Isolation and Screening of Microorganisms for R-(+)-Limonene and (−)-β-Pinene Biotransformation

This work is focused on the biotransformation of R-(+)-limonene and (?)-β-pinene to bioflavor production. To carry out the present study, 405 microorganisms were tested for their ability to bioconvert the substrates. From the isolated microorganisms, 193 were selected in the prescreening using mineral medium for limonene degradation. At the screening step, eight strains were able to convert R-(+)-limonene and 15 to transform (?)-β-pinene, both in α-terpineol. The highest concentration in α-terpineol from R-(+)-limonene was about 3,450 mg/L for Penicillium sp. isolated from eucalyptus steam. From (?)-β-pinene, the highest product concentration of 675.5 mg/L was achieved using an Aspergillus sp. strain isolated from orange tree stem.     

Microbial Oxidation of (-)-α-pinene to Verbenol Production by Newly Isolated Strains

Verbenol is a bicyclicbicycle secondary allylic alcohol, with pronounced camphor and mint flavor notes, mainly used as food flavoring. This compound is also used to control harmful insects, and hence has potential for using in agriculture, and is an intermediate in the synthesis of valuable perfume and medicinal substances. This work is focused on the microbial oxidation of (-)-α-pinene to verbenol production. To carry out the present study, 405 microorganisms were tested for their ability to bioconvert the substrate. From the isolated microorganisms, 193 were selected in the pre-screening using mineral medium for limonene degradation. At the screening step, 31 strains were able to convert (-)-α-pinene in verbenol. The highest concentration in verbenol from (-)-α-pinene was about 125.6 mg/L for yeast isolated from orange juice industrial residue.     

Screening of Pectinase-Producing Microorganisms with Polygalacturonase Activity

The aim of this work was to perform the screening of microorganisms, previously isolated from samples of agro-industrial waste and belonging to the culture collection of our laboratory, able to produce polygalacturonases (PG). A total of 107 microorganisms, 92 newly isolated and 15 pre-identified, were selected as potential producers of enzymes with PG activity. From these microorganisms, 20 strains were able to synthesize PG with activities above 3 U mL⁻¹. After the kinetic study, the enzyme activity was increased up to 13 times and the microorganism identified as Aspergillus niger ATCC 9642 and the newly isolated W23, W43, and D2 (Penicillium sp.) after 24 h of fermentation led to PG activities of 30, 41, 43, and 45 U mL⁻¹, respectively. The RAPD analysis demonstrated that the selected strains differs genetically, indicating that no duplication of strains among them in the experiments for polygalacturonases production was verified.     

Studies on the genetic variation of the green unicellular alga Haematococcus pluvialis (Chlorophyceae) obtained from different geographical locations using ISSR and RAPD molecular marker

Haematococcus pluvialis (Flotow) is a unicellular green alga, which is considered to be the best astaxanthin-producing organism. Molecular markers are suitable tools for the purpose of finding out genetic variations in organisms; however there have been no studies conducted on ISSR or RAPD molecular markers for this organism. The DNA of 10 different strains of H. pluvialis (four strains from Iran, two strains from Finland, one strain from Switzerland and three strains from the USA) was extracted. A genetic similarity study was carried out using 14 ISSR and 12 RAPD primers. Moreover, the molecular weights of the bands produced ranged from 0.14 to 3.4 Kb. The PCA and dendrogram clustered the H. pluvialis strains into various groups according to their geographical origin. The lowest genetic similarity was between the Iran2 and USA2 strains (0.08) and the highest genetic similarity was between Finland1 and Finland2 (0.64). The maximum numbers of bands produced by the ISSR and RAPD primers were 35 and 6 bands, respectively. The results showed that ISSR and RAPD markers are useful for genetic diversity studies of Haematococcus as they showed geographical discrimination.     

