Selection of DNA Aptamers for Root Exudate l-Serine Using Multiple Selection Strategies.

Agricultural biosensing can aid decisions about crop health and maintenance, because crops release root exudates that can inform about their status. l-Serine has been found to be indicative of nitrogen uptake in wheat and canola. The development of a biosensor for l-serine could allow farmers to monitor crop nutrient demands more precisely. The development of robust l-serine-binding DNA aptamers is described. Because small molecules can be challenging targets for Systematic Evolution of Ligands by EXponential enrichment (SELEX), three separate DNA libraries were used for SELEX experiments. A l-homocysteine aptamer was randomized to create a starting library for a l-serine selection (randomized SELEX). The final selection rounds of the l-homocysteine selection were also used as a starting library for l-serine (redirected SELEX). Finally, an original DNA library was used (original SELEX). All three SELEX experiments produced l-serine-binding aptamers with micromolar affinity, with Red.1 aptamer having a Kd of 7.9 ± 3.6 µM. Truncation improved the binding affinity to 5.2 ± 2.7 µM, and from this sequence, a Spiegelmer with improved nuclease resistance was created with a Kd of 2.0 ± 0.8 µM. This l-serine-binding Spiegelmer has the affinity and stability to be incorporated into aptamer-based biosensors for agricultural applications.

Influence of inorganic additives on wheat straw composting: Characterization and structural composition of organic matter derived from the process.

Metallic oxides and clay minerals have gained increasing interest as additives of composting due to their influence in greenhouse gas emissions reduction and their effectivity in the stabilization of carbon both in compost and soils, leading to a cleaner compost production and potentially C sequestrant amendments. In this study, wheat straw (WS) was co-composted with iron oxide and allophanic soil and their influence on WS composting and composition of the end-products was evaluated. WS compost and their humic like-substances (HS) fraction were characterized by chemical and spectroscopic analyzes. After 126 days of process, the elemental composition showed slight differences of the N content for compost and HS, where the C/N atomic ratio tended to decrease relative to the initial material (WS; ~130). This trend was more pronounced in the HS from co-composted treatments (<30). The addition of inorganic materials increased the total acidity and phenolic-OH group contents (~15 and 14 mEq g-1 respectively, iron oxide treatment) relative to the treatment without inorganic additives. Nevertheless, the FTIR and solid-state 13CNMR spectroscopy barely support the wet chemical analysis and revealed a similar final composition between all the studied compost treatments. These results suggest that the incorporation of these materials as compost additives had no major effect on the spectroscopic features of the end-products, however, critical changes of the properties such as the extractability, functionality and composition of HS were revealed by traditional methods. In conclusion, the supply of metal oxides and clays could impact the aerobic composting of WS favorizing the stabilization of certain C pools and adsorptive properties of the end-products, that is of importance in production of amendments suitable for being used in degraded and contaminated soils. Nevertheless, under the experimental conditions of our research C stabilization apparently depends of other mechanisms that still need to be elucidate.

Influence of saprophytic fungi and inorganic additives on enzyme activities and chemical properties of the biodegradation process of wheat straw for the production of organo-mineral amendments.

Cellulose and lignin as main components of crop residues have a significant influence on composting operations and composition of the final products. Both are strongly associated, and lignin can be considered an important barrier during the biodegradation process of lignocellulosic materials. Saprophytic fungi are efficient lignin degraders due to their complex enzymatic system. Therefore, the influence of the inoculation of saprophytic fungi (Coriolopsis rigida, Pleurotus ostreatus, Trichoderma harzianum and Trametes versicolor) and the supply of inorganic additives (Al2O3, Fe2O3 and allophanic soil) that promote the stabilization of carbon (C), were analyzed in the biodegradation of wheat straw (WS). The activity of Laccase (LAC), manganese peroxidase (MnP) and ß-glucosidase and changes in temperature, pH and E4/E6 ratio were analyzed in a biodegradation process of 126 days. The activity of LAC, MnP and the E4/E6 ratio were significantly influenced and increased (enzymes) by fungi species, inorganic additives, and time of inorganic material addition, as well as their interactions (p < 0.05). The WS inoculated with T. versicolor showed the highest average activities for LAC, MnP and ß-glucosidase (2000, 220 UL-1 and 400 µmol pNP g-1 h-1 respectively). Furthermore, the addition of Al2O3 and Fe2O3 increased all the activities regarded to the decomposition of WS and influenced the changes associated with the stabilization of OM in composted WS. In conclusion, the inoculation of WS with T. versicolor in combination with metal oxides improved the enzyme related to the biodegradation process of WS favorizing its stabilization in the medium time, which is of importance in the composting of residues with high C/N ratio.

