Cytotoxic and Antibiotic Potential of Secondary Metabolites from the Lichen Umbilicaria muhlenbergii.

OBJECTIVE: Lichens are emerging as a promising natural source of bioactivities of pharmaceutical interest. The present study aims to contribute to the knowledge of the lichen Umbilicaria muhlenbergii as a potential source of pharmaceutically relevant anticancer and antibiotic lichen chemicals. METHODS: The crude acetone extract of U. muhlenbergii exhibited 13.3 µg mL-1 cytotoxic activity (EC50) against breast cancer cells (MCF-7), as compared to a cisplatin positive control with EC50 of 5.8 µg mL-1. The antibiotic activity of the crude extract against a gram-positive Staphylococcus aureus was 22.5 µg mL-1 as MIC. Using silica gel 60 (SG60) column chromatography, the crude extract was then separated into eight fractions, which were further evaluated for their anticancer activities against MCF-7 cells. By means of propidium iodide flow cytometry, two of the eight SG60 fractions were found to cause cell cycle arrest in MCF-7 cells (73.14% of cells) at the G2 phase, which is indicative of apoptosis and inhibition of cellular proliferation. RESULTS: Identification of chemical constituents present in these two SG60 fractions was carried out with Thin-Layer Chromatography (TLC) and a lichen metabolite database (Wintabolites). The two fractions (SG60-5 and SG60-6) were found to contain compounds belonging to the chemical families depsides, depsidones, anthraquinones, and xanthones. DISCUSSION: The SG60-5 and SG60-6 fractions were further fractionated with Sephadex LH-20. Over 15% of the 46 LH-20 fractions obtained from the SG60-5 fraction caused 100% cell death, whereas 32% of the LH-20 fractions derived from SG60 6 fraction reduced cell survival to below 20%. CONCLUSION: This work extends the evaluation of the cytotoxic and antibiotic activities of lichen secondary metabolites to the species U. muhlenbergii. It presents encouraging results of pharmaceutical interest that set up lichens as an effective source of new bioactive natural products. Further investigations are underway to reveal the full biopharmaceutical potential of U. muhlenbergii.

Lichenochemicals: extraction, purification, characterization, and application as potential anticancer agents.

Introduction: To date, over 1,000 lichen secondary metabolites have been identified. Despite their promising cytotoxic properties, the number of literature reports on anticancer evaluation of lichenochemicals is limited. As cancer prevalence among the human population increases, there is growing interest in lichens as a natural source of secondary metabolites for anti-cancer drug discovery and development.Areas covered: The lack of significant progress in lichen anticancer research is due to the low levels of cytotoxic compounds contained in lichens, the technical difficulties associated with their isolation and characterization, and the insufficient understanding of their mechanism of action on different cancer cell lines. In this review, the authors discuss these challenges and provide systematically organized information on the limitations and advantages of commonly used and newly developed methods for lichen exploration and screening of lichen secondary metabolites for their anticancer potential.Expert opinion: Recent research activities have demonstrated that lichen secondary metabolites possess chemotherapeutic properties. A systematic and multidisciplinary approach is required to advance lichen research and improve our understanding of the mechanisms responsible for the potent cytotoxic properties of lichenochemicals. More efforts need to focus on screening and discovery of new lichen-derived compounds with unique anticancer properties.

Sustainable Production of Cellulose-Based Hydrogels with Superb Absorbing Potential in Physiological Saline.

Nowadays, most of the commonly used superabsorbent polymers (SAPs) are derived from synthetic polymers, particularly acrylic acid and its copolymers made with acrylamide. Here, we describe a novel and environmentally friendly aqueous-based process for fabrication of a new, natural, cellulose-based SAP (hydrogel). In this two-step process, cellulose was first reacted with sodium monochloroacetate (MCA) to obtain carboxymethyl cellulose (CMC) and then cross-linked with epichlorohydrin (ECH). In distilled water (d-water), the water retention value (WRV) of the newly fabricated hydrogels reached 725 g d-water/g gel, which is significantly greater than any other commercially available superabsorbent cellulose-based material (WRV of 10-100 g/g) and comparable to the commercial synthetic (polyacrylate) SAP gels (WRV of up to 1000 g/g). In saline water (s-water; 0.9% NaCl), the maximum WRV attained was 118 g s-water/g gel, which exceeds more than 2-fold the WRV of commercial gels (40-50 g/g). Compositional analysis was carried out to determine the amount of carboxyl groups and average molecular mass, and the parameters for hydrogel preparation were optimized. The natural SAP was characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The hydrogels showed good re-swelling properties losing only 5-10% of their capabilities to reabsorb d-water when reused in four consecutive cycles. Because of their superior swelling properties in physiological saline, the new hydrogels can compete with their synthetic counterparts in applications such as high-value hygiene and biomedical products.

