Inhibition of Erwinia amylovora by Bacillus nakamurai.

A variety of potential inhibitors were tested for the first time for the suppression of Erwinia amylovora, the causal agent of fire blight in apples and pears. Strain variability was evident in susceptibility to inhibitors among five independently isolated virulent strains of E. amylovora. However, most strains were susceptible to culture supernatants from strains of Bacillus spp., and particularly to the recently described species B. nakamurai. Minimal inhibitory concentrations (MICs) were 5-20% (vol/vol) of culture supernatant from B. nakamurai against all five strains of E. amylovora. Although Bacillus species have been previously reported to produce lipopeptide inhibitors of E. amylovora, matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and column chromatography indicated that the inhibitor from B. nakamurai was not a lipopeptide, but rather a novel inhibitor.

Inhibition of Streptococcus mutans and S. sobrinus biofilms by liamocins from Aureobasidium pullulans.

The aim of this study was to determine if the novel anti-streptococcal inhibitors, liamocins, also inhibit biofilm formation by S. mutans and S. sobrinus. S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 were tested for biofilm formation in a rapid microtiter plate (MTP) assay and the effects of added liamocins were determined. This assay measures relative biofilm growth on pin lids. Results were verified in a biofilm flow cell assay, using hydroxyapatite-coated coupons to simulate dental material. Planktonic cultures of S. mutans and S. sobrinus were inhibited by 0.1 mg liamocins/ml. When liamocins were added after the adhesion phase in a rapid microtiter plate assay, S. mutans was inhibited 53% by 5 mg liamocins/ml, while S. sobrinus was more sensitive, showing 100% inhibition at 0.5 mg liamocins/ml. When liamocins were added during the adhesion phase, biofilms of S. mutans showed 78% inhibition at 3.0 mg liamocins/ml. In a biofilm flow cell assay, liamocins added after the adhesion phase at 0.5 mg liamocins/ml inhibited biofilms of S. sobrinus, and appeared to remove biofilms over time. Liamocins were shown for the first time to inhibit biofilm formation by S. mutans and S. sobrinus. Since liamocins are specific for Streptococcus spp., they are potential new inhibitors of oral streptococcal biofilms that should not affect normal oral microflora.

Valorization of egg shell as a detoxifying and buffering agent for efficient polymalic acid production by Aureobasidium pullulans NRRL Y-2311-1 from barley straw hydrolysate.

Stepwise formulation of a versatile and cost-effective medium based on barley straw hydrolysate and egg shell for efficient polymalic acid production by A. pullulans NRRL Y-2311-1 was carried out for the first time. The strain did not grow and produce polymalic acid when dilute acid pretreated barley straw hydrolysate (total fermentable sugars: 94.60 g/L; furfural: 1.01 g/L; hydroxymethylfurfural: 0.55 g/L; acetic acid: 5.06 g/L) was directly used in medium formulation without detoxification (e.g. charcoal pretreatment). When CaCO3 in the medium formulation was substituted with egg shell powder, efficient production of polymalic acid was achieved without a detoxification step. Utilization of 40 g/L of egg shell powder led to 43.54 g polymalic acid production per L with the productivity of 0.30 g/L/h and yield of 0.48 g/g. The bioprocess strategy used in this study can also be utilized for mass production of several other industrially important microbial organic acids and biomaterials.

Inhibition of Lactobacillus biofilm growth in fuel ethanol fermentations by Bacillus.

Commercial fuel ethanol fermentations suffer from microbial contaminants, particularly species of Lactobacillus that may persist as antibiotic-resistant biofilms. In this study, culture supernatants from 54 strains of Bacillus known to produce lipopeptides were tested for inhibition of biofilm formation by Lactobacillus fermentum, L. plantarum, and L. brevis strains previously isolated as biofilm-forming contaminants of a commercial fuel ethanol facility. Eleven Bacillus strains inhibited biofilm formation by all three strains by at least 65% of controls. None of these strains inhibited Saccharomyces cerevisiae. Three strains also significantly inhibited planktonic cultures of Lactobacillus. Culture supernatants from B. nakamurai strain NRRL B-41091 were particularly effective. Inhibition was bacteriostatic rather than bacteriocidal, and appeared to be specific for strains of Lactobacillus. Furthermore, the inhibitor from B. nakamurai was shown to prevent stuck fermentations in a corn mash model fermentation system of S. cerevisiae contaminated with L. fermentum.