Chemical characterization of fatty acids, alkanes, n-diols and alkyl esters produced by a mixed culture of Trichoderma koningii and Penicillium janthinellum grown aerobically on undecanoic acid, potatoe dextrose and their mixture.

Little is known about the mixed fungal synthesis of high-value aliphatics derived from the metabolism of simple and complex carbon substrates. Trichoderma koningii and Penicillium janthinellum were fed with undecanoic acid (UDA), potatoe dextrose broth (PDB), and their mixture. Pyrolysis Field Ionization Mass Spectrometry (Py-FIMS) together with (1)H and (13)C Nuclear Magnetic Resonance (NMR) characterized CHCl3 soluble aliphatics in the fungal cell culture. Data from NMR and Py-FIMS analysis were complementary to each other. On average, the mixed fungal species produced mostly fatty acids (28% of total ion intensity, TII) > alkanes (2% of TII) > n-diols (2% of TII) > and alkyl esters (0.8% of TII) when fed with UDA, PDB or UDA+PDB. The cell culture accumulated aliphatics extracellularly, although most of the identified compounds accumulated intracellularly. The mixed fungal culture produced high-value chemicals from the metabolic conversion of simple and complex carbon substrates.

Metabolism of n-C10:0 and n-C11:0 fatty acids by Trichoderma koningii, Penicillium janthinellum and their mixed culture: I. Biomass and CO2 production, and allocation of intracellular lipids.

The capacity of two soil fungi, Trichoderma koningii and Penicillium janthinellum, to oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and store intracellular lipids during growth is unknown. This article reports for the first time the metabolism of decanoic acid (DA, C10:0), undecanoic acid (UDA, n-C11:0), a mixture of the acids (UDA+DA) and a mixture of UDA+ potato dextrose broth (PDB) by T. koningii and P. janthinellum and their mixed culture. A control PDB complex substrate was used as a substrate control treatment. The fungal cultures were assayed for their capacity to: (1) oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and (2) store lipids intracellularly during growth. On all four fatty acid substrates, the mixed T. koningii and P. janthinellum culture produced more biomass and CO2 than the individual fungal cultures. Per 150 mL culture, the mixed species culture grown on UDA+PDB and on PDB alone produced the most biomass (7,567 mg and 11,425 mg, respectively). When grown in DA, the mixed species culture produced the least amount of biomass (6,400 mg), a quantity that was lower than those obtained in UDA (7,550 mg) or UDA+DA (7,270 mg). Amounts of CO2 produced ranged from 210 mg under DA to 618 mg under PDB, and these amounts were highly correlated with biomass (r(2) = 0.99). Fluorescence microscopy of stained lipids in the mixed fungal cell cultures growing during the exponential phase demonstrated larger fungal cells and higher accumulation of lipids in membranes and storage bodies than those observed during the lag and stationary phases. T. koningii and P. janthinellum grown on n-C10:0 and n-C11:0 fatty acids produced lower amounts of biomass and CO2, but stored higher amounts of intracellular lipids, than when grown on PDB alone.

Metabolism of nC11 fatty acid fed to Trichoderma koningii and Penicillium janthinellum II: Production of intracellular and extracellular lipids.

Little is known about the fungal metabolism of nC10 and nC11 fatty acids and their conversion into lipids. A mixed batch culture of soil fungi, T. koningii and P. janthinellum, was grown on undecanoic acid (UDA), a mixture of UDA and potato dextrose broth (UDA+PDB), and PDB alone to examine their metabolic conversion during growth. We quantified seven intracellular and extracellular lipid classes using Iatroscan thin-layer chromatography with flame ionization detection (TLC-FID). Gas chromatography with flame ionization detection (GC-FID) was used to quantify 42 individual fatty acids. Per 150 mL culture, the mixed fungal culture grown on UDA+PDB produced the highest amount of intracellular (531 mg) and extracellular (14.7 mg) lipids during the exponential phase. The content of total intracellular lipids represented 25% of the total biomass-carbon, or 10% of the total biomass dry weight produced. Fatty acids made up the largest class of intracellular lipids (457 mg/150 mL culture) and they were synthesized at a rate of 2.4 mg/h during the exponential phase, and decomposed at a rate of 1.8 mg/h during the stationary phase, when UDA+PDB was the carbon source. Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and vaccenic acid (C18:1) accounted for >80% of the total intracellular fatty acids. During exponential growth on UDA+PDB, hydrocarbons were the largest pool of all extracellular lipids (6.5 mg), and intracellularly they were synthesized at a rate of 64 µg/h. The mixed fungal species culture of T. koningii and P. janthinellum produced many lipids for potential use as industrial feedstocks or bioproducts in biorefineries.

