Abundant and stable char residues in soils: implications for soil fertility and carbon sequestration.

Large-scale soil application of biochar may enhance soil fertility, increasing crop production for the growing human population, while also sequestering atmospheric carbon. But reaching these beneficial outcomes requires an understanding of the relationships among biochar's structure, stability, and contribution to soil fertility. Using quantitative (13)C nuclear magnetic resonance (NMR) spectroscopy, we show that Terra Preta soils (fertile anthropogenic dark earths in Amazonia that were enriched with char >800 years ago) consist predominantly of char residues composed of ~6 fused aromatic rings substituted by COO(-) groups that significantly increase the soils' cation-exchange capacity and thus the retention of plant nutrients. We also show that highly productive, grassland-derived soils in the U.S. (Mollisols) contain char (generated by presettlement fires) that is structurally comparable to char in the Terra Preta soils and much more abundant than previously thought (~40-50% of organic C). Our findings indicate that these oxidized char residues represent a particularly stable, abundant, and fertility-enhancing form of soil organic matter.

Spatial and temporal gene expression of Fn-type II and cysteine-rich secretory proteins in the reproductive tracts and ejaculated sperm of Chinese Meishan pigs.

Fibronectin type II and cysteine-rich secretory proteins have been well studied in the murine and human. The present study evaluated CRISP1, CRISP2, CRISP3 and Fn-type II (ELSPBP1 and pB1) gene expression patterns in ejaculated sperm and reproductive tracts of Chinese Meishan pigs from birth to day 150 of age. In ejaculated sperm, except for ELSPBP1, all others genes studied were detectable. In sexually mature boars and gilts, CRISP1 gene was expressed strongly in whole epididymides, moderate in prostate and weak in seminal vesicle. CRISP2 gene represented extensive distribution along reproductive tracts with highest abundance in testis. CRISP3 gene was expressed highly in prostate and bulbourethral gland, but weakly in testis. ELSPBP1 gene was expressed with highest abundance in cauda epididymides, moderate in corpus epididymides and weak in seminal vesicle and prostate. pB1 mRNA expression was also abundant along reproductive tracts. During the sexual development of boars after birth, these genes showed different expression patterns. CRISP1 and CRISP3 gene expression was high on day 1 and maintained until day 150, while CRISP2 expression was detectable on day 60 with high abundance and maintained until day 90 and dropped on day 150. ELSPBP1 showed low expression at birth and increased significantly on day 30 (p < 0.05) and then kept static until day 150. pB1 gene displayed moderate expression from birth to day 30 and increased significantly on day 60 (p < 0.05) and maintained at high level until day 150. Collectively, CRISP and Fn-type II genes were expressed extensively along genital tracts, and most of them showed mRNA signal in ejaculated sperm. The expression of CRISP1 and CRISP3 genes in Meishan boar was not age-dependent, while CRISP2 and pB1 gene expression was parallel with sexual development. Their unique gene expression patterns may shed light on the mechanism for the high prolificacy of Meishan pigs.

Chemical structures of corn stover and its residue after dilute acid prehydrolysis and enzymatic hydrolysis: insight into factors limiting enzymatic hydrolysis.

Advanced solid-state NMR techniques and wet chemical analyses were applied to investigate untreated corn stover (UCS) and its residues after dilute acid prehydrolysis (DAP) and enzymatic hydrolysis (RES) to provide evidence for the limitations to the effectiveness of enzyme hydrolysis. Advanced solid-state NMR spectral-editing techniques as well as 1H-13C two-dimensional heteronuclear correlation NMR (2D HETCOR) were employed. Our results indicated that dilute acid prehydrolysis selectively removed amorphous carbohydrates, increased aromatic CH/other protonated -C═C- and enriched alkyl CH and CH2 components. Cinnamic acids were increased, and proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated in RES. 2D HETCOR experiments indicated a close association between lignin and the residual carbohydrates. Ketones/aldehydes were not detected in the DAP, in contrast to a report in which an appreciable amount of ketones/aldehydes was generated from the acid pretreatment of a purified cellulose in the literature. This suggested that acid pretreatment may modify the structure of purified cellulose more than biomass and that biomass may be a better substrate than model biopolymers and compounds for assessing structural changes that occur with industrial processing. On the basis of NMR and wet chemical analyses, we found the following factors could cause the limitations to the effectiveness of enzymatic hydrolysis: (1) chemical modification of carbohydrates limited the biologically degradable carbohydrates available; (2) cinnamic acids in the residue accumulated; (3) accessibility was potentially limited due to the close association of carbohydrates with lignin; and (4) proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated.

A comparative study of oxidation of Cr(III) in aqueous ions, complex ions and insoluble compounds by manganese-bearing mineral (birnessite).

Manganese oxides are considered to be main oxidants resulting in transformation of Cr(III) to Cr(VI) in soils. Oxidation of aqueous Cr(III), Cr(III)-EDTA and insoluble species of Cr(III), such as Cr(OH)3, CrFe(OH)6 and CrPO4, by delta-MnO2 was investigated in batch reaction systems at 25 degrees C and different pH values to predict the potential for Cr(III) oxidation in soil environments. Results indicate that Cr(III) can be rapidly oxidized to Cr(VI) at the beginning of the reaction; however, Mn(II) is produced and fills the adsorption sites on the manganese oxide surface. As a result, produced Mn(II) greatly slows Cr(III) oxidation by delta-MnO(2). Lower pH and higher concentration of manganese oxide markedly enhance the rate and extent of aqueous Cr(III) oxidation. The oxidation of Cr(III)-EDTA by manganese oxide is significantly affected by the chelating time between Cr(III) and EDTA and the molar ratio of EDTA to Cr(III). The formed complex ions of Cr(III)-EDTA are hardly oxidized by manganese oxide and no Cr(VI) was detected in a pH range of 5-6. The rate and extent of oxidation of Cr(OH)3 and CrFe(OH)6 by manganese oxide decrease with pH increasing from 2 to 4. No release of Cr(VI) was observed in the suspension of CrFe(OH)6 and manganese oxide at pH 4 and in the suspension of CrPO4 and manganese oxide at all pH levels tested. The results demonstrate that the order of stability of Cr(III) in these precipitates is CrPO4>CrFe(OH)6>Cr(OH)3 in the presence of manganese oxide.

Fe(III) photocatalytic reduction of Cr(VI) by low-molecular-weight organic acids with alpha-OH.

The photochemical reduction of Cr(VI) by four low-molecular-weight organic acids (tartaric acid, citric acid, malic acid, and n-butyric acid) in the presence of either dissolved Fe(III) in dilute aqueous solution or adsorbed Fe(III) on clay mineral surfaces (kaolinite, montmorillonite and illite) was investigated using batch reactors at a pH range from 3.5 to 4.5 at 25 degrees C. The results indicate that Fe(III) photocatalytic reduction of Cr(VI) by organic acids with alpha-OH is extremely fast. During a reaction period when less than 80% initial Cr(VI) was consumed, the reaction can be described as pseudo-first-order with respect to Cr(VI) when organic acid in excess. By plotting ln[Cr(VI)] as a function of reaction time, rate constants of Cr(VI) reduction by organic acids are obtained. The rate constants involving the four acids are in the order: tartaric acid (with 2 carboxylic groups and 2 alpha-OH groups)>citric acid (with 3 carboxylic groups and 1 alpha-OH group) approximately malic acid (with 2 carboxylic groups and 1 alpha-OH group)>>n-butyric acid (with 1 carboxylic group and no alpha-OH group). This order suggests that the number of alpha-OH but not the number of carboxylic groups is an important determinant of kinetics. With light, the reduction of Cr(VI) by citric acid is accelerated by clay minerals. The enhancement of Cr(VI) reduction is attributed to the catalysis of Fe(III) adsorbed on clay mineral surfaces. However, such an acceleration is markedly suppressed by introducing NaF into the reaction system since NaF forms a complex with Fe(III). It is concluded that the complex formation between Fe(III) and organic acid is a key step for the photocatalytic reduction of Cr(VI) in the presence of Fe(III) and organic acids with alpha-OH.

Effect of magnetic particles on NMR spectra of Murchison meteorite organic matter and a polymer-based model system.

Organic matter from the Murchison meteorite shows pronounced spinning sidebands of the (1)H MAS NMR spectrum and exhibits a large bulk magnetization of 0.75emicro/g extrapolated to 94kOe at 300K. By comparison with data of diamagnetic polystyrene and laponite clay mixed with ferrimagnetic gamma-Fe(2)O(3) nano-particles, we show that the spinning sidebands arise from a combination of dipolar couplings of a given (1)H to magnetic particles, seen in a backscattered-electron image, and to other protons. Signal loss and significant broadening of protonated-carbon peaks in (13)C MAS NMR spectra of polystyrene with Fe(2)O(3) nano-particles is demonstrated, and implications for (13)C NMR spectroscopy of Murchison meteorite are discussed.

Absence of mobile carbohydrate domains in dry humic substances proven by NMR, and implications for organic-contaminant sorption models.

The mobility and domain structure of various standard humic substances have been investigated by 1H and 1H-13C solid-state nuclear magnetic resonance (NMR) experiments. In four dry humic acids, a fulvic acid, a natural organic matter sample, and a whole peat sample, segments that undergo fast, large-amplitude motions account for <9% of the sample. This disproves a previous suggestion, based on 1H NMR data, that flexible domains, presumably carbohydrates, make up >40% of various humic acids; these putative mobile domains were also linked to dual-mode sorption. In particular, neither the polar alkyl (carbohydrate) nor the aromatic components show any fast, large-amplitude mobility. A small fraction of mobile nonpolar alkyl segments identified by us before is the only component undergoing large-amplitude motions, apart from absorbed water that we observe in humic acids exposed to ambient air. 1H-13C wide-line separation NMR shows that, contrary to previous suggestions, the dipolar couplings in the aromatic regions are smaller than in the polar alkyl segments, most likely due to differences in local 1H densities. Series of 1H-13C heteronuclear correlation experiments with 1H spin diffusion reveal close proximity of aromatic and polar alkyl segments in several humic acids, precluding carbohydrate domains on a scale of > 1-nm diameter. In the standard peat humic acid, nonpolar aromatic segments also do not form sorption domains of significant size, while nonpolar aliphatic domains, which we had previously shown to correlate with sorption capacity, have been confirmed.

Methylene spectral editing in solid-state 13C NMR by three-spin coherence selection.

A robust new solid-state nuclear magnetic resonance (NMR) method for selecting CH2 signals in magic-angle spinning (MAS) 13C NMR spectra is presented. Heteronuclear dipolar evolution for a duration of 0.043 ms, under MREV-8 homonuclear proton decoupling, converts 13C magnetization of CH2 groups into two- and three-spin coherences. The CH2 selection in the SIJ (C H H) spin system is based on the three-spin coherence S(x)I(z)J(z), which is distinguished from 13C magnetization (S(x)) by a 1H 0 degrees/90 degrees pulse consisting of two 45 degrees pulses. The two-spin coherences of the type S(y)I(z) are removed by a 13C 90 degrees x-pulse. The three-spin coherence is reconverted into magnetization during the remainder of the rotation period, still under MREV-8 decoupling. The required elimination of 13C chemical-shift precession is achieved by a prefocusing 180 degrees pulse bracketed by two rotation periods. The selection of the desired three-spin coherence has an efficiency of 13% theoretically and of 8% experimentally relative to the standard CP/MAS spectrum. However, long-range couplings also produce some three-spin coherences of methine (CH) carbons. Therefore, the length of the 13C pulse flipping the two-spin coherences is increased by 12% to slightly invert the CH signals arising from two-spin coherences and thus cancel the signal from long-range three-spin coherences. The signal intensity in this cleaner spectrum is 6% relative to the regular CP/TOSS spectrum. The only residual signal is from methyl groups, which are suppressed at least sixfold relative to the CH2 peaks. The experiment is demonstrated on cholesteryl acetate and applied to two humic acids.

Accurate quantification of aromaticity and nonprotonated aromatic carbon fraction in natural organic matter by 13C solid-state nuclear magnetic resonance.

An improved approach for accurately determining the aromatic carbon fraction (fa) and nonprotonated aromatic carbon fraction (faN) in natural organic matter by solid-state 13C NMR is described. Quantitative peak areas are obtained from direct polarization 13C nuclear magnetic resonance (NMR) under high-speed magic angle spinning (MAS). The problem of overlap between aromatic and alkyl carbon resonances around 90-120 ppm in 13C NMR spectra is solved by a 13C chemical shift anisotropy (CSA) filter technique. After correction for residual spinning sidebands, an accurate value of the aromaticity fa is obtained. To obtain a quantitative faN fraction, dipolar dephasing was adapted for high-speed MAS 13C NMR; the separation of the signals of nonprotonated alkyl and aromatic carbons was achieved by CSA filtering plus dipolar dephasing. The method is demonstrated on a peat humic acid, yielding fa = 45 +/- 2% and faN = (0.64 +/- 0.07) x 45%.

Separation of aromatic-carbon 13C NMR signals from di-oxygenated alkyl bands by a chemical-shift-anisotropy filter.

Selection of alkyl-carbon and suppression of aromatic-carbon 13C nuclear magnetic resonance (NMR) signals has been achieved by exploiting the symmetry-based, systematic difference in their 13C chemical-shift anisotropies (CSAs). Simple three- or five-pulse CSA-recoupling sequences with "gamma-integral" cleanly suppress the signals of all sp2- and sp-hybridized carbons. The chemical-shift-anisotropy-based dephasing is particularly useful for distinguishing the signals of di-oxygenated alkyl (O-C-O) carbons, found for instance as anomeric carbons in carbohydrates, from bands of aromatic carbons with similar 13C isotropic chemical shifts. The alkyl signals are detected with an efficiency of > 60%, with little differential dephasing. Combined with C-H dipolar dephasing, the CSA filter can identify ketal (unprotonated O-C-O) carbons unambiguously for the first time. Conversely, after short cross polarization and the CSA filter, O-CH-O (acetal) carbon signals are observed selectively. The methods are demonstrated on various model compounds and applied to a humic acid.

Nitrogen-bonded aromatics in soil organic matter and their implications for a yield decline in intensive rice cropping.

Previous research has shown that long-term intensive cropping of irrigated lowland rice has led to significant grain-yield declines in field trials. The yield decline was attributed to decreased availability of soil nitrogen, which is held mostly in the soil organic matter. By advanced solid-state NMR spectroscopy, we have detected significant amounts of amide nitrogen directly bonded to aromatic rings in a humic acid fraction extracted from a continually submerged, triple-cropped rice soil. Because nitrogen bonded to aromatics is not readily plant-available, this observation can explain the yield decline. Quantitative (13)C NMR combined with advanced spectral editing showed that this humic acid is rich in lignin derivatives (>45% of all carbon), whereas the corresponding humic acid fraction extracted from an aerobic, single-cropped rice soil contains less lignin and less nitrogen bonded to aromatics.

Nuclear magnetic resonance and diffuse-reflectance infrared Fourier transform spectroscopy of biosolids-derived biocolloidal organic matter.

We extracted the acid-soluble portion of municipal biosolids, fractionated it by both molecular weight (MW) and hydrophobicity, and used various solid-state nuclear magnetic resonance (NMR) methods and diffuse-reflectance infrared Fourier transform (DRIFT) spectroscopy to characterize the fractions. Spectroscopic characterization of the MW components of the biosolids-derived organic matter fractions revealed the presence of functionally distinct groups of compounds. Quantitative 13C NMR, CH spectral editing, and several two-dimensional NMR experiments show that the high-MW hydrophilic fraction in particular is structurally simple, consisting predominantly of N-acetylated polysaccharides, perhaps derived from bacterial peptidoglycans. In the high-MW hydrophobic fraction, aromatic compounds were present in addition to the N-acetylated polysaccharides. Infrared spectroscopy confirmed that hydrophilic fractions were dominated by carbohydrates and indicated that the lower-MW fractions lacked amide moieties. Complementary interpretations of the DRIFT and NMR spectra improved our knowledge of the components separated by this fractionation scheme, allowing better characterization of biosolids organic matter. Moreover, fractionation based on both MW and hydrophobicity may prove useful in detailed characterization of the structure of biosolids-derived organic matter and other similarly heterogeneous natural organic matter in soils and sediments.

Recoupled long-range C-H dipolar dephasing in solid-state NMR, and its use for spectral selection of fused aromatic rings.

This work introduces a simple new solid-state 13C NMR method for distinguishing various types of aromatic residues, e.g. those of lignin from fused rings of charcoal. It is based on long-range dipolar dephasing, which is achieved by recoupling of long-range C-H dipolar interactions, using two 1H 180 degrees pulses per rotation period. This speeds up dephasing of unprotonated carbon signals approximately threefold compared to standard dipolar dephasing without recoupling and thus provides much more efficient differential dephasing. It also reduces the effects of spinning-speed dependent effective proton-proton dipolar couplings on the heteronuclear dephasing. Signals of unprotonated carbons with two or more protons at a two-bond distance dephase to <3% within less than 0.9 ms, significantly faster than those of aromatic sites separated from the nearest proton by three or more bonds. Differential dephasing among different unprotonated carbons is demonstrated in a substituted anthraquinone and 3-methoxy benzamide. The data yield a calibration curve for converting the dephasing rates into estimated distances from the carbon to the nearest protons. This can be used for peak assignment in heavily substituted or fused aromatic molecules. Compared to lignin, slow dephasing is observed for the aromatic carbons in wood charcoal, and even slower for inorganic carbonate. Direct 13C polarization is used on these structurally complex samples to prevent loss of the signals of interest, which by design originate from carbons that are distant from protons and therefore crosspolarize poorly. In natural organic matter such as humic acids, this combination of recoupled dipolar dephasing and direct polarization at 7-kHz MAS enables selective observation of signals from fused rings that are characteristic of charcoal.

Efficient CH-group selection and identification in 13C solid-state NMR by dipolar DEPT and 1H chemical-shift filtering.

A new spectral-editing technique for solid-state nuclear magnetic resonance (NMR), based principally on the different dipolar-dephasing properties of CH and CH(2) multiple-quantum (MQ) coherence, yields pure C-H spectra with overall efficiencies of up to 14%. The selection is based on dephasing of methylene heteronuclear MQ coherence by the second proton and can be considered essentially as a solid-state, slow-magic-angle-spinning version of the distortionless enhancement by polarization transfer (DEPT) experiment. A short dipolar transfer and inverse gated decoupling suppress quaternary-carbon resonances, and T(1)-filtering reduces methyl signals. Applications to amorphous polymers with long, flexible sidegroups demonstrate excellent suppression of the signals of partially mobile methylene groups, consistent with simulations and superior to existing methods. CH selection in various model compounds and a humic acid confirms the robust nature and good sensitivity of the technique. Distinction of NCH and CCH groups, which have overlapping (13)C chemical-shift ranges, is achieved by combining dipolar DEPT with (1)H isotropic-chemical-shift filtering. In the humic acid, this permits unequivocal assignment of the methine resonance near 53 ppm to NCH groups.

Selective dephasing of OH and NH proton magnetization based on (1)H chemical-shift anisotropy recoupling.

A method for selectively suppressing the signals of OH and NH protons in (1)H combined rotation and multiple-pulse spectroscopy (CRAMPS) and in (1)H-(13)C heteronuclear correlation (HETCOR) solid-state NMR spectra is presented. It permits distinction of overlapping CH and OH/NH proton signals, based on the selective dephasing of the magnetization of OH and NH protons by their relatively large (1)H chemical-shift anisotropies. For NH protons, the (14)N-(1)H dipolar coupling also contributes significantly to this dephasing. The dephasing is achieved by a new combination of heteronuclear recoupling of these anisotropies with (1)H homonuclear dipolar decoupling. Since the 180 degrees pulses traditionally used for heteronuclear dipolar and chemical-shift anisotropy recoupling would result in undesirable homonuclear dephasing of proton magnetization, instead the necessary inversion of the chemical-shift Hamiltonian every half rotation period is achieved by inverting the phases of all the pulses in the HW8 multiple-pulse sequence. In the HETCOR experiments, carefully timed (13)C 180 degrees pulses remove the strong dipolar coupling to the nearby (13)C spin. The suppression of NH and OH peaks is demonstrated on crystalline model compounds. The technique in combination with HETCOR NMR is applied to identify the CONH and NH-CH groups in chitin and to distinguish NH and aromatic proton peaks in a peat humin.

A robust technique for two-dimensional separation of undistorted chemical-shift anisotropy powder patterns in magic-angle-spinning NMR.

A robust magic-angle-spinning experiment for separating undistorted, quasi-static chemical-shift powder patterns is presented. It is derived from the technique of R. Tycko, G. Dabbagh, and P. Mirau (1989, J. Magn. Reson. 85, 265), but uses 360 degrees instead of 180 degrees pulses. In combination with a suitable phase sequence, this makes the spectral patterns very insensitive to pulse-length errors and other imperfections, as demonstrated both experimentally and theoretically. This method, termed separation of undistorted powder patterns by effortless recoupling (SUPER), can be used at standard spinning speeds, between 2.5 and 5 kHz, and with standard radiofrequency power levels (in particular, for protons, a decoupling field strength gammaB(1)/2pi of less than 80 kHz). No significant artifacts are observed even for samples extending to the ends of the radiofrequency coil. The method has been applied to samples containing various sp(2)- and sp(3)-hybridized carbon sites. Even for the methylene groups in polyethylene, a system with very strong C-H and H-H dipolar couplings and only moderate chemical-shift anisotropy (CSA), a useful CSA powder pattern has been obtained. With a CSA scaling factor of 0.155, accuracies of +/-5, +/-3, and +/-1 ppm of the principal values can be achieved for protonated aromatic carbons, unprotonated sp(2)-hybridized groups, and aliphatic sites, respectively. Examples of CSA-based assignment of COOC vs other COO or CON groups, and of aromatic vs olefinic C=C carbons are shown, for both small molecules and polymers.

Correlation of poly(methylene)-rich amorphous aliphatic domains in humic substances with sorption of a nonpolar organic contaminant phenanthrene.

The structural makeup of natural organic matter plays a major role in regulating its capacity to retain nonionic hydrophobic organic compounds (HOCs). We used a model HOC--phenanthrene--to investigate the correlations between sorption capacity, specifically the modified Freundlich coefficient (K'f), and compositional data of humic acids, humins, and a peat obtained from quantitative 13C solid-state NMR spectroscopy. A positive correlation between K'f and the weight fraction of amorphous poly(methylene) in the sorbents was observed. In contrast, the correlation between phenanthrene sorption capacity and aromaticity or polarity indices of the sorbents was insignificant. The nonpolar aliphatic carbon fraction, excluding poly(methylene), