Melinda S. Nunnally

Characterization of a novel modified alternan

Abstract A novel modified alternan, produced by using newly isolated strains of Penicillium sp., was physically characterized. High molecular weight native alternan was progressively modified to lower molecular weight heterodisperse forms, associated with a reduction in absorbance at 225 nm, light scattering, and opalescence. Methylation analysis indicated that modified alternan has a linkage pattern similar to that of native alternan. The solution viscosity properties of modified alternan resemble those of ultrasonicated alternan and commercial gum arabic. However, alternan lacks the emulsification capacity of gum arabic. Alternan solutions are stable for at least 7 days under all conditions tested, from 4 to 70 °C and from pH 3–9. Dry preparations of alternan are bright white powders that are not highly hygroscopic. Thus, modified alternan is promising for further development as a gum arabic substitute, particularly in food applications requiring a low-viscosity bulking agent.

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.

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 MICs (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 novel Penicillium spp.

Four strains identified as Penicillium spp. were isolated from soil samples based on their capacity to modify the unique polysaccharide, alternan. Spores from these isolates germinated in medium containing alternan and reduced the apparent molecular weight of alternan as determined by high-performance size exclusion chromatography and viscometry. However, the fungi exhibited limited growth on alternan and did not consume the substrate. The rheological properties of the modified alternan resembled those of commercial gum arabic. Thus, treatment of native alternan with spores from these Penicillium spp. strains constitutes a simple bioconversion method to quantitatively produce novel and potentially useful modified alternan. Journal of Industrial Microbiology & Biotechnology (2002) 29, 177–180 doi:10.1038/sj.jim.7000272

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

Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a β-1,3-linked backbone and β-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 β-glucanase activity were identified as Penicillium simplicissimum, Penicillium crustosum, and Hypocrea nigricans. β-glucanases also showed activity against the similar β-glucans, laminarin and curdlan. By comparison, commercial β-glucanase from Trichoderma longibrachiatum and laminarinase from Trichoderma sp. showed lower specific activities toward schizophyllan than most of the novel isolates. β-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 β-glucanase from P. simplicissimum and P. crustosum demonstrated the presence of multiple active endoglucanase species, including a 20-25 kD enzyme from P. simplicissimum.

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.

Simplified process for preparation of schizophyllan solutions for biomaterial applications.

ABSTRACT 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.

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.