Wheat straw biomass: a resource for high-value chemicals.

Two methods are proposed for increasing the commercial value of wheat straw based on its chemical constituents. The first method involves the determination and extraction of the major organic components of wheat straw, and the second involves those found and extracted in the aqueous and viscous biooils derived from the straw by fast pyrolysis. We used pyrolysis-field ionization mass spectrometry to identify the fine chemicals, which have high commercial values. The most abundant organic compounds in the wheat straw and biooil used as precursors for green chemicals are N-heterocycles (16 to 29% of the Total Ion Intensities, TII) and fatty acids (19 to 26% of TIIs), followed by phenols and lignins (12 to 23% of TIIs). Other important precursors were carbohydrates and amino acids (1 to 8% TIIs), n-alkyl benzenes (3 to 5% of TIIs), and diols (4 to 9% TIIs). Steroids and flavonoids represented 1 to 5% of TIIs in the three materials. Examples of valuable chemical compounds that can be extracted from the wheat straw and biooils are m/z 256, 270, 278, 280, 282 and 284, which are the n-C16 and n-C17 fatty acids respectively, and the C18:3, C18:2 and C18:1 unsaturated fatty acids. In particular, the C18:2 (linoleic acid) is present at a concentration of 1.7% of TIIs. Pyrazole, pyrazine, pyridine, indoles, quinolines, carbazoles, and their identified derivatives are found in relatively high concentrations (1 to 8% of TIIs). Other useful compounds are sterols such as m/z 412 (stigmasterol), m/z 414 (ß-sitosterol), and steroids such m/z 394 (stigmastatriene), m/z 398 (stigmastene) and m/z 410 (stigmastadienone). Relative to the wheat straw, the relative concentration of all flavonoids such as m/z 222 (flavone) and m/z 224 (flavonone) doubled in the biooils. The conversion of wheat straw by fast pyrolysis, followed by chemical characterization with mass spectrometry, and extraction of fine chemicals, opens up new possibilities for increasing the monetary value of crop residues.

Production of a refined biooil derived by fast pyrolysis of chicken manure with chemical and physical characteristics close to those of fossil fuels.

The chemical and physical properties of raw biooils prevent their direct use in combustion engines. We processed raw pyrolytic biooil derived from chicken manure to yield a colorless refined biooil with diesel qualities. Chemical characterization of the refined biooil involved elemental and several spectroscopic analyses. The physical measurements employed were viscosity, density and heat of combustion. The elemental composition (% wt/wt) of the refined biooil was 82.7 % C, 15.3 % H, 0.2 % N and 1.8 % O, no S. Its viscosity was 0.006 Pa.s and a heat of combustion of 43 MJ kg(-1). The refined biooil fraction contains n-alkanes, ranging from n-C(14) to n-C(27), alkenes varying from C(10:1) to C(22:1), and long-chain alcohols. The refined biooil makes a good diesel fuel due to its chemical and physical properties.

Separation and identification of heterocyclic nitrogen compounds in biooil derived by fast pyrolysis of chicken manure.

N-heterocyclics were separated from a biooil, generated by the pyrolysis of chicken manures by column chromatography over neutral alumina and silica, and identified by Pyrolysis Field Ionization Mass Spectrometry (Py-FIMS) and Electrospray Ionization Mass Spectrometry (ESI-MS). Identities of chemical structures, whose presence was indicated by ESI-MS, were confirmed by comparing the Collision-Induced Dissociations (CID's) mass spectra of unknown and standards. The following seven base structures were identified: pyrazine, benzoquinoline, carbazole, phenylpyridine, indole, pyrazole and pyridine. Available hydrogens bonded to ring carbons and nitrogens on the seven N-heterocyclics were increasingly substituted by alkyl groups, mainly methylene groups (m/z 14) to yield mono-, di-, tri- methyl N-heterocyclics. In some instances, longer alkyl chains, such as ethyl, propyl, up to heptyl groups were the substituents.

Phylogenetic analysis of methanotrophic communities in cover soils of a landfill in Ontario.