A novel, cost-effective and eco-friendly method for preparation of textile fibers from cellulosic pulps.

Although cellulosic fibers are increasingly used in textile products, current methods for production of cellulose-based textiles suffer certain economic and/or environmental drawbacks. We have developed a new, cost-effective and environmentally-friendly (CS2-free) process that overcomes some of the shortcomings of existing technologies. The process is based on a modified method for periodate oxidation of cellulose that is then cross-linked with chitosan and extruded to obtain cellulosic fibers in the form of textile fibers. The produced fibers have low content of aldehyde groups (∼2mmol/g cellulose) and water retention values of 1.5-2.0g/g fibers. The new process makes use of both hardwood and softwood pulps, and offers significant yield advantages over the use of dissolving pulp as a raw material. The mechanical, water absorbency and morphological properties of the new textile fibers and their potential applications are discussed. The potential techno-economic and environmental benefits of the process are summarized.

Biological activities of undescribed North American lichen species.

BACKGROUND: Lichens provide a large array of compounds with the potential for pharmaceutical development. In the present study, extracts from three previously undescribed North American lichen species were examined for antioxidant, antibacterial and anticancer activities. RESULTS: The results from this study demonstrated the following: (i) Acarospora socialis ethanol extract exhibited significant DPPH antioxidant scavenging activities, which were concentration dependent; (ii) acetone and ethyl acetate extracts of Xanthoparmelia mexicana inhibited Gram-positive bacteria but had no effect on Gram-negative bacteria; X. mexicana acetone extract yielded a minimum inhibitory concentration (MIC) of 20.9 µg mL-1 against Staphylococcus aureus, and 41.9 µg mL-1 against Enterococcus faecalis; (iii) acetone extract of Lobothallia alphoplaca inhibited growth of cultured breast cancer MCF-7 cells with an effective concentration (EC50 ) of 87 µg mL-1 ; the MCF-7 cell cycle appears arrested in the G2 phase, whereas the DNA synthesis cell cycle (S) may be inhibited. CONCLUSION: New lichen species that possess strong biological activities have been identified. These lichens comprise secondary metabolites that possess antioxidant, antibacterial and anticancer properties. © 2017 Society of Chemical Industry.

Growth and metabolic profiling of the novel thermophilic bacterium Thermoanaerobacter sp. strain YS13.

A strictly anaerobic, thermophilic bacterium, designated strain YS13, was isolated from a geothermal hot spring. Phylogenetic analysis using the 16S rRNA genes and cpn60 UT genes suggested strain YS13 as a species of Thermoanaerobacter. Using cellobiose or xylose as carbon source, YS13 was able to grow over a wide range of temperatures (45-70 °C), and pHs (pH 5.0-9.0), with optimum growth at 65 °C and pH 7.0. Metabolic profiling on cellobiose, glucose, or xylose in 1191 medium showed that H2, CO2, ethanol, acetate, and lactate were the major metabolites. Lactate was the predominant end product from glucose or cellobiose fermentations, whereas H2 and acetate were the dominant end products from xylose fermentation. The metabolic balance shifted away from ethanol to H2, acetate, and lactate when YS13 was grown on cellobiose as temperatures increased from 45 to 70 °C. When YS13 was grown on xylose, a metabolic shift from lactate to H2, CO2, and acetate was observed in cultures as the temperature of incubation increased from 45 to 65 °C, whereas a shift from ethanol and CO2 to H2, acetate, and lactate was observed in cultures incubated at 70 °C.

Draft Genome Sequence of Thermoanaerobacter sp. Strain YS13, a Novel Thermophilic Bacterium.

