Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens

Aims: A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents. This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus. Methods and Results: To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B. amyloliquefaciens strain. Assays using B. amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity. The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations. Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect. The results are supported by site‐directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production. Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application. Conclusions: We conclude that the iturin family of lipopeptides are vital in the antagonism of B. amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested. Significance and Impact of the Study: We elucidated the principal mechanism used by B. amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits.     

Optimization of antifungal lipopeptide production from Bacillus sp. BH072 by response surface methodology

Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH₄Cl, urea, and ammonium citrate); and metal ions (Zn²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ca²⁺, and K⁺). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg²⁺ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg²⁺. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.     

Comparing Methods for Identifying Bacillus Strains Capable of Producing the Antifungal Lipopeptide Iturin A

Lipopeptides represent a unique class of bioactive microbial secondary metabolites, and iturin A shows attractive antibiotic properties among them. This study compares three methods, such as yeast/fungal growth inhibition assay, quantitative high-performance liquid chromatography (HPLC) and polymerase chain reaction (PCR) for identifying a number of Bacillus species that produce iturin A. We examined the feasibility of screening iturin A-producing Bacillus strains by PCR using specific primers for ituD and lpa-14 amplification. Twenty standard strains and 120 field-collected Bacillus spp. isolates were tested in this study. Four B. subtilis and one B. circulan strains from ATCC, and B. amyloliquefaciens B128, a known iturin A producer, exhibited positive results. Of the 120 field-collected isolates, 42 strains were positive. The potential of producing iturin A by these PCR-positive strains were then confirmed by conventional methods such as fungal growth inhibition assay and HPLC analysis. The consistency between results of PCR, HPLC, and fungal growth inhibition assay suggests that the PCR method could be used as an alternative tool for fast screening of iturin A-producing Bacillus strains from the environment. This is the first report of detecting iturin A production from B. circulans.     

Direct Bio-Utilization of Untreated Rapeseed Meal for Effective Iturin A Production by Bacillus subtilis in Submerged Fermentation

The feasibility of using untreated rapeseed meal as a nitrogen source for iturin A production by Bacillus subtilis 3–10 in submerged fermentation was first evaluated by comparison with two different commercial nitrogen sources of peptone and ammonium nitrate. A significant promoting effect of rapeseed meal on iturin A production was observed and the maximum iturin A concentration of 0.60 g/L was reached at 70 h, which was 20% and 8.0 fold higher than that produced from peptone and ammonium nitrate media, respectively. It was shown that rapeseed meal had a positive induction effect on protease secretion, contributing to the release of soluble protein from low water solubility solid rapeseed meal for an effective supply of available nitrogen during fermentation. Moreover, compared to raw rapeseed meal, the remaining residue following fermentation could be used as a more suitable supplementary protein source for animal feed because of the great decrease of major anti-nutritional components including sinapine, glucosinolate and its degradation products of isothiocyanate and oxazolidine thione. The results obtained from this study demonstrate the potential of direct utilization of low cost rapeseed meal as a nitrogen source for commercial production of iturin A and other secondary metabolites by Bacillus subtilis.     

Medium optimization of antifungal lipopeptide,iturin A,production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in solid-state fermentation by response surface methodology

Iturin A, a lipopeptide antibiotic produced by Bacillus subtilis RB14-CS, suppresses the growth of various plant pathogens. Here, enhancement of iturin A production in solid-state fermentation (SSF) on okara, a soybean curd residue produced during tofu manufacturing, was accomplished using statistical experimental design. Primary experiments showed that the concentrations of carbon and nitrogen sources were the main factors capable of enhancing iturin A production, whereas initial pH, initial water content, temperature, relative humidity, and volume of inoculum were only minor factors. Glucose and soybean meal were the most effective among tested carbon and nitrogen sources, respectively. Based on these preliminary findings, response surface methodology was applied to predict the optimum amounts of the carbon and nitrogen sources in the medium. The maximum iturin A concentration was 5,591 μg/g initial wet okara under optimized condition. Subsequent experiments confirmed that iturin A production was significantly improved under the predicted optimal medium conditions. The SSF product generated under the optimized conditions exhibited significantly higher suppressive effect on the damping-off of tomato caused by Rhizoctonia solani K-1 compared with the product generated under the non-optimized conditions.     