We examined the methanotrophs in the Trail Road Landfill soils, Ottawa, Ontario, through cultivation-independent molecular assay and the culturing approach. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified methanotroph-specific 16S rDNA gene fragments revealed a more diverse type I (RuMP pathway) methanotrophic community than type II (serine pathway) in 17 soil samples taken along a 50 m transect. The type II methanotrophic community was less diverse, with the dominance of Methylocystis in almost all samples, and clustering with high similarity (85%-88%). Also, the results showed that the C/N ratio of soil organic matter could significantly affect the methanotrophic community structures. The DGGE results were supported by sequence analysis of cloned pmoA. Members of the genera Methylobacter (type I), Methylocaldum (type X), and Methylocystis (type II) appeared to be the dominant methanotrophs. From methanotrophic enrichments, we isolated type I Methylobacter sp., and 3 type II Methylocystis spp.,which appeared to be one of the dominant bacteria species in the soil sample from which isolates were obtained.

Chemical composition of acid-base fractions separated from biooil derived by fast pyrolysis of chicken manure.

Our earlier investigations on the chemical composition of biooils derived by the fast pyrolysis of chicken manure revealed the presence of more than 500 compounds. In order to simplify this heterogeneous and complex chemical system, we produced four biooil fractions namely strongly acidic fraction A, weakly acidic fraction B, basic fraction C and neutral fraction D on the basis of their solubilities in aqueous solutions at different pHs. The yield (wt/wt.%) for fraction A was 3%, for fraction B 21.3%, for fraction C 2.4% and for fraction D 32.4%, respectively. The four fractions were analyzed by elemental analyses, Fourier Transform infrared spectrophotometry (FTIR), (1)H and (13)C nuclear magnetic spectroscopy (NMR), and electrospray ionization mass spectrometry (ESI-MS). The major components of the four fractions were saturated and unsaturated fatty acids, N-heterocyclics, phenols, sterols, diols and alkylbenzenes. The pH separation system produced fractions of enhanced chemical homogeneity.

The conversion of chicken manure to bio-oil by fast pyrolysis. III. Analyses of chicken manure, bio-oils and char by Py-FIMS and Py-FDMS.

Fast pyrolysis of chicken manure produced the following three fractions: bio-oil Fraction I, bio-oil Fraction II, and a char. In a previous investigation we analyzed each of the four materials by curie-point pyrolysis-gas chromatography/mass spectrometry (CpPy-FDMS). The objective of this article is to report on the analyses of the same chicken manure and the three fractions derived from it by fast pyrolysis. We now used pyrolysis-field ionization mass spectrometry (Py-FIMS) to characterize the three fractions. In addition, the two bio-oil materials were analyzed by pyrolysis-field desorption mass spectrometry (Py-FDMS). The use of both Py-FIMS and Py-FDMS produced signals over significantly wider mass ranges than did CpPy-GC/MS, and so allowed us to identify considerably larger numbers of constituents in each material. Individual compounds identified in the mass spectra were classified into the following twelve compound classes: (a) low molecular weight compounds (< m/z 62); (b) carbohydrates; (c) phenols + lignin monomers; (d) lignin dimers; (e) n-alkylbenzenes; (f) N-heterocyclics; (g) n-fatty acids; (h) n-alkanes; (i) alkenes; (j) sterols; (k) n-diols and (l) high molecular weight compounds (> m/z 562). Of special interest were the high abundances of low-molecular weight compounds in the two bio-oils which constituted close to one half of the two bio-oils. Prominent among these compounds were water, ammonia, acetic acid, acetamide, propyl radical, formamide and hydrogen cyanide. The main quantitative differences between the two bio-oils was that bio-oil Fraction I, as analyzed by the two mass spectrometric methods, contained lower concentrations of low-molecular weight compounds, carbohydrates, and N-heterocyclics than bio-oil Fraction II but was richer in lignin dimers, n-alkylbenzenes and aliphatics (n-fatty acids, n-alkanes, alkenes, and n-diols). Of special interest were the N-heterocyclics in the two bio-oils such as pyrazole, pyrazoline, substituted pyrroles, pyridine and substituted pyridines, substituted methoxazole, substituted pyrazines, indole and substituted indoles. Fatty acids in all four materials ranged from n-C(9) to n-C(33), alkanes from n-C(9) to n-C(40), alkenes from C(10:1) to C(40:1) and diols from n-C(7) to n-C(29). The chicken manure, bio-oil Fraction I, and char each contained about 4% sterols with cholesterol, ethylcholestriene, ergosterol, ethylcholestene, ethylcholesterol and beta -sitosterol as major components. Semi-quantitative estimates of the total materials identified by Py-FIMS were: chicken manure: 61.1%; bio-oil Fraction I: 81.3%; bio-oil Fraction II: 78.6%; char: 61.3%; and by Py-FDMS were: bio-oil Fraction I: 65.4%; bio-oil Fraction II: 70.0%.