Here, we report the draft genome sequence of Thermoanerobacter sp. YS13, isolated from a geothermal hot spring in Yellowstone National Park, which consists of 2,713,030 bp with a mean G+C content of 34.05%. A total of 2,779 genes, including 2,707 protein-coding genes, 12 rRNAs, and 59 tRNAs were identified.

Fungal demethylation of Kraft lignin.

Demethylation of industrial lignin has been for long coveted as a pathway to the production of an abundant natural substitute for fossil-oil derived phenol. In an attempt to possibly identify a novel Kraft lignin-demethylating enzyme, we surveyed a collection of fungi by using selected ion flow tube-mass spectrometry (SIFT-MS). This method readily identifies methanol resulting from lignin demethylation activity. Absidia cylindrospora, and unidentified Cylindrocladium sp. and Aspergillus sp. were shown to metabolize lignin via different pathways, based on the HPLC analysis of lignin fragments. Of these three, Cylindrocladium and Aspergillus were shown to retain most of the lignin intact after 3 weeks in culture, while removing about 40% of the available methoxy groups. Our results demonstrate that after optimization of culture and lignin recovery methods, biological modification of Kraft lignin may be a feasible pathway to obtaining demethylated lignin for further industrial use.

Production of lactic acid using a new homofermentative Enterococcus faecalis isolate.

Lactic acid is an intermediate-volume specialty chemical for a wide range of food and industrial applications such as pharmaceuticals, cosmetics and chemical syntheses. Although lactic acid production has been well documented, improved production parameters that lead to reduced production costs are always of interest in industrial developments. In this study, we describe the production of lactic acid at high concentration, yield and volumetric productivity utilizing a novel homofermentative, facultative anaerobe Enterococcus faecalis CBRD01. The highest concentration of 182 g lactic acid l(-1) was achieved after 38 h of fed-batch fermentation on glucose. The bacterial isolate utilized only 2-13% of carbon for its growth and energy metabolism, while 87-98% of carbon was converted to lactic acid at an overall volumetric productivity of 5 g l(-1) h(-1). At 13 h of fermentation, the volumetric productivity of lactate production reached 10.3 g l(-1) h(-1), which is the highest ever reported for microbial production of lactic acid. The lactic acid produced was of high purity as formation of other metabolites was less than 0.1%. The present investigation demonstrates a new opportunity for enhanced production of lactic acid with potential for reduced purification costs.

Metabolic engineering as a tool for enhanced lactic acid production.

Metabolic engineering is a powerful biotechnological tool that finds, among others, increased use in constructing microbial strains for higher lactic acid productivity, lower costs and reduced pollution. Engineering the metabolic pathways has concentrated on improving the lactic acid fermentation parameters, enhancing the acid tolerance of production organisms and their abilities to utilize a broad range of substrates, including fermentable biomass-derived sugars. Recent efforts have focused on metabolic engineering of lactic acid bacteria as they produce high yields and have a small genome size that facilitates their genetic manipulation. We summarize here the current trends in metabolic engineering techniques and strategies for manipulating lactic acid producing organisms developed to address and overcome major challenges in the lactic acid production process.

Kenaf biomass biodecomposition by basidiomycetes and actinobacteria in submerged fermentation for production of carbohydrates and phenolic compounds.

The efficiency and dynamics of simultaneous kenaf biomass decomposition by basidiomycetous fungi and actinobacteria were investigated. After 8weeks of incubation, up to 34wt.% of the kenaf biomass was degraded, with the combination of fungi and bacteria being the most efficient. Lignin decomposition accounted for ∼20% of the observed biomass reduction, regardless of the culture used. The remaining 80% of biomass degradation was due to carbohydrate based polymers. Major monosaccharides were produced in tangible yields (26-38%) at different times. Glucose, fructose and xylose were then fully consumed by day 25 while some galactose persisted until day 45. Once monosaccharides were depleted, the production of laccase, manganese-dependent peroxidase and lignin peroxidase enzymes, essential for lignin decomposition, was induced. The products of lignin biodecomposition were shown to be water-soluble and characterized by thermal desorption-pyrolysis-gas chromatography.

Draft Genome Sequence of a New Homofermentative, Lactic Acid-Producing Enterococcus faecalis Isolate, CBRD01.

