Modification of Deoxynivalenol by a Fungal Laccase Paired with Redox Mediator TEMPO.

Mycotoxins such as deoxynivalenol introduce a health risk to the food supply and are costly to manage or avoid. Technologies for reducing or eliminating the toxicity of deoxynivalenol could be useful in a variety of processes, such as in preserving the value as animal feed of byproducts of ethanol production. We characterized transformation products of deoxynivalenol that were formed by the combination of a fungal laccase paired with the chemical mediator 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), using chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Alcohol groups at the C3 and C15 positions of deoxynivalenol were oxidized to ketones, and the chemical mediator became covalently linked to the C4 position. Conditions experienced during gas chromatography led to the dissociation of TEMPO, forming 3,15-diketodeoxynivalenol. Understanding the range of possible modifications to deoxynivalenol and other trichothecenes is a necessary step toward effective remediation of contaminated grain.

Recombinant bacteriophage LysKB317 endolysin mitigates Lactobacillus infection of corn mash fermentations.

BACKGROUND: Commercial ethanol fermentation facilities traditionally rely on antibiotics for bacterial contamination control. Here we demonstrate an alternative approach to treat contamination using a novel peptidoglycan hydrolase (LysKB317) isolated from a bacteriophage, EcoSau. This endolysin was specially selected against Lactobacillus strains that were isolated as contaminants from a fuel ethanol plant. The LysKB317 gene was recombinantly expressed in Escherichia coli as a 33 kDa purified enzyme. RESULTS: In turbidity reduction assays, the recombinant enzyme was subjected to a panel of 32 bacterial strains and was active against 28 bacterial strains representing 1 species of Acetobacter, 8 species of Lactobacillus, 1 species of Pediococcus, 3 species of Streptococcus, and 1 species of Weissella. The activity of LysKB317 was optimal around pH 6, but it has broad activity and stability from pH 4.5-7.5 up to at least 48 h. Maximum activity was observed at 50 °C up to at least 72 h. In addition, LysKB317 was stable in 30% ethanol up to at least 72 h. In experimentally infected corn mash fermentations, 1 µM endolysin reduced bacterial load by 3-log fold change, while 0.01 µM reduced bacteria by 2-log fold change. Concentration of fermentation products (ethanol, residual glucose, lactic acid, and acetic acids) for infected cultures treated with ≥ 0.01 µM LysKB317 was similar to uncontaminated controls. CONCLUSION: Exogenously added LysKB317 endolysin is functional in conditions typically found in fuel ethanol fermentations tanks and may be developed as an alternative to antibiotics for contamination control during fuel ethanol fermentations.

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.

Microbial-derived products as potential new antimicrobials.

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

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.

Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli.

UNLABELLED: A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-ß-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. IMPORTANCE: The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid, plus the generally regarded as safe status of lactobacilli, which therefore have less regulatory concerns, LfFE from the probiotic L. fermentum reported in this work can be directly used for increased production of high-value hydroxycinnamates and ferulic acid from natural or synthetic carbon sources.

Fate of virginiamycin through the fuel ethanol production process.

Antibiotics are frequently used to prevent and treat bacterial contamination of commercial fuel ethanol fermentations, but there is concern that antibiotic residues may persist in the distillers grains coproducts. A study to evaluate the fate of virginiamycin during the ethanol production process was conducted in the pilot plant facilities at the National Corn to Ethanol Research Center, Edwardsville, IL. Three 15,000-liter fermentor runs were performed: one with no antibiotic (F1), one dosed with 2 parts per million (ppm) of a commercial virginiamycin product (F2), and one dosed at 20 ppm of virginiamycin product (F3). Fermentor samples, distillers dried grains with solubles (DDGS), and process intermediates (whole stillage, thin stillage, syrup, and wet cake) were collected from each run and analyzed for virginiamycin M and virginiamycin S using a liquid chromatography-mass spectrometry method. Virginiamycin M was detected in all process intermediates of the F3 run. On a dry-weight basis, virginiamycin M concentrations decreased approximately 97 %, from 41 µg/g in the fermentor to 1.4 µg/g in the DDGS. Using a disc plate bioassay, antibiotic activity was detected in DDGS from both the F2 and F3 runs, with values of 0.69 µg virginiamycin equivalent/g sample and 8.9 µg/g, respectively. No antibiotic activity (<0.6 µg/g) was detected in any of the F1 samples or in the fermentor and process intermediate samples from the F2 run. These results demonstrate that low concentrations of biologically active antibiotic may persist in distillers grains coproducts produced from fermentations treated with virginiamycin.

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.

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates.

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.

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.

Antibacterial activity of a cell wall hydrolase from Lactobacillus paracasei NRRL B-50314 produced by recombinant Bacillus megaterium.

The cell-free supernatant (CFS) from Lactobacillus paracasei NRRL B-50314 culture has been previously reported as containing antibacterial activity against a wide variety of Gram-positive bacteria. The CFS protein gel slice corresponding to antibacterial activities was subjected to trypsin digestion and ion trap MASS (Gel/LC-MS/MS) analysis. BlastP search of the resulted IQAVISIAEQQIGKP sequence led to a hypothetical cell-wall associated hydrolase (designated as CWH here) from Lactobacillus paracasei ATCC 25302. Further analyses of CWH revealed that the IQAVISIAEQQIGKP belongs to a highly conserved region of the NlpC/P60 superfamily. The L. paracasei NRRL B-50314 CWH gene, cloned in pStrepHIS1525CWH477, was introduced into Bacillus megaterium MS 941. The production of CWH477 protein was induced by xylose. The CWH477 protein was purified by using NiNTA column, and elution fraction E2 showed highest antibacterial activity. This study and bioinformatics analyses suggested that the antibacterial activity of CWH could originate from its cell wall degrading enzymatic function.