Comparison of three DNA marker systems for assessing genetic diversity in Asian arowana (Scleropages formosus)

Three DNA marker systems -- random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellites -- were used to estimate the genetic diversity in Asian arowana (Scleropages formosus) by genotyping fish individuals from three different sources. Parallel application of the three DNA marker systems allowed us to compare their utility for the analysis of genetic diversity. Microsatellites displayed the highest expected heterozygosity, whereas the values obtained by RAPD and AFLP were much lower. Multiplex ratio and marker index were higher for AFLP than for RAPD or microsatellites. Weak correlation was detected between genetic similarity estimated from data obtained with the three DNA marker systems: estimates from RAPD and AFLP data turned out to be higher than those from microsatellites. On the other hand genetic similarity was higher in the red variety than in the green one, especially when tested with microsatellites. Based on the genetic distance matrices calculated from microsatellite analysis, all red individuals were clustered into one group, whereas only a subset of them was clustered when either RAPD or AFLP was used. This indicated that the microsatellite system detected population subdivision more efficiently than either RAPD or AFLP.     

Pros and cons of analytical methods to quantify surrogate contaminants from the challenge test in recycled polyethylene terephthalate

Different analytical methods were optimized and applied to quantify certain surrogate contaminants (toluene, chlorobenzene, phenol, limonene and benzophenone) in samples of contaminated and recycled flakes and virgin pellets of polyethylene terephthalate (PET) coming from the industrial challenge test. A screening analysis of the PET samples was carried out by direct solid-phase microextraction (SPME) in headspace mode (HS). The methods developed and used for quantitative analysis were a) total dissolution of PET samples in dichloroacetic acid and analysis by HS-SPME coupled to gas chromatography–mass spectrometry (GC–MS) and, b) dichloromethane extraction and analysis by GC–MS. The concentration of all surrogates in the contaminated PET flakes analyzed by HS-SPME method was lower than expected according to information provided by the supplier. Dichloroacetic acid interacted with the surrogates, resulting in a tremendous decrease of limonene concentration. The degradation compounds from limonene were identified. Dichloromethane extraction and GC–MS analysis evidenced the highest values of analytes in these PET samples. Based on the foregoing data, the efficiency of the recycling process was evaluated, whereby the removal of 99.9% of the surrogates proceeding from the contaminated flakes was confirmed.     

Genetic and Phytochemical Analysis of Armillaria mellea by RAPD,ISSR, and HPLC

Abstract

Molecular genetic and phytochemical methods were combined to investigate 17 Chinese strains of Armillaria mellea. Ten random amplified polymorphic DNA (RAPD) primers yielded 106 bands, of which 94 were polymorphic; 80 out of the 84 bands produced by nine inter-simple sequence repeats (ISSR) markers were polymorphic. Contents of water-soluble constituents of mycelia were characterized by high-performance liquid-chromatography–diode array detection (HPLC-DAD) analyses. Cluster analyses of the genetic and phytochemical variation revealed that Armillaria mellea exhibited four chemotypes and four clusters from phytochemical contents. Phylogenetic groupings displayed in tree plots calculated from the RAPD and ISSR data matrix correlated with the geographical origin of the fungi materials. Genetic profiles partially correlated with the chemotypes. Phytochemical contents mainly correlated with the strains. A method based on RAPD and HPLC is described to establish genetic and chemical quality control of Armillaria mellea simultaneously. Ten RAPD primers and a coefficient of >0.95 can be used in authentication of Armillaria mellea. The fingerprints obtained with HPLC consist of 22 common peaks within 45 min. This method could be readily utilized as a quality-control method for pharmaceutical-grade Armillaria mellea.     

Biotransformation of (−)β-pinene by Aspergillus niger ATCC 9642

The main objective of this work was to investigate the biotransformations of (−)α-pinene, (−)β-pinene, and (+) limonene by Aspergillus niger ATCC 9642. The culture conditions involved—concentration of cosolvent (EtOH), substrate applied, and sequential addition of substrates—were investigated. Adaptation of the precultures with small amounts of substrate was also studied. The experiments were performed in conical flasks with liquid cultures. This strain of A. niger was able to convert only (−)β-pinene into α-terpineol. An optimum conversion of (−)β-pinene into α-terpineol of about 4% was obtained when the substrate was applied as a diluted solution in EtOH and sequential addition of substrate was used.     