Production of casein hydrolysates by extracellular acid proteinases of immobilized Humicola lutea cells

Casein hydrolysis was studied during the cultivation of immobilized Humicola lutea cells producing acid proteinases. By monitoring the cultivation with time, various casein hydrolysates could be obtained, from partially modified proteins (yield 80%) with improved emulsion properties to peptones (yield > 50%) with a degree of hydrolysis >40%. The casein from the fermentation medium appeared to be simultaneously a nitrogen source, an inducer of proteinase biosynthesis, and a substrate for the production of casein hydrolysates. Casein (4%) and glucose (2%) ensured optimal cultivation conditions. The fungal cells, immobilized in calcium alginate beads, required a short cultivation time and demonstrated comparable hydrolysis of casein during five to seven reuses in batch mode. Correspondence to: B. Tchorbanov     

Methane production from rice straw pretreated by a mixture of acetic–propionic acid

Rice straw was treated with a mixed solution of acetic acid and propionic acid to enhance its biodegradability. The effect of acid concentration, pretreatment time, and the ratio of solid to liquid on the delignification performance of rice straw were investigated. It was found that the optimal conditions for hydrolysis were 0.75 mol/L acid concentration, 2 h pretreatment time and 1:20 solid to liquid ratio. Batch methane fermentation of untreated rice straw, pretreated rice straw, and the hydrolysates (the liquid fraction) of pretreatment were conducted at 35 °C for 30 days, and the results indicated that methane production of rice straw can be enhanced by dilute organic acid pretreatment. Moreover, most of the acid in hydrolysates can also be converted into methane gas.     

Statistical optimization of antifungal iturin A production from Bacillus amyloliquefaciens RHNK22 using agro-industrial wastes

Biosurfactants are secondary metabolites with surface active properties and have wide application in agriculture, industrial and therapeutic products. The present study was aimed to screen bacteria for the production of biosurfactant, its characterization and development of a cost effective media formulation for iturin A production. A total of 100 bacterial isolates were isolated from different rhizosphere soil samples by enrichment culture method and screened for biosurfactant activity. Twenty isolates were selected for further studies based on their biosurfactant activity [emulsification index (EI%), emulsification assay (EA), surface tension (ST) reduction] and antagonistic activity. Among them one potential isolate Bacillus sp. RHNK22 showed good EI% and EA with different hydrocarbons tested in this study. Using biochemical methods and 16S rRNA gene sequence, it was identified as Bacillus amyloliquefaciens. Presence of iturin A in RHNK22 was identified by gene specific primers and confirmed as iturin A by FTIR and HPLC. B. amyloliquefaciens RHNK22 exhibited good surface active properties and antifungal activity against Sclerotium rolfsii and Macrophomina phaseolina. For cost-effective production of iturin A, 16 different agro-industrial wastes were screened as substrates, and Sunflower oil cake (SOC) was favouring high iturin A production. Further, using response surface methodology (RSM) model, there was a 3-fold increase in iturin A production (using SOC 4%, inoculum size 1%, at pH 6.0 and 37 °C temperature in 48 h). This is the first report on using SOC as a substrate for iturin A production.     

Dilute acid pretreatment of barley straw and its saccharification and fermentation

In this study, the optimization of the major factors for efficient dilute acid pretreatment (DAP) of Korean barley straw was conducted by response surface method (RSM). In addition, saccharification of the optimized pretreated barley straw as well as fermentation of solubilized hemicellulose and enzymatic hydrolysates was performed for bioethanol production. The factors optimized by RSM were concentration of sulfuric acid, reaction time and temperature. Optimization experiments were carried out within the scope of 0.16 ∼ 1.84% sulfuric acid, 10 ∼ 20 min of reaction time, and 116 ∼ 183°C of temperature using a statistical program, and optimal conditions (1.16% of sulfuric acid, 16.9 min of reaction time, and 150°C) were determined based on reliable statistical indicators. The predicted value at stationary point and the experimental value were 81.38 and 80.66%, respectively. Saccharification was performed at 50°C using Celluclast (cellulase) and Novozyme 188 (β-glucosidase) as biocatalysts in an enzyme loading test. Conversion of the saccharification process was approximately 65%. In addition, fermentation of glucose after saccharification and solubilization of xylose solution by DAP were performed using Saccharomyces cerevisiae and Pichia stipitis at 30°C and 200 rpm for 12 h.     