Adsorption of urease on PE-MCM-41 and its catalytic effect on hydrolysis of urea.

Pore-expanded MCM-41 (PE-MCM-41) silica exhibits a unique combination of high specific surface area (ca. 1000 m(2)/g), pore size (up to 25 nm) and pore volume (up to 3.5 cm(3)/g). As such, this material is highly suitable for the adsorption of large biomolecules. The current study focused primarily on the application of PE-MCM-41 material as suitable host for urease (nickel-based large metalloenzyme) in controlled hydrolysis of urea. Urease adsorbed on PE-MCM-41, regular MCM-41 and silica gel (SGA) were used as catalysts for urea hydrolysis reaction. Adsorption studies of urease on these materials from aqueous solution at pH 7.2 revealed that the adsorption capacity of PE-MCM-41 (102 mg/g) is significantly higher than that of MCM-41 (56 mg/g) and SGA (21 mg/g). The equilibrium adsorption data were well fitted using the Langmuir-Freundlich model. Furthermore, the kinetic study revealed that the uptake of urease follow the pseudo-first order kinetics. The in vitro urea hydrolysis reaction on pristine urease and different urease-loaded catalysts showed that the rate of hydrolysis reaction is significantly slower on U/PE-MCM-41 compared to that of bulk urease and urease on MCM-41 and SGA. This technique could be an alternative means to the use of urease inhibitors to control the ammonia release from urea fertilizer.

The conversion of chicken manure to biooil by fast pyrolysis I. Analyses of chicken manure, biooils and char by 13C and 1H NMR and FTIR spectrophotometry.

Fast pyrolysis of chicken manure produced two biooils (Fractions I and II) and a residual char. All four materials were analyzed by chemical methods, 13C and 1H Nuclear Magnetic Resonance Spectrometry (13C and 1H NMR), and Fourier Transform Infrared Spectrosphotometry (FTIR). The char showed the highest C content and the highest aromaticity. Of the two biooils Fraction II was higher in C, yield and calorific value but lower in N than Fraction I. The S and ash content of the two biooil fractions were low. The Cross Polarization Magic Angle Spinning (CP-MAS) 13C NMR spectrum of the initial chicken manure showed it to be rich in cellulose, which was a major component of sawdust used as bedding material. Nuclear Magnetic Resonance (NMR) spectra of the two biooils indicated that Fraction I was less aromatic than Fraction II. Among the aromatics in the two biooils, we were able to tentatively identify N-heterocyclics like indoles, pyridines, and pyrazines. FTIR spectra were generally in agreement with the NMR data. FTIR spectra of both biooils showed the presence of both primary and secondary amides and primary amines as well as N-heterocyclics such as pyridines, quinolines, and pyrimidines. The FTIR spectrum of the char resembled that of the initial chicken manure except that the concentration of carbohydrates was lower.

The conversion of chicken manure to biooil by fast pyrolysis II. Analysis of chicken manure, biooils, and char by curie-point pyrolysis-gas chromatography/mass spectrometry (Cp Py-GC/MS).

The initial chicken manure and the three fractions derived from it by fast pyrolysis, that is, the two biooils Fractions I and II as well as the residual char were analyzed by Curie-point pyrolysis-gas chromatography/mass spectrometry (Cp Py-GC/MS). The individual compounds identified were grouped into the following six compound classes: (a) N-heterocyclics; (b) substituted furans; (c) phenol and substituted phenols; (d) benzene and substituted benzenes; (e) carbocyclics; and (f) aliphatics. Of special interest were the relatively high concentrations of N-heterocyclics in biooil Fraction II which was obtained in the highest yield and had the highest calorific value. Prominent N-heterocyclics in biooil Fraction II were methyl-and ethyl-substituted pyrroles, pyridines, pyrimidine, pyrazines, and pteridine. Also noteworthy was the high abundance of aliphatics in biooil Fraction I and the char. The alkanes and alkenes in biooil Fraction I ranged from n-C7 to n-C18 and C7:1 to C18:1, respectively, and those in the char from n-C7 to n-C19 and C7:1 to C19:1, respectively. The N-heterocyclics in the two biooil Fractions came from the chicken manure, from proteinaceous materials during fast pyrolysis or were formed during the fast pyrolysis manure conversion by the Maillard reaction which involved the formation of N-heterocyclics by amino acids interacting with sugars.