Resolving bacterial contamination of fuel ethanol fermentations with beneficial bacteria - An alternative to antibiotic treatment.

Fuel ethanol fermentations are not performed under aseptic conditions and microbial contamination reduces yields and can lead to costly "stuck fermentations". Antibiotics are commonly used to combat contaminants, but these may persist in the distillers grains co-product. Among contaminants, it is known that certain strains of lactic acid bacteria are capable of causing stuck fermentations, while other strains appear to be harmless. However, it was not previously known whether or how these strains interact one with another. In this study, more than 500 harmless strains of lactic acid bacteria were tested in a model system in combination with strains that cause stuck fermentations. Among these harmless strains, a group of beneficial strains was identified that restored ethanol production to near normal levels. Such beneficial strains may serve as an alternative approach to the use of antibiotics in fuel ethanol production.

Inactivation of virginiamycin by Aureobasidium pullulans.

OBJECTIVE: To test the inactivation of the antibiotic, virginiamycin, by laccase-induced culture supernatants of Aureobasidium pullulans. RESULTS: Fourteen strains of A. pullulans from phylogenetic clade 7 were tested for laccase production. Three laccase-producing strains from this group and three previously identified strains from clade 5 were compared for inactivation of virginiamycin. Laccase-induced culture supernatants from clade 7 strains were more effective at inactivation of virginiamycin, particularly at 50 °C. Clade 7 strain NRRL Y-2567 inactivated 6 µg virginiamycin/ml within 24 h. HPLC analyses indicated that virginiamycin was degraded by A. pullulans. CONCLUSIONS: A. pullulans has the potential for the bioremediation of virginiamycin-contaminated materials, such as distiller's dry grains with solubles (DDGS) animal feed produced from corn-based fuel ethanol production.

Reduced-molecular-weight derivatives of frost grape polysaccharide.

A new Type II arabinogalactan was recently described as an abundant gum exudate from stems of wild frost grape (Vitus riparia Michx.). The purpose of the current study is to more thoroughly characterize the physical properties of this frost grape polysaccharide (FGP), and develop methods to modify the molecular weight of FGP for potential new applications. Specifically, native FGP was modified by heat treatment, digestion with the enzyme L-arabinosidase, and ultrasonication. Results showed that native FGP was progressively and irreversibly denatured by heat treatment, while the polymer remained largely resistant to enzymatic digestion. However, ultrasonication reduced the molecular weight of FGP from 1.6×107Da to about 3.0×105Da. Reduced-molecular-weight FGP exhibited modified solution viscosity properties, which could be useful in food and prebiotic applications.

Polyols, not sugars, determine the structural diversity of anti-streptococcal liamocins produced by Aureobasidium pullulans strain NRRL 50380.

Liamocins are polyol lipids produced by the fungus Aureobasidium pullulans, and have selective antibacterial activity against Streptococcus species. Liamocins produced by A. pullulans strain NRRL 50380 on sucrose medium have a d-mannitol head group ester-linked to 3,5-dihydroxydecanoate acyl chains, three or four of which are joined together by 1,5-polyester bonds (liamocins Man-A1 and Man-B1), and similar 3'-O-acetylated analogs (Man-A2 and Man-B2). However, other types of liamocins are produced depending on the choice of strain and growth conditions. In the current study, growth on different polyols, but not sugars, resulted in considerable structural variation, including liamocins with d-galactitol (dulcitol), d-sorbitol (glucitol), d- and l-arabitol, d-xylitol, l-threitol and glycerol head groups. The head groups of liamocins produced on arabitol were shown to be entirely composed of d-arabitol. These liamocin variants were structurally characterized by NMR and MS, and tested for antibacterial activity. The new liamocin variants also had selective activity against Streptococcus. Liamocin structural variants are novel antibacterials against Streptococcus sp. that merit further investigation.