Production of lactic acid and ethanol by Rhizopus oryzae integrated with cassava pulp hydrolysis

Cassava pulp was hydrolyzed with acids or enzymes. A high glucose concentration (>100 g/L) was obtained from the hydrolysis with 1 N HCl at 121 °C, 15 min or with cellulase and amylases. While a high glucose yield (>0.85 g/g dry pulp) was obtained from the hydrolysis with HCl, enzymatic hydrolysis yielded only 0.4 g glucose/g dry pulp. These hydrolysates were used as the carbon source in fermentation by Rhizopus oryzae NRRL395. R. oryzae could not grow in media containing the hydrolysates treated with 1.5 N H₂SO₄ or 2 N H₃PO₄, but no significant growth inhibition was found with the hydrolysates from HCl (1 N) and enzyme treatments. Higher ethanol yield and productivity were observed from fermentation with the hydrolysates when compared with those from fermentation with glucose in which lactic acid was the main product. This was because the extra organic nitrogen in the hydrolysates promoted cell growth and ethanol production.     

Production and characterization of β-glucosidase from Aspergillus niger fermentation: Application for organic fraction of municipal solid waste hydrolysis and methane enhancement

The anaerobic digestion of the organic fraction of municipal solid waste (OFMSW) is currently an attractive treatment process with energy production in the form of biogas. Hydrolysis is the rate-limiting step for the anaerobic digestion of solid wastes. Thus, in the present study fungal enzymatic pretreatment of OFMSW was applied to enhance biogas production. Two enzyme cocktails rich on β-glucosidase were produced from submerged fermentation of Aspergillus niger on basal medium using OFMSW as carbon source and urea (Urea cocktail) and Ulva rigida as nitrogen source (Ulva cocktail). Ulva cocktail displayed an important effect on OFMSW solubilization. Therefore, an increase of reducing sugar concentration about 60% was obtained which was in correlation with chemical oxygen demand (COD) increase. The performance of enzymatic pretreatment on anaerobic digestion of OFMSW was studied by conducting biochemical methane potential tests. Results showed that the enzymatic pretreatment improved methane yield of OFMSW even at high solid concentration. High methane yield about 500 ml/g total volatile solid was obtained, which corresponds up to 68% enhancement over the control.     

Nutritional requirements ofLysobacter lactamgenus for the production of cephabacins

Summary To optimize the fermentation medium for the production of new cephem compounds, cephabacins, by an eubacteriaLysobacter lactamgenus IFO 14,288, the effects of medium components on cephabacin production were investigated. Supplementation of glucose as a sole carbon source in liquid media was the best for the antibiotic production as well as for the cell growth. Casamino acid was the best nitrogen source for antibiotic biosynthesis and cell growth among nitrogen sources tested, and this strain could utilize sulfate or thiosulfate as a sulfur source. No significant effects of growth factors (vitamins) on the antibiotic production and cell growth were observed, but ferrous, magnesium and nickel ions slightly enhanced the cephabacin production.     

Influence of iturin A on mycelial weight and aflatoxin production byAspergillus flavus andAspergillus parasiticus in shake culture

Iturin A, a peptidolipid produced byBacillus subtilis, inhibits growth of a large number of fungi. In this study, the effects of iturin A were evaluated on nine isolates ofA. flavus and seven isolates ofA. parasiticus in liquid shake culture. The mycelial dry weight of theA. flavus isolates was not significantly influenced by iturin A, however, there was a significant reduction in mycelial dry weight for two of theA. parasiticus isolates. Aflatoxin production was significantly reduced in five of theA. flavus isolates and three of the six aflatoxigenicA. parasiticus isolates. For the other seven isolates, aflatoxin levels were either unchanged or significantly increased in the presence of iturin A. These results indicate that iturin A does not consistently reduce growth or aflatoxin production of these fungi in pure culture.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases

Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.     

