High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production.

BACKGROUND: Because the model yeast Yarrowia lipolytica can synthesize and store lipids in quantities up to 20 % of its dry weight, it is a promising microorganism for oil production at an industrial scale. Typically, optimization of the lipid production process is performed in the laboratory and later scaled up for industrial production. However, the scale-up process can be complicated by genetic modifications that are optimized for one set of growing conditions can confer a less-than-optimal phenotype in a different environment. To address this issue, small cultivation systems have been developed that mimic the conditions in benchtop bioreactors. In this work, we used one such microbioreactor system, the BioLector, to develop high-throughput fermentation procedures that optimize growth and lipid accumulation in Y. lipolytica. Using this system, we were able to monitor lipid and biomass production in real time throughout the culture duration. RESULTS: The BioLector can monitor the growth of Y. lipolytica in real time by evaluating scattered light; this produced accurate measurements until cultures reached an equivalent of OD600nm = 115 and a cell dry weight of 100 g L(-1). In addition, a lipid-specific fluorescent probe was applied which reliably monitored lipid production up to a concentration of 12 g L(-1). Through screening various growing conditions, we determined that a carbon/nitrogen ratio of 35 was the most efficient for lipid production. Further screening showed that ammonium chloride and glycerol were the most valuable nitrogen and carbon sources, respectively, for growth and lipid production. Moreover, a carbon concentration above 1 M appeared to impair growth and lipid accumulation. Finally, we used these optimized conditions to screen engineered strains of Y. lipolytica with high lipid-accumulation capability. The growth and lipid content of the strains cultivated in the BioLector were compared to those grown in benchtop bioreactors. CONCLUSION: To our knowledge, this is the first time that the BioLector has been used to track lipid production in real time and to monitor the growth of Y. lipolytica. The present study also showed the efficacy of the BioLector in screening growing conditions and engineered strains prior to scale-up. The method described here could be applied to other oleaginous microorganisms.

Mycosubtilin and surfactin are efficient, low ecotoxicity molecules for the biocontrol of lettuce downy mildew.

The use of surfactin and mycosubtilin as an eco-friendly alternative to control lettuce downy mildew caused by the obligate pathogen Bremia lactucae was investigated. Preliminary ecotoxicity evaluations obtained from three different tests revealed the rather low toxicity of these lipopeptides separately or in combination. The EC50 (concentration estimated to cause a 50 % response by the exposed test organisms) was about 100 mg L(-1) in Microtox assays and 6 mg L(-1) in Daphnia magna immobilization tests for mycosubtilin and 125 mg L(-1) and 25 mg L(-1) for surfactin, respectively. The toxicity of the mixture mycosubtilin/surfactin (1:1, w/w) was close to that obtained with mycosubtilin alone. In addition, the very low phytotoxic effect of these lipopeptides has been observed on germination and root growth of garden cress Lepidium sativum L. While a surfactin treatment did not influence the development of B. lactucae on lettuce plantlets, treatment with 100 mg L(-1) of mycosubtilin produced about seven times more healthy plantlets than the control samples, indicating that mycosubtilin strongly reduced the development of B. lactucae. The mixture mycosubtilin/surfactin (50:50 mg L(-1)) gave the same result on B. lactucae development as 100 mg L(-1) of mycosubtilin. The results of ecotoxicity as well as those obtained in biocontrol experiments indicated that the presence of surfactin enhances the biological activities of mycosubtilin. Mycosubtilin and surfactin were thus found to be efficient compounds against lettuce downy mildew, with low toxicity compared to the toxicity values of chemical pesticides. This is the first time that Bacillus lipopeptides have been tested in vivo against an obligate pathogen and that ecotoxic values have been given for surfactin and mycosubtilin.

Characterization of Bacillus velezensis 32a metabolites and their synergistic bioactivity against crown gall disease.