Production of anti-streptococcal liamocins from agricultural biomass by Aureobasidium pullulans.

Liamocins are unique heavier-than-water "oils" produced by certain strains of the fungus Aureobasidium pullulans. Liamocins have antibacterial activity with specificity for Streptococcus sp. Previous studies reported that liamocin yields were highest from strains of A. pullulans belonging to phylogenetic clades 8, 9, and 11, cultured on medium containing sucrose. In this study, 27 strains from these clades were examined for the first time for production of liamocins from agricultural biomass substrates. Liamocin yields were highest from strains in phylogenetic clade 11, and yields were higher from cultures grown on sucrose than from those grown on pretreated wheat straw. However, when supplementary enzymes (cellulase, ß-glucosidase, and xylanase) were added, liamocin production on pretreated wheat straw was equivalent to that on sucrose. Liamocins produced from wheat straw were free of the melanin contamination common in sucrose-grown cultures. Furthermore, MALDI-TOF MS analysis showed that liamocins produced from wheat straw were under-acetylated, resulting in higher proportions of the mannitol A1 and B1 species of liamocin, the latter of which has the highest biological activity against Streptococcus sp.

Bacillus nakamurai sp. nov., a black-pigment-producing strain.

Two isolates of a Gram-stain-positive, strictly aerobic, motile, rod-shaped, endospore-forming bacterium were identified during a survey of the Bacillus diversity of the Agriculture Research Service Culture Collection. These strains were originally isolated from soil and have a phenotype of producing a dark pigment on tryptic soy agar. Phylogenetic analysis of the 16S rRNA gene indicated that these strains were related most closely to Bacillus subtilis subsp. inaquosorum (99.7 % similarity) and Bacillus axarquiensis (99.7 %). In phenotypic characterization, the novel strains were found to grow between 17 and 50 °C and can tolerate up to 9 % (w/v) NaCl. Furthermore, the strains grew in media of pH 5.5-10 (optimal growth at pH 7.0-8.0). The predominant cellular fatty acids were anteiso-C15 : 0 (34.8 %) and iso-C15 : 0 (21.9 %). The cell-wall peptidoglycan contained meso-diaminopimelic acid. A draft genome of both strains was completed. The DNA G+C content was 43.8 mol%. A phylogenomic analysis on the core genome of these two new strains and all members of the Bacillus subtilis group revealed these two strains formed a distinct monophyletic clade with the nearest neighbour Bacillus amyloliquefaciens. DNA-DNA relatedness studies using in silico DNA-DNA hybridizations showed the two strains were conspecific (93.8 %), while values with all other species (<31.5 %) were well below the species threshold of 70 %. Based on the consensus of phylogenetic and phenotypic analyses, these strains are considered to represent a novel species within the genus Bacillus, for which the name Bacillus nakamurai sp. nov. is proposed, with type strain NRRL B-41091T (=CCUG 68786T).

Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase.

OBJECTIVE: The objective was to phylogenetically classify diverse strains of Aureobasidium pullulans and determine their production of feruloyl esterase. RESULTS: Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-ß-1,4-xylanase overproduction were associated with phylogenetic clade 10 and particularly clade 8. Literature strains used for pullulan production all belonged to clade 7. These strains and 36 previously classified strains were tested for feruloyl esterase production, which was found to be associated with phylogenetic clades 4, 11, and particularly clade 8. Clade 8 strains NRRL 58552 and NRRL 62041 produced the highest levels of feruloyl esterase among strains tested. CONCLUSIONS: Production of both xylanase and feruloyl esterase are associated with A. pullulans strains in phylogenetic clade 8, which is thus a promising source of enzymes with potential biotechnological applications.

Simplified process for preparation of schizophyllan solutions for biomaterial applications.