Direct fermentation of potato starch in wastewater to lactic acid by<Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

The fungal species ofRhizopus oryzae 2062 has the capacity to carry out a single stage fermentation process for lactic acid production from potato starch wastewater. Starch hydrolysis, reducing sugar accumulation, biomass formation, and lactic acid production were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/L at pH 6.0 and 30°C was favourable for starch fermentation, resulting in a lactic acid yield of 78.3%–85.5% associated with 1.5–2.0 g/L fungal biomass produced in 36 h of fermentation.     

The influence of process parameters in production of lipopeptide iturin A using aerated packed bed bioreactors in solid-state fermentation

The strain Bacillus iso 1 co-produces the lipopeptide iturin A and biopolymer poly-γ-glutamic acid (γ-PGA) in solid-state fermentation of substrate consisting of soybean meal, wheat bran with rice husks as an inert support. The effects of pressure drop, oxygen consumption, medium permeability and temperature profile were studied in an aerated packed bed bioreactor to produce iturin A, diameter of which was 50 mm and bed height 300 mm. The highest concentrations of iturin A and γ-PGA were 5.58 and 3.58 g/kg-dry substrate, respectively, at 0.4 L/min after 96 h of fermentation. The low oxygen uptake rates, being 23.34 and 22.56 mg O₂/kg-dry solid substrate for each air flow rate tested generated 5.75 W/kg-dry substrate that increased the fermentation temperature at 3.7 °C. The highest pressure drop was 561 Pa/m at 0.8 L/min in 24 h. This is the highest concentration of iturin A produced to date in an aerated packed bed bioreactor in solid-state fermentation. The results can be useful to design strategies to scale-up process of iturin A in aerated packed bed bioreactors. Low concentration of γ-PGA affected seriously pressure drop, decreasing the viability of the process due to generation of huge pressure gradients with volumetric air flow rates. Also, the low oxygenation favored the iturin A production due to the reduction of free void by γ-PGA production, and finally, the low oxygen consumption generated low metabolic heat. The results show that it must control the pressure gradients to scale-up the process of iturin A production.     

Succinic acid production from wheat using a biorefining strategy

The biosynthesis of succinic acid from wheat flour was investigated in a two-stage bio-process. In the first stage, wheat flour was converted into a generic microbial feedstock either by fungal fermentation alone or by combining fungal fermentation for enzyme and fungal bio-mass production with subsequent flour hydrolysis and fungal autolysis. In the second stage, the generic feedstock was converted into succinic acid by bacterial fermentation by Actinobacillus succinogenes. Direct fermentation of the generic feedstock produced by fungal fermentation alone resulted in a lower succinic acid production, probably due to the low glucose and nitrogen concentrations in the fungal broth filtrate. In the second feedstock production strategy, flour hydrolysis conducted by mixing fungal broth filtrate with wheat flour generated a glucose-rich stream, while the fungal bio-mass was subjected to autolysis for the production of a nutrient-rich stream. The possibility of replacing a commercial semi-defined medium by these two streams was investigated sequentially. A. succinogenes fermentation using only the wheat-derived feedstock resulted in a succinic acid concentration of almost 16 g l^–1 with an overall yield of 0.19 g succinic acid per g wheat flour. These results show that a wheat-based bio-refinery employing coupled fungal fermentation and subsequent flour hydrolysis and fungal autolysis can lead to a bacterial feedstock for the efficient production of succinic acid.     

Pilot-scale production of carboxymethylcellulase from rice hull by Bacillus amyloliquefaciens DL-3

Optimal conditions for pilot-scale production of the carboxymethylcellulase (CMCase) by Bacillus amyloliquefaciens DL-3 were investigated. The best carbon and nitrogen sources for the production of CMCase by B. amyloliquefaciens DL-3 were found to be rice hull and peptone and their optimal concentrations were 5.0 and 0.20% (w/v), respectively. Optimal temperature and initial pH for the production of CMCase were 37°C and 6.8. Optimal agitation speed and aeration rate for the production of CMCase were 300 rpm and 1.0 vvm in a 7 L bioreactor, which were different from those for the cell growth of B. amyloliquefaciens DL-3. The highest productions of CMCase by B. amyloliquefaciens DL-3 from 5.0% (w/v) rice hull as a carbon source under optimal conditions in a 7 or 100 L bioreactor were 220 and 367 U/mL, respectively.