High throughput screening and QSAR-3D/CoMFA: useful tools to design predictive models of substrate specificity for biocatalysts

After a hierarchical microbial screening process, new microorganisms have been discovered that act as biocatalysts for the stereoselective oxidation of secondary alcohols or for ketone reduction. Oxidation activity is more widespread in yeasts and bacteria, while actinomycetes, filamentous fungi and yeasts present the highest reduction activities. QSAR-3D/CoMFA is an adequate technique to design predictive models of the biocatalysts' activity. In this paper CoMFA models are designed to compare the activities of the biocatalysts selected for the oxidation of alcohols and for the reduction of ketones, starting from the results obtained during the screening process. These models are useful for learning about the activity of these microorganisms and to compare the substrate specificity requirements between alcohol oxidation and ketone reduction biocatalysts.     

AlIII‐Catalysed Formation of Poly(limonene)carbonate: DFT Analysis of the Origin of Stereoregularity

Amino‐triphenolate derived Al^III complexes combined with suitable nucleophiles have been investigated as binary catalysts for the coupling of limonene oxide and carbon dioxide to afford alternating polycarbonates. These catalysts are able to produce stereoregular, perfectly alternating trans‐polymers from cis‐limonene oxide, whereas the pure trans isomer and cis/trans mixture give rise to lower degrees of stereoregularity. The best Al^III catalyst shows the potential to mediate the conversion of both stereoisomers of limonene oxide with high conversion levels of up to 71 % under neat conditions, indicating the high degree of robustness and atom‐efficiency of this catalytic process. Computational studies have revealed unique features of the binary catalyst system, among which is the preferred nucleophilic attack on the quaternary carbon centre in the limonene oxide substrate.     

Essential oils of Retama raetam from Libya: chemical composition and antimicrobial activity

Retama raetam (Forssk) Webb & Berthel is well known in the folk medicine of North and East Mediterranean regions for the treatment of microbial infections. The powdered leaves are used to heal circumcision wounds and used as an antiseptic for wounds, skin rash and pruritus. In this study, to validate this antiseptic property, the chemical composition and antimicrobial activity of the essential oil from the flowers of R. raetam was evaluated. The oil was obtained using hydrodistillation and was analysed by gas chromatography-mass spectrometry. The antibacterial activity was achieved using disc diffusion and broth dilution assay against six bacteria species. Analysis of the essential oil revealed the presence of β-linalool (51%), 2-decen-1-ol (6.6%) and limonene (7.4%) as the major components. The results showed significant activity against microorganisms, especially Staphylococcus aureus, with inhibition zones and minimal inhibitory concentration values in the range of 5.0 mm and 3.0 mg mL?1, respectively. The results on the antibacterial activity provide scientific support for the use of these plants in traditional herbal preparations.     

Chemical composition and antimicrobial activity of the essential oil from Ambrosia trifida L

The essential oil obtained by steam distillation of dried aerial parts of Ambrosia trifida L. from Northeast China was analyzed by GC and GC-MS. The essential oil yield based on dried plant material was 0.12% and thirty-five compounds (corresponding to 86.7% of the total weight) were identified. The main components were: bornyl acetate (15.5%), borneol (8.5%), caryophyllene oxide (8.3%), alpha-pinene (8.0%), germacrene D (6.3%), beta-caryophyllene (4.6%), trans-carveol (2.9%), beta-myrcene (2.6%), camphor (2.4%) and limonene (3.2%). A. trifida essential oil demonstrated bactericidal and fungicidal activity against six bacterial strains and two fungal strains, using the agar diffusion method.     

Detection of Variation in Long-Term Micropropagated Mature Pistachio via DNA-Based Molecular Markers

Determination of genetic stability of in vitro-grown plantlets is needed for safe and large-scale production of mature trees. In this study, genetic variation of long-term micropropagated mature pistachio developed through direct shoot bud regeneration using apical buds (protocol A) and in vitro-derived leaves (protocol B) was assessed via DNA-based molecular markers. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) were employed, and the obtained PIC values from RAPD (0.226), ISSR (0.220), and AFLP (0.241) showed that micropropagation of pistachio for different periods of time resulted in “reasonable polymorphism” among donor plant and its 18 clones. Mantel’s test showed a consistence polymorphism level between marker systems based on similarity matrices. In conclusion, this is the first study on occurrence of genetic variability in long-term micropropagated mature pistachio plantlets. The obtained results clearly indicated that different marker approaches used in this study are reliable for assessing tissue culture-induced variations in long-term cultured pistachio plantlets.     

