potential for industrial use according tothe recent reports on its germicidal activity against pathogenicGram-positive bacteria and plant pathogenic fungi and therebyfusaricidins are in increasing demand (Ryu et al., 2006; Razaet al., 2009). So this information will aid in developing fermenta-tion technology for maximum antibiotic production under labora-tory and commercial fermentation conditions along with alreadyreported information (Raza et al., 2010).

Acknowledgements

This work was financially supported by the Agricultural Minis-try of China (201103004) and National Nature Science Foundationof China (40871126). We would like to thank Professor WarrenDick at The Ohio State University, USA for his help in careful andcritical correction and edition of this manuscript.

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