We report here the draft genome sequence of the novel homofermentative Enterococcus faecalis isolate CBRD01, which is capable of high lactic acid productivity and yields, with minimal nutritional requirements. The genome is 2.8 Mbp, with 37% G+C, and contains genes for two lactate dehydrogenase (LDH) enzymes found in related organisms.

Consolidated bioprocessing of untreated switchgrass to hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus DSM 8903.

The abilities of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus DSM 8903 to ferment switchgrass (SWG), microcrystalline cellulose (MCC) and glucose to hydrogen (H2) in one-step were examined. Hydrogen production from glucose reached the theoretical maximum for dark fermentation of 4 mol H2/mol glucose. The H2 yield on MCC and SWG after 6 days of fermentation was 23.2 mmol H2/L or 9.4 mmol H2/g MCC and 14.3 mmol H2/L or 11.2 mmol H2/g SWG, respectively. The rate of H2 formation however was higher on MCC (0.7 mmol/Lh) than SWG (0.1 mmol/Lh). C. saccharolyticus DSM 8903 was able to produce H2 directly from mechanically-comminuted SWG without any physicochemical or biological pretreatment. Combining four processing steps (pretreatment, enzyme production, saccharification and fermentation) into a single biorefinery operation makes C. saccharolyticus DSM 8903 a promising candidate for consolidated bioprocessing (CBP) of lignocellulosic biomass.

Biopharmaceutical potential of lichens.

CONTEXT: Lichens are composite organisms consisting of a symbiotic association of a fungus (the mycobiont) with a photosynthetic partner (the phytobiont), usually either a green alga or cyanobacterium. The morphology, physiology and biochemistry of lichens are very different from those of the isolated fungus and alga in culture. Lichens occur in some of the most extreme environments on the Earth and may be useful to scientists in many commercial applications. OBJECTIVE: Over the past 2 decades, there has been a renewed and growing interest in lichens as a source of novel, pharmacologically active biomolecules. This review summarizes the past and current research and development trends in the characterization and use of lichens and their bioactive compounds in traditional medicine and other biopharmaceutical applications of commercial interest. METHODS: The present review contains 10 illustrations and 188 references compiled from major databases including Science Direct, Chemical Abstracts, PubMed and Directory of Open Access Journals. RESULTS: Lichen morphology, symbiosis, diversity and bioactivities including enzyme inhibitory, antimicrobial, antifungal, antiviral, anticancer, anti-insecticidal and antioxidant actions were reviewed and summarized. Recent progress in lichens and lichen-forming fungi was discussed with emphasis on their potential to accelerate commercialization of lichen-based products. CONCLUSIONS: Lichens are an untapped source of biological activities of industrial importance and their potential is yet to be fully explored and utilized. Lichen-derived bioactive compounds hold great promise for biopharmaceutical applications as antimicrobial, antioxidant and cytotoxic agents and in the development of new formulations or technologies for the benefit of human life.

Tight controlled expression and secretion of Lactobacillus brevis SlpA in Lactococcus lactis.

Prokaryotes commonly present outer cell wall structures composed of a crystalline array of proteinaceous subunits, known as surface layers (S-layers). The ORF encoding the S-layer protein (SlpA) of Lactobacillus brevis was cloned into Lactococcus lactis under the transcriptional control of the xylose-inducible expression system (XIES). SlpA was secreted into the extracellular medium, as determined by immunoblotting, and assays on the kinetics of SlpA production revealed that repression of the system with glucose did not require the depletion of xylose from the medium that allows transitory ORF expression. The successful use of XIES to express S-layer proteins in the versatile and generally recognized as safe species L. lactis opens new possibilities for an efficient production and isolation of SlpA S-layer protein for its various applications in biotechnology and importantly as an antigen-carrying vehicle.

Bioprocessing of agricultural residues to ethanol utilizing a cellulolytic extremophile.

A recently discovered thermophilic isolate, Geobacillus sp. R7, was shown to produce a thermostable cellulase with a high hydrolytic potential when grown on extrusion-pretreated agricultural residues such corn stover and prairie cord grass. At 70°C and 15-20% solids, the thermostable cellulase was able to partially liquefy solid biomass only after 36 h of hydrolysis time. The hydrolytic capabilities of Geobacillus sp. R7 cellulase were comparable to those of a commercial cellulase. Fermentation of the enzymatic hydrolyzates with Saccharomyces cerevisiae ATCC 24860 produced ethanol yields of 0.45-0.50 g ethanol/g glucose with more than 99% glucose utilization. It was further demonstrated that Geobacillus sp. R7 can ferment the lignocellulosic substrates to ethanol in a single step that could facilitate the development of a consolidated bioprocessing as an alternative approach for bioethanol production with outstanding potential for cost reductions.

Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains.

The composition of thermophilic (60 degrees C) mixed cellulose-degrading enrichment culture initiated from compost samples was examined by constructing a 16S rRNA gene clone library and the presence of sequences related to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria were identified. Eight isolates capable of degrading cellulose, carboxymethyl cellulose (CMC), or ponderosa pine sawdust were identified as belonging to the genera Geobacillus, Thermobacillus, Cohnella, and Thermus. A compost isolate WSUCF1 (Geobacillus sp.) was selected based on its higher growth rate and cellulase activity compared to others in liquid minimal medium containing cellulose as a source of carbon and energy. Strain WSUCF1 and a previously isolated thermophilic cellulose-degrading deep gold mine strain DUSELR13 (Bacillus sp.) were examined for their enzyme properties and kinetics. The optimal pH for carboxymethyl cellulase (CMCase) activity was 5.0 for both isolates. The optimum temperatures for CMCase of WSUCFI and DUSELR13 were 70 and 75 degrees C, respectively. For CMC, the DUSELR13 and WSUCF1 CMCases had K(m) values of 3.11 and 1.08mg/ml, respectively. Most remarkably, WSUCF1 and DUSELR13 retained 89% and 78% of the initial CMCase activities, respectively, after incubation at 70 degrees C for 1day. These thermostable enzymes would facilitate development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process to convert lignocellulosic biomass into biofuels.

Effect of cultivation pH and agitation rate on growth and xylanase production by Aspergillus oryzae in spent sulphite liquor.

The effects of cultivation pH and agitation rate on growth and extracellular xylanase production by Aspergillus oryzae NRRL 3485 were investigated in bioreactor cultures using spent sulphite liquor (SSL) and oats spelts xylan as respective carbon substrates. Xylanase production by this fungus was greatly affected by the culture pH, with pH 7.5 resulting in a high extracellular xylanase activity in the SSL-based medium as well as in a complex medium with xylan as carbon substrate. This effect, therefore, was not solely due to growth inhibition at the lower pH values by the acetic acid in the SSL. The xylanase activity in the SSL medium peaked at 199 U ml(-1) at pH 7.5 with a corresponding maximum specific growth rate of 0.39 h(-1). By contrast, the maximum extracellular beta-xylosidase activity pf 0.36 U ml(-1) was recorded at pH 4.0. Three low molecular weight xylanase isozymes were secreted at all pH values within the range of pH 4-8, whereas cellulase activity on both carbon substrates was negligible. Impeller tip velocities within the range of 1.56-3.12 m s(-1) had no marked effect, either on the xylanase activity, or on the maximum volumetric rate of xylanase production. These results also demonstrated that SSL constituted a suitable carbon feedstock as well as inducer for xylanase production in aerobic submerged culture by this strain of A. oryzae.

Xylanase production by fungal strains on spent sulphite liquor.

Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and D: -xylose as carbon substrates. An SSLc-based medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml(-1), respectively; these values were higher than those obtained on xylan or D: -xylose with the same fungal strains. The highest xylanase activity of 322 U ml(-1) was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55 degrees C, respectively. The A. oryzae xylanolytic activity was stable at 50 degrees C over the pH range 4.5-10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20-30% without sacrificing brightness.

Mechanisms prevalent during bioremediation of wastewaters from the pulp and paper industry.

Bioremediation of wastewaters represents an important treatment methodology, especially when examined against the backdrop of ever-stricter legislation that is evolving in order to regulate effluent release into the environment. It has been reported that bioremediation specifically holds promise in solving environmental problems. Crucial questions surrounding the treatment of effluents include: efficiency of the process, economic feasibility, legal requirements, and the mechanisms involved in the remediation process. Of all these issues mentioned, the last requires special attention. This paper investigates these matters and focuses on techniques that are currently employed to determine the efficiency of bioremediation and mechanisms involved therein. The physiological significance of biosorption is also examined, as this subject has not been fully addressed in previous publications.