Highly Valuable Polyunsaturated Fatty Acids from Microalgae: Strategies to Improve Their Yields and Their Potential Exploitation in Aquaculture

Microalgae have a great potential for the production of healthy food and feed supplements. Their ability to convert carbon into high-value compounds and to be cultured in large scale without interfering with crop cultivation makes these photosynthetic microorganisms promising for the sustainable production of lipids. In particular, microalgae represent an alternative source of polyunsaturated fatty acids (PUFAs), whose consumption is related to various health benefits for humans and animals. In recent years, several strategies to improve PUFAs’ production in microalgae have been investigated. Such strategies include selecting the best performing species and strains and the optimization of culturing conditions, with special emphasis on the different cultivation systems and the effect of different abiotic factors on PUFAs’ accumulation in microalgae. Moreover, developments and results obtained through the most modern genetic and metabolic engineering techniques are described, focusing on the strategies that lead to an increased lipid production or an altered PUFAs’ profile. Additionally, we provide an overview of biotechnological applications of PUFAs derived from microalgae as safe and sustainable organisms, such as aquafeed and food ingredients, and of the main techniques (and their related issues) for PUFAs’ extraction and purification from microalgal biomass.     

Cell immobilization and xylitol production using sugarcane bagasse as raw material

Sugarcane bagasse pretreated by three different procedures (with 2% [v/v] polyethyleneimine (PEI), with 2% [w/v] NaOH, or with a sequence of NaOH and PEI) was used as cell immobilization carrier for xylitol production byCandida guilliermondii yeast. Fermentations using these pretreated carriers were performed in semidefined medium and in a hydrolysate medium produced from sugarcane bagasse hemicellulose. Sugarcane bagasse pretreated with NaOH was the best carrier obtained with respect to immobilization efficiency, because it was able to immobilize a major quantity of cells (0.30 g of cells/g of bagasse). Fermentation in semidefined medium using the NaOH-pretreated carrier attained a high efficiency of xylose-to-xylitol bioconversion (96% of the theoretical value). From hydrolysate medium, the bioconversion efficiency was lower (63%), probably owing to the presence of other substances in the medium that caused an inadequate mass transfer to the cells. In this fermentation medium, better results with relation to xylitol production were obtained by using PEI-pretreated carrier (xylose-to-xylitol bioconversion of 81% of the theoretical and volumetric productivity of 0.43 g/[L·h]). The results showed that sugarcane bagasse is a low-cost material with great potential for use as cell immobilization carrier in the fermentative process for xylitol production.     

Application of a process friendly morpholine synthesis to (S,S)-Reboxetine

We report our results on the construction of a morpholine ring system from the corresponding epoxide and amino alcohol. From this study, we were able to convert a previous four-step synthesis into a more efficient two-step process.     

Biotransformation of (R)-(+)- and (S)-(-)-citronellol by Aspergillus sp. and Penicillium sp., and the use of solid-phase microextraction for screening

The biotransformation of (R)-(+)- and (S)-(-)-citronellol by fungi was studied. For screening experiments, solid-phase microextraction (SPME) was used as analytical sampling technique. It was found that sporulated surface cultures of Aspergillus niger were able to convert the substrate into cis- and trans-rose oxides and nerol oxide. The relative contents in the headspace SPME extract of the three bioconversion products cis- and trans-rose oxide and nerol oxide were up to 54, 21 and 12%, respectively. Rose oxide is found in minor amounts in some essential oils, such as Bulgarian rose oil and geranium oil and contributes to its unique odor. It is one of the most important fragrance materials in perfumery in creating rosy notes. Other bioconversion products were 6-methyl-5-hepten-2-one, 6-methyl-5-hepten-2-ol, limonene, terpinolene, linalool and alpha-terpineol. These bioconversion reactions were confirmed by sporulated surface cultures on larger scale and sampling by dynamic headspace sweep and steam distillation solvent extraction. The same conversions were noticed with A. tubingensis and Penicillium roqueforti. This bioconversion was enantioselective since more of the chiral cis- than trans-rose oxide was obtained (cisitrans ratio up to 95/5). Submerged liquid cultures of P. roqueforti yielded two unidentified metabolites after conversion of citronellol (yield up to 5%). The stability and acid-catalyzed conversion of citronellol was also investigated. No chemical oxidation or auto-oxidation products were detected in acidified liquid control broths up to pH 3.5. However, when control tests were run with solid media, acid-catalyzed conversion of the substrate to small amounts of cis- and trans-rose oxides, nerol oxide, linalool and alpha-terpineol was observed at pH 3.5 and when heat treatment (steam distillation solvent extraction) was applied.     