Schizophyllan is a biopolymer commercially produced for pharmaceutical and cosmetics uses. However, schizophyllan also has potential biomaterial applications. Schizophyllan is conventionally produced from glucose and recovered by diafiltration and ultrafiltration to produce a highly purified product. Here we demonstrate a simplified process for preparation of schizophyllan solutions for biomaterial applications. Schizophyllan was produced in 1.5-L bioreactors from distiller's dried grains with solubles (DDGS), an abundant coproduct of dry grind fuel ethanol production. Downstream processing eliminated filtration and concentration steps, providing solutions containing 4.2 ± 0.3 g schizophyllan/L. Solutions contained high-molecular-weight schizophyllan and exhibited viscosity properties similar to those of commercial schizophyllan. Schizophyllan solutions showed promise as a component of biolubricants in friction and wear tests and by dynamic surface and interfacial tension measurements.

Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation.

Bacterial contaminants can inhibit ethanol production in biofuel fermentations, and even result in stuck fermentations. Contaminants may persist in production facilities by forming recalcitrant biofilms. A two-year longitudinal study was conducted of bacterial contaminants from a Midwestern dry grind corn fuel ethanol facility. Among eight sites sampled in the facility, the combined liquefaction stream and yeast propagation tank were consistently contaminated, leading to contamination of early fermentation tanks. Among 768 contaminants isolated, 92% were identified as Lactobacillus sp., with the most abundant species being Lactobacillus plantarum, Lactobacillus casei, Lactobacillus mucosae, and Lactobacillus fermentum. Seven percent of total isolates showed the ability to form biofilms in pure cultures, and 22% showed the capacity to significantly inhibit ethanol production. However, these traits were not correlated. Ethanol inhibition appeared to be related to acetic acid production by contaminants, particularly by obligately heterofermentative species such as L. fermentum and L. mucosae.

Production of novel types of antibacterial liamocins by diverse strains of Aureobasidium pullulans grown on different culture media.

OBJECTIVES: To compare production of antibacterial liamocins (polyol lipids) by diverse strains of Aureobasidium pullulans grown on different culture media. RESULTS: Liamocins produced by strains of A. pullulans have potential agricultural and pharmaceutical applications as antibacterials with specificity against Streptococcus spp. Six strains of A. pullulans were characterized for liamocin production on four different culture media. The choice of strain and culture medium affected growth, liamocin yields, and production of contaminating pigments. Best growth and highest liamocin yields were obtained using A. pullulans strain NRRL 50384 grown on a sucrose basal medium. Unexpectedly, the choice of strain and culture medium also affected the structure of liamocins produced, providing novel types of liamocins. Liamocins varied not only in the ratios of trimer and tetramer polyester tail groups, but also in the nature of the polyol headgroup, which could include mannitol, arabitol, or glycerol. CONCLUSIONS: The ability to conveniently produce novel types of liamocins in good yields will provide novel antibacterials for applied uses, and facilitate structure-function studies on the mechanism of antibacterial activity.

Enzymatic modification of schizophyllan.

An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of ß-endoglucanase with specificity towards schizophyllan. Concentrated enzyme preparations from these strains showed specific activities of 1.7-4.3 U ß-glucanase/mg protein. Using dilutions of these enzymes in time course digestions, schizophyllan was progressively modified to reduced molecular weight species. Glucose and oligosaccharides were found only in the more complete digestions, and thus modified schizophyllan can be produced quantitatively, without loss, to small molecules. Permethylation analysis confirmed that modified schizophyllan retains the fundamental linkage structure of native schizophyllan. Modified schizophyllan species showed progressively reduced viscosity profiles, and all exhibited pseudoplasticity in response to shear thinning.

Inhibitors of biofilm formation by biofuel fermentation contaminants.

Biofuel fermentation contaminants such as Lactobacillus sp. may persist in production facilities by forming recalcitrant biofilms. In this study, biofilm-forming strains of Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus plantarum were isolated and characterized from a dry-grind fuel ethanol plant. A variety of potential biofilm inhibitors were tested, including microbial polysaccharides, commercial enzymes, ferric ammonium citrate, liamocins, phage endolysin, xylitol, and culture supernatants from Bacillus sp. A commercial enzyme mixture (Novozyme 188) and culture supernatants from Bacillus subtilis strains ALT3A and RPT-82412 were identified as the most promising biofilm inhibitors. In biofilm flow cells, these inhibitors reduced the density of viable biofilm cells by 0.8-0.9 log cfu/cm(2). Unlike B. subtilis strain RPT-82412, B. subtilis strain ALT3A and Novozyme 188 did not inhibit planktonic growth of Lactobacillus sp. MALDI-TOF mass spectra showed the production of surfactin-like molecules by both B. subtilis strains, and the coproduction of iturin-like molecules by strain RPT-82412.

Aureobasidium pullulans as a source of liamocins (heavy oils) with anticancer activity.

Liamocins are structurally unique, heavier-than-water "oils" produced by certain strains of Aureobasidium pullulans. The aim of the current study is to identify new sources of liamocins and evaluate their potential as anticancer agents. Nine strains of A. pullulans from phylogenetic clades 8, 9, and 11 were examined for the first time for production of liamocins. Strains in these clades have only been isolated from tropical environments, and all strains tested here were from various locations in Thailand. Strains RSU 9, RSU 21, and RSU 29, all from clade 11, produced from 7.0 to 8.6 g liamocins/l from medium containing 5 % sucrose. These are the highest yields of liamocins that we have found thus far. These strains also produced from 9.4 to 17 g pullulan/l. The structural identity of liamocins was confirmed by matrix-assisted laser desorption/ionization mass spectrometry; differential spectra were obtained in which the dominant ion was either at about m/z 805.5 or m/z 949.6, consistent with the structure of liamocins. Liamocins from A. pullulans strains RSU 9 and RSU 21 inhibited two human breast cancer cell lines and a human cervical cancer cell line (IC50 values of 32.2 ± 1.4 to 63.1 ± 2.4 µg liamocins/ml) but were not toxic to a normal cell line. Liamocins weakly inhibited a strain of Enterococcus faecalis, but did not inhibit strains of Lactobacillus fermentum, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Thus, A. pullulans phylogenetic clade 11 is a promising source of liamocins, and these compounds merit further examination as potential anticancer agents.

Production of schizophyllan from distiller's dried grains with solubles by diverse strains of Schizophyllum commune.

Eleven diverse strains of Schizophyllan commune were examined for production of the biopolymer schizophyllan from agricultural biomass. Strains were grown in malt extract (ME) basal medium containing 1% (w/v) distiller's dried grains with solubles (DDGS), an abundant coproduct of fuel ethanol production by the dry grind process. Ten of 11 strains tested produced more than 2 g schizophyllan/L. Two strains, ATCC 20165 and CBS 266.60, produced more than 10 g schizophyllan/L. Schizophyllan from these strains was similar to commercial product in terms of solution viscosity, molecular weight, and surface tension properties, suggesting that they would be equivalent in biomaterial applications.

Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium tyrobutyricum strain RPT-4213.

A novel Clostridium tyrobutyricum strain RPT-4213 was found producing butyrate under strict anaerobic conditions. This strain produced 9.47 g L(-1) butyric acid from MRS media (0.48 g/g glucose). RPT-4213 was also used to ferment dilute acid pretreated hydrolysates including wheat straw (WSH), corn fiber (CFH), corn stover (CSH), rice hull (RHH), and switchgrass (SGH). Results indicated that 50% WSH with a Clostridia medium (Ct) produced the most butyric acid (8.06 g L(-1), 0.46 g/g glucose), followed by 50% SGH with Ct (6.01 g L(-1), 0.44 g/g glucose), however, 50% CSH Ct showed growth inhibition. RPT-4213 was then used in pH-controlled bioreactor fermentations using 60% WSH and SGH, with a dilute (0.5×) Ct medium, resulting 9.87 g L(-1) butyric acid in WSH (yield 0.44 g/g) and 7.05 g L(-1) butyric acid in SGH (yield 0.42 g/g). The titer and productivity could be improved through process engineering.