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.

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.

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.

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.

Novel sources of ß-glucanase for the enzymatic degradation of schizophyllan.

Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a ß-1,3-linked backbone and ß-1,6-linked side chains of single glucose units at every other residue. Schizophyllan is commercially produced for pharmaceutical and cosmetics uses. However, surprisingly little information is available on the biodegradation of schizophyllan. Enzymes that attack schizophyllan could be useful for controlled modifications of the polymer for novel applications. Enrichment cultures were used to isolate 20 novel fungal strains from soil samples, capable of growing on schizophyllan as a sole carbon source. Three additional strains were isolated as contaminants of stored schizophyllan solutions. Strains showing the highest levels of ß-glucanase activity were identified as Penicillium simplicissimum, Penicillium crustosum, and Hypocrea nigricans. ß-glucanases also showed activity against the similar ß-glucans, laminarin and curdlan. By comparison, commercial ß-glucanase from Trichoderma longibrachiatum and laminarinase from Trichoderma sp. showed lower specific activities toward schizophyllan than most of the novel isolates. ß-glucanases from P. simplicissimum and H. nigricans exhibited temperature optima of 60°C and 50°C against schizophyllan, respectively, with broad pH optima around pH 5.0. Partial purifications of ß-glucanase from P. simplicissimum and P. crustosum demonstrated the presence of multiple active endoglucanase species, including a 20-25 kD enzyme from P. simplicissimum.

Utilization of agricultural biomass in the production of the biopolymer schizophyllan.

Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a ß-1,3-linked backbone and ß-1,6-linked side chains of single glucose units at every other residue. Schizophyllan is commercially produced for pharmaceutical and cosmetics uses. However, the unique physical properties of schizophyllan suggest that it may have biomaterials applications. Schizophyllan is conventionally produced by submerged culture fermentation using glucose as a carbon source. This study demonstrates for the first time the efficient utilization of agricultural biomass substrates, particularly distiller's dried grains with solubles, for schizophyllan production. Sugar composition analysis, NMR, and permethylation linkage analysis confirmed that the recovered product was schizophyllan. Schizophyllan produced from agricultural residues was of a high molecular weight and exhibited solution viscosity properties similar to those of commercially produced material. Utilization of biomass substrates could reduce the cost of schizophyllan production and provide a new value-added bioproduct for integrated biorefineries of the future.

Rapid evaluation of the antibiotic susceptibility of fuel ethanol contaminant biofilms.

Bacterial contaminants from commercial fuel ethanol production facilities were previously shown to form biofilms as mixed cultures under laboratory conditions. In this study, a rapid assay was developed to simultaneously compare isolates for their ability to form biofilms as pure cultures. A total of 10 strains were isolated from a dry-grind fuel ethanol plant that routinely doses with virginiamycin. These were identified by sequence analysis as six strains of Lactobacillus fermentum, two strains of L. johnsonii, and one strain each of L. mucosae and L. amylovorus. Isolates exhibited a range of susceptibility to virginiamycin in a planktonic assay, with MIC's (minimum inhibitory concentration) of ≤0.5-16 µg/ml. Even though all strains were isolated from a mixed culture biofilm, they varied greatly in their ability to form biofilms as pure cultures. Surprisingly, growth as biofilms did not appear to provide resistance to virginiamycin, even if biofilms were grown for 144 h prior to antibiotic challenge.

Modification of alternan by dextranase.

Alternan is a unique glucan with a backbone structure of alternating alpha-(1 --> 6) and alpha-(1 --> 3) linkages. Previously, we isolated strains of Penicillium sp. that modify native, high molecular weight alternan in a novel bioconversion process to a lower molecular weight form with solution viscosity properties similar to those of commercial gum arabic. The mechanism of this modification was unknown. Here, we report that these Penicillium sp. strains secrete dextranase during germination on alternan. Furthermore, alternan is modified in vitro by commercial dextranases, and dextranase-modified alternan appears to be identical to bioconversion-modified alternan. This is surprising, since alternan has long been considered to be resistant to dextranase. Results suggest that native alternan may have localized regions of consecutive alpha-(1 --> 6) linkages that serve as substrates for dextranase. Dextranase treatment of native alternan, particularly with GRAS enzymes, may have practical advantages for the production of modified alternan as a gum arabic substitute.

Co-production of schizophyllan and arabinoxylan from corn fiber.

Schizophyllum commune strain ATCC 38548 grew well on a medium containing alkaline H2O2 -pretreated corn fiber as a sole carbon source, and clarified the culture medium within 7 days. The strain preferentially utilized the starch component of corn fiber for growth and production of schizophyllan. Culture supernatants contained approx. 50 mg schizophyllan and 200 mg arabinoxylan per g corn fiber. These polysaccharides were recovered separately by differential precipitation with ethanol.