Growth of lignocellulosic-fermenting fungi on different substrates under low oxygenation conditions

Four soil fungi able to grow under low oxygenation conditions were selected and used in studies to determine the production of enzymes that promote the degradation of lignocellulosic materials. The capacity of these fungi to ferment such materials was also investigated. The fungi were grown in sugarcane bagasse and sawdust at final concentrations of 4 and 10%, as the carbon sources. The strains were cultivated under microaerophilic and combined conditions of oxygenation (aerobic followed by microaerophilic conditions). The results obtained with the basidiomycete specie, Trichocladium canadense, Geotrichum sp., and Fusarium sp. suggest that they prefer lower oxygen concentration for growth and enzyme production. Lignocellulolytic activities were detected in all strains but varied with the carbon source used. The highest levels of these activities were produced by the Basidiomycete specie and Fusarium sp. Ethanol and other nongaseous fermentation products were detected following high-performance liquid chromatography analysis using a supelcogel C-610H column, demonstrating the fermentative capability of these strains. In view of their ability to produce enzymes necessary for the breakdown of lignocellulosic materials and to utilize most of the degradation products for growth, these strains have a great potential for biotechnological application.     

Fermentation of Biologically Pretreated Wheat Straw for Ethanol Production: Comparison of Fermentative Microorganisms and Process Configurations

The pretreatment of lignocellulosic biomass with white-rot fungi to produce bioethanol is an environmentally friendly alternative to the commonly used physico-chemical processes. After biological pretreatment, a solid substrate composed of cellulose, hemicellulose and lignin, the two latter with a composition lower than that of the initial substrate, is obtained. In this study, six microorganisms and four process configurations were utilised to ferment a hydrolysate obtained from wheat straw pretreated with the white-rot fungus Irpex lacteus. To enhance total sugars utilisation, five of these microorganisms are able to metabolise, in addition to glucose, most of the pentoses obtained after the hydrolysis of wheat straw by the application of a mixture of hemicellulolytic and cellulolytic enzymes. The highest overall ethanol yield was obtained with the yeast Pachysolen tannophilus. Its application in combination with the best process configuration yielded 163 mg ethanol per gram of raw wheat straw, which was between 23 and 35 % greater than the yields typically obtained with a conventional bioethanol process, in which wheat straw is pretreated using steam explosion and fermented with the yeast Saccharomyces cerevisiae.     

Hydroxy lactones with the gem-dimethylcyclohexane system – Synthesis and antimicrobial activity

Two new lactones comprising the gem-dimethylcyclohexane ring: 2-chloro-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one and 2-bromo-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one as well as the already known 2-iodo-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one, were obtained from (6,6-dimethylcyclohex-2-en-1-yl)acetic acid. These lactones were used as substrates for the screening of biotransformation by whole cells of nine fungal strains (Fusarium species, Syncephalastrum racemosum and Cunninghamella japonica). Some of these microorganisms (mainly Fusarium species) transformed all three lactones during the hydrolytic dehalogenation into 2-hydroxy-5,5-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one. It is worth noting that two microorganisms (Fusarium culmorum and Fusarium scirpi) converted iodolactone with very high enantioselectivity (75.1% and 91.6%, respectively). The (+) isomer of hydroxy lactone was preferred. At the last step the hydroxy lactone obtained during biotransformation was examined for its biological activity against bacteria, yeasts and fungi. It was found that this compound inhibits growth of some tested microorganisms.