Crown gall disease caused by Agrobacterium tumefaciens is considered to be the main bacterial threat of stone fruit plants in Mediterranean countries. In a previous study, Bacillus velezensis strain 32a was isolated from Tunisian rhizosphere soil and revealed high antagonistic potential against A. tumefaciens strains. In order to better characterize the antagonistic activity of this strain against this important plant pathogen, the production of secondary metabolites was analyzed using liquid chromatography coupled with mass spectrometry. The results revealed the production of different compounds identified as surfactins, fengycins, iturins and bacillibactin belonging to the lipopeptide group, three polyketides (macrolactins, oxydifficidin and bacillaenes), bacilysin and its chlorinated derivative; chlorotetaine. The involvement of lipopeptides in this antagonistic activity was ruled out by performing agar and broth dilution tests with pure molecules. Thus, the construction of B. velezensis 32a mutants defective in polyketides and bacilysin biosynthesis and their antagonistic activity was performed and compared to a set of derivative mutants of a comparable strain, B. velezensis GA1. The defective difficidin mutants (△dfnA and △dfnD) were unable to inhibit the growth of A. tumefaciens, indicating the high-level contribution of difficidin in the antagonism process. While the macrolactin deficient mutant (∆mlnA) slightly decreased the activity, suggesting a synergetic effect with difficidin. Remarkably, the mutant △dhbC only deficient in bacillibactin production showed significant reduction in its capacity to inhibit the growth of Agrobacterium.Taken collectively, our results showed the strong synergetic effect of difficidin and macrolactins and the significant implication of siderophore to manage crown gall disease.

Synthetic Flavonoid BrCl-Flav-An Alternative Solution to Combat ESKAPE Pathogens.

ESKAPE pathogens are considered as global threats to human health. The discovery of new molecules for which these pathogens have not yet developed resistance is a high medical priority. Synthetic flavonoids are good candidates for developing new antimicrobials. Therefore, we report here the potent in vitro antibacterial activity of BrCl-flav, a representative of a new class of synthetic tricyclic flavonoids. Minimum inhibitory/bactericidal concentration, time kill and biofilm formation assays were employed to evaluate the antibacterial potential of BrCl-flav. The mechanism of action was investigated using fluorescence and scanning electron microscopy. A checkerboard assay was used to study the effect of the tested compound in combination with antibiotics. Our results showed that BrCl-flav displayed important inhibitory activity against all tested clinical isolates, with MICs ranging between 0.24 and 125 µg/mL. A total kill effect was recorded after only 1 h of exposing Enterococcus faecium cells to BrCl-flav. Additionally, BrCl-flav displayed important biofilm disruption potential against Acinetobacter baumannii. Those effects were induced by membrane integrity damage. BrCl-flav expressed synergistic activity in combination with penicillin against a MRSA strain. Based on the potent antibacterial activity, low cytotoxicity and pro-inflammatory effect, BrCl-flav has good potential for developing new effective drugs against ESKAPE pathogens.

Assessment of Lipopeptide Mixtures Produced by Bacillus subtilis as Biocontrol Products against Apple Scab (Venturia inaequalis).

Apple scab is an important disease conventionally controlled by chemical fungicides, which should be replaced by more environmentally friendly alternatives. One of these alternatives could be the use of lipopeptides produced by Bacillus subtilis. The objective of this work is to study the action of the three families of lipopeptides and different mixtures of them in vitro and in vivo against Venturia inaequalis. Firstly, the antifungal activity of mycosubtilin/surfactin and fengycin/surfactin mixtures was determined in vitro by measuring the median inhibitory concentration. Then, the best lipopeptide mixture ratio was produced using Design of Experiment (DoE) to optimize the composition of the culture medium. Finally, the lipopeptides mixtures efficiency against V. inaequalis was assessed in orchards as well as the evaluation of the persistence of lipopeptides on apple. In vitro tests show that the use of fengycin or mycosubtilin alone is as effective as a mixture, with the 50-50% fengycin/surfactin mixture being the most effective. Optimization of culture medium for the production of fengycin/surfactin mixture shows that the best composition is glycerol coupled with glutamic acid. Finally, lipopeptides showed in vivo antifungal efficiency against V. inaequalis regardless of the mixture used with a 70% reduction in the incidence of scab for both mixtures (fengycin/surfactin or mycosubtilin/surfactin). The reproducibility of the results over the two trial campaigns was significantly better with the mycosubtilin/surfactin mixture. The use of B. subtilis lipopeptides to control this disease is very promising.

Photoinactivation of Staphylococcus aureus and Vibrio parahaemolyticus in the model aquatic microcosm: effect of light intensity and dissolved biodegradable organic compound.