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.

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.

Lipase production by diverse phylogenetic clades of Aureobasidium pullulans.

Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2-0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains in clade 9, which exhibit a dark olivaceous pigment, produced the highest levels of lipase, with strain NRRL 62034 yielding 0.57 U lipase/ml. By comparison, Candida cylindracea strain NRRL Y-17506 produced 0.05 U lipase/ml under identical conditions. A. pullulans strain NRRL 62034 reached maximal lipase levels in 5 days on lipase induction medium, while A. pullulans strain NRRL Y-2311-1 and strains in clades 4 and 10 were highest after 6 days. A. pullulans strain NRRL Y-2311-1 and strains in clade 9 produced two extracellular proteins in common, at >50 and <37 kDa.

Laccases from Aureobasidium pullulans.

Laccases are polyphenol oxidases (EC 1.10.3.2) that have numerous industrial and bioremediation applications. Laccases are well known as lignin-degrading enzymes, but these enzymes can play numerous other roles in fungi. In this study, 41 strains of the fungus Aureobasidium pullulans were examined for laccase production. Enzymes from A. pullulans were distinct from those from lignin-degrading fungi and associated with pigment production. Laccases from strains in phylogenetic clade 5, which produced a dark vinaceous pigment, exhibited a temperature optimum of 50-60°C and were stable for an hour at 50°C, unlike enzymes from the lignin-degrading fungi Trametes versicolor and Pycnoporus cinnabarinus. Laccase purified from A. pullulans strain NRRL 50381, a representative of clade 5, was glycosylated but had a molecular weight of 60-70kDa after Endo H treatment. Laccase purified from strain NRRL Y-2568, which produced a dark olivaceous pigment, was also glycosylated, but had a molecular weight of greater than 100kDa after Endo H treatment.

Automated UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 and selection for microaerophilic growth and ethanol production at elevated temperature on biomass sugars.

The yeast Kluyveromyces marxianus is a potential microbial catalyst for fuel ethanol production from a wide range of biomass substrates. To improve its growth and ethanol yield at elevated temperature under microaerophilic conditions, K. marxianus NRRL Y-1109 was irradiated with UV-C using automated protocols on a robotic platform for picking and spreading irradiated cultures and for processing the resulting plates. The plates were incubated under anaerobic conditions on xylose or glucose for 5 mo at 46 °C. Two K. marxianus mutant strains (designated 7-1 and 8-1) survived and were isolated from the glucose plates. Both mutant strains, but not wild type, grew aerobically on glucose at 47 °C. All strains grew anaerobically at 46 °C on glucose, galactose, galacturonic acid, and pectin; however, only 7-1 grew anaerobically on xylose at 46 °C. Saccharomyces cerevisiae NRRL Y-2403 did not grow at 46 °C on any of these substrates. With glucose as a carbon source, ethanol yield after 3 d at 46 °C was higher for 8-1 than for wild type (0.51 and 0.43 g ethanol/g glucose, respectively). With galacturonic acid as a carbon source, the ethanol yield after 7 d at 46 °C was higher for 7-1 than for wild type (0.48 and 0.34 g ethanol/g galacturonic acid, respectively). These mutant strains have potential application in fuel ethanol production at elevated temperature from sugar constituents of starch, sucrose, pectin, and cellulosic biomass.

Combinatorial evaluation of laccase-mediator system in the oxidation of veratryl alcohol.

Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both commercially prepared and crude extracts, for their ability to oxidize veratryl alcohol in the presence of various solvents and mediators. Screening revealed complete conversion of veratryl alcohol to veratraldehyde catalyzed by a crude preparation of the laccase from Trametes versicolor ATCC 11235 and the mediator TEMPO in 20 % (v/v) tert-butanol.

The production of glucans via glucansucrases from Lactobacillus satsumensis isolated from a fermented beverage starter culture.

Several starter cultures used in the production of fermented beverages were screened for lactic acid bacteria that produced water-insoluble polysaccharides from sucrose. The strain producing the greatest amount was identified as Lactobacillus satsumensis by its 16S RNA sequence and was deposited in the ARS culture collection as NRRL B-59839. This strain produced at least two α-D-glucans from sucrose. One was a water-soluble dextran, consisting of predominantly α-(1 → 6)-linked D-glucose units, and the other was a water-insoluble glucan containing both α-(1 → 6)-linked and α-(1 → 3)-linked D-glucose units. The culture fluid was found to contain glucansucrases responsible for the two glucans, and no significant level of fructansucrase was detected. Glucansucrase activity was not present in the culture fluid when the bacteria were grown on glucose, fructose, or raffinose as the carbon source. Although the water-soluble glucans produced by cell-free enzyme and by cell suspensions were essentially identical, the same was not true for the water-insoluble glucans. The water-insoluble glucan produced by cell-free culture fluid contained a higher proportion of α-(1 → 3)-linked D-glucose units than the water-insoluble glucan produced by cell suspensions.