The impact of light (1,000 × 100,000 lx) on the inactivation of S. aureus and V. parahaemolyticus has been assessed under different concentrations of dissolved biodegradable organic compound (BOC) at pH 7.0. First, a gradual decrease in the number of cultivable cells was observed. Secondly, a cell reactivation was observed and it was marked in the absence of BOC. In the absence of BOC, the lowest value of cell inhibition rate (CIR) during the first 3 h was 0.138 h(-1) for S. aureus and 0.218 h(-1) for V. parahaemolyticus. In the presence of 10,100 and 1,000 mg/l of BOC, it was 0.196 h(-1), 0.243 h(-1) and 0.257 h(-1) for S. aureus respectively, and 0.285 h(-1), 0.306 h(-1) and 0.409 h(-1) for V. parahaemolyticus respectively. The CIRs values of each bacterial species significantly varied (P<0.001) with the changes in BOC concentration. In most cases, no significant difference was noted in the CIRs of both species when they were under the same light intensity and BOC. Nevertheless, it seems important to consider the impact of dissolved BOC during the treatment of bacterial polluted water.

Involvement of cell shape and flagella in the bacterial retention during percolation of contaminated water through soil columns in tropical region.

Microorganisms' retention in soil contributes to the natural purification of groundwater. Bacteria found in groundwater are generally of various shapes. The aim of this study was to assess the importance of cell shape and flagella in bacterial retention during polluted water percolation through two soil columns CA and CB, in the equatorial region in Central Africa. Percolation tests were carried out using different water loads samples which were contaminated by Escherichia coli (straight rods, peritrichous flagella), Vibrio parahaemolyticus (rods bacteria, polar flagella), and Staphylococcus saprophyticus (spherical, free-flagellum). It has been noted that showed that through soil column CA, the mean values of cells retention ratios (T(R)) varied with bacteria species considered, and from one applied water load sample to another. E. coli T(R) and that of S. saprophyticus were not significantly different (P> 0.05) for the two soil columns. V. parahaemolyticus T(R) significantly differed from that of E. coli and S. saprophyticus through soil column CA (P< 0.01) when the highest water load was applied, and through soil column CB (P< 0.05) for each of water load applied. A relative hierarchical arrangement of retained cells based on the T(R) showed that V. parahaemolyticus was less retained through the 2 soil columns. S. saprophyticus in most cases was more retained than others. The physical properties of the bacterial cell must be taken into consideration when evaluating the transfer of bacteriological pollutants towards groundwater.

Agrobacterium tumefaciens C58 presence affects Bacillus velezensis 32a ecological fitness in the tomato rhizosphere.

The persistence of pathogenic Agrobacterium strains as soil-associated saprophytes may cause an inconsistency in the efficacy of the biocontrol inoculants under field condition. The study of the interaction occurring in the rhizosphere between the beneficial and the pathogenic microbes is thus interesting for the development of effective biopesticides for the management of crown gall disease. However, very little is still known about the influence of these complex interactions on the biocontrol determinants of beneficial bacteria, especially Bacillus strains. This study aimed to evaluate the effect of the soil borne pathogen Agrobacterium tumefaciens C58 on root colonization and lipopeptide production by Bacillus velezensis strain 32a during interaction with tomato plants. Results show that the presence of A. tumefaciens C58 positively impacted the root colonization level of the Bacillus strain. However, negative impact on surfactin production was observed in Agrobacterium-treated seedling, compared with control. Further investigation suggests that these modulations are due to a modified tomato root exudate composition during the tripartite interaction. Thus, this work contributes to enhance the knowledge on the impact of interspecies interaction on the ecological fitness of Bacillus cells living in the rhizosphere.

Enhanced growth and ß-galactosidase production on Escherichia coli using oxygen vectors.

The addition of n-dodecane (between 1-3%) to the Escherichia coli fermentation broth in a mechanically agitated and aerated bioreactor revealed improved DO (dissolved oxygen) levels induced during fermentation which lead to an increase in biomass productivity and faster glucose consumption. The maximum values for enzyme activity (increased with 43% compared with the control) and k L a (the volumetric mass transfer coefficient) were obtained for the addition of 2% v/v n-dodecane in the bioreactor, due to the fact that oxygen limitation during the exponential growth phase of the bacterium can repress ß-galactosidase production. The oxygen vector addition increased the available dissolved oxygen and activated a redox-sensitive regulation and an elevated intracellular oxidative metabolism that lead to the enhancement in E. coli biomass accumulation and a more accurate protein folding of ß-galactosidase that would increase its activity. In addition to the experimental analysis, a complex model, developed using an improved version of Bacterial Foraging Algorithm and Artificial Neural Networks, was proposed, with a good average absolute value (6.2% in the training phase and 7.28% in the testing phase) between the process dynamic and the predictions generated by the model.

Bovine hemoglobin: an attractive source of antibacterial peptides.

A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the alpha chains of hemoglobin and 6 peptides were derived from the beta chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration.

Biofilm formation is determinant in tomato rhizosphere colonization by Bacillus velezensis FZB42.

In this work, the behavior in tomato rhizosphere of Bacillus velezensis FZB42 was analyzed taking into account the surfactin production, the use of tomato roots exudate as substrates, and the biofilm formation. B. velezensis FZB42 and B. amyloliquefaciens S499 have a similar capability to colonize tomato rhizosphere. Little difference in this colonization was observed with surfactin non producing B. velezensis FZB42 mutant strains. B. velezensis is able to grow in the presence of root exudate and used preferentially sucrose, maltose, glutamic, and malic acids as carbon sources. A mutant enable to produce exopolysaccharide (EPS-) was constructed to demonstrate the main importance of biofilm formation on rhizosphere colonization. This mutant had completely lost its ability to form biofilm whatever the substrate present in the culture medium and was unable to efficiently colonize tomato rhizosphere.

Lipopeptides produced by Bacillus subtilis as new biocontrol products against fusariosis in ornamental plants.

In this study, we have investigated the effects of three lipopeptides (fengycin, surfactin and mycosubtilin) produced by different strains of Bacillus subtilis against the phytopathogenic fungi Fusarium oxysporum f. sp. iridacearum, which affects the ornamental bulb plant populations of Iris sp. The antifungal effects were tested using minimum inhibitory concentration assay, determination of mycelium growth and spore germination inhibition rates. Also, in vivo tests on infected rhizomes and scanning electron microscopy were employed. Mycosubtilin alone and in combination with fengycin or/and surfactin showed potent inhibitory activity at concentrations as low as 5 µg ml-1 which is 100 times lower compared to Topsin M, a common chemical fungicide frequently used against fusariosis in ornamental plants. An enhancement of mycosubtilin antifungal activity was observed when it was used in combination with surfactin due to a synergistic effect. At a concentration of 20 µg ml-1, mycosubtilin inhibited the growth of the mycelium up to 49% and the spore germination ability up to 26% in comparison to control. In addition, significant changes on the macro- and micro-morphology have been observed. The antifungal activity is related to the inhibition of spore germination and the irreversible damage of the hyphae cell wall. To the best of our knowledge, this is the first attempt to propose the lipopeptides as biopesticides against the fusariosis of ornamental plants.

Biocontrol of the wheat pathogen Zymoseptoria tritici using cyclic lipopeptides from Bacillus subtilis.

Innovation toward ecofriendly plant protection products compatible with sustainable agriculture and healthy food is today strongly encouraged. Here, we assessed the biocontrol activity of three cyclic lipopeptides from Bacillus subtilis (mycosubtilin, M; surfactin, S; fengycin, F) and two mixtures (M + S and M + S + F) on wheat against Zymoseptoria tritici, the main pathogen on this crop. Foliar application of these biomolecules at a 100-mg L-1 concentration on the wheat cultivars Dinosor and Alixan, 2 days before fungal inoculation, provided significant reductions of disease severity. The best protection levels were recorded with the M-containing formulations (up to 82% disease reduction with M + S on Dinosor), while S and F treatments resulted in lower but significant disease reductions. In vitro and in planta investigations revealed that M-based formulations inhibit fungal growth, with half-maximal inhibitory concentrations of 1.4 mg L-1 for both M and M + S and 4.5 mg L-1 for M + S + F, thus revealing that the observed efficacy of these products may rely mainly on antifungal property. By contrast, S and F had no direct activity on the pathogen, hence suggesting that these lipopeptides act on wheat against Z. tritici as resistance inducers rather than as biofungicides. This study highlighted the efficacy of several lipopeptides from B. subtilis to biocontrol Z. tritici through likely distinct and biomolecule-dependent modes of action.

Isolation and characterization of four antibacterial peptides from bovine hemoglobin.

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.

Burkholderia genome mining for nonribosomal peptide synthetases reveals a great potential for novel siderophores and lipopeptides synthesis.

Burkholderia is an important genus encompassing a variety of species, including pathogenic strains as well as strains that promote plant growth. We have carried out a global strategy, which combined two complementary approaches. The first one is genome guided with deep analysis of genome sequences and the second one is assay guided with experiments to support the predictions obtained in silico. This efficient screening for new secondary metabolites, performed on 48 gapless genomes of Burkholderia species, revealed a total of 161 clusters containing nonribosomal peptide synthetases (NRPSs), with the potential to synthesize at least 11 novel products. Most of them are siderophores or lipopeptides, two classes of products with potential application in biocontrol. The strategy led to the identification, for the first time, of the cluster for cepaciachelin biosynthesis in the genome of Burkholderia ambifaria AMMD and a cluster corresponding to a new malleobactin-like siderophore, called phymabactin, was identified in Burkholderia phymatum STM815 genome. In both cases, the siderophore was produced when the strain was grown in iron-limited conditions. Elsewhere, the cluster for the antifungal burkholdin was detected in the genome of B. ambifaria AMMD and also Burkholderia sp. KJ006. Burkholderia pseudomallei strains harbor the genetic potential to produce a novel lipopeptide called burkhomycin, containing a peptidyl moiety of 12 monomers. A mixture of lipopeptides produced by Burkholderia rhizoxinica lowered the surface tension of the supernatant from 70 to 27 mN·m(-1) . The production of nonribosomal secondary metabolites seems related to the three phylogenetic groups obtained from 16S rRNA sequences. Moreover, the genome-mining approach gave new insights into the nonribosomal synthesis exemplified by the identification of dual C/E domains in lipopeptide NRPSs, up to now essentially found in Pseudomonas strains.

New antibacterial peptide derived from bovine hemoglobin.

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.

Characterization of Candida famata Isolated from Poultry Feces for Possible Probiotic Applications.

We studied here the yeast content of poultry feces, collected randomly from a French farm located in the north of the country. Thus, 81 yeast colonies were isolated and clustered into 22 distinct groups using the rep-PCR method. A single colony was taken from each group and identified using biochemical (ID 32C system) and molecular (sequencing of the D1/D2 domain of 26S rDNA and ITS1-5.8-ITS2 rDNA region) methods. Both methods led to the identification of Candida famata species. One isolate of C. famata strains, named strain Y5, was further studied for its cytotoxicity, adhesion, and surface properties, hemolytic activity, and its survival in simulated gastric and intestine environments. The data obtained advocate the probiotic potential of this isolate.

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine.

Non-self-recognition of microorganisms partly relies on the perception of microbe-associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA-regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long-lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP-non-producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.

Kinetic study of the appearance of an anti-bacterial peptide in the course of bovine haemoglobin peptic hydrolysis.

The kinetics of the alpha (1-23) peptide, which is the first anti-bacterial peptide to be isolated from a haemoglobin hydrolysate, was studied in the course of peptic hydrolysis at pH 4.5 and 23 degrees C in an homogeneous-phase system. A one-step reversed-phase HPLC coupled with photodiode array detector method was applied to identify and isolate this anti-bacterial peptide. The kinetics of peptide appearance were investigated in acetate buffer alone and in urea as a haemoglobin-denaturing agent. Two different mechanisms, 'one-by-one' for native haemoglobin hydrolysis and 'zipper' for denatured haemoglobin hydrolysis, were observed. Whatever the haemoglobin state, native or denatured, and whatever the hydrolytic mechanism, one-by-one or zipper, the anti-bacterial alpha (1-23) peptide is a transient peptide. To prepare the alpha (1-23) peptide it is suitable to hydrolyse haemoglobin in the presence of urea at a corrected degree of hydrolysis (DH(c)) of 13.5%. The amount of peptide produced in the presence of urea was twice as high as for the hydrolysis of native haemoglobin. The yields of alpha (1-23) peptide with respect to haemoglobin at the optimal DH(c) values were 55 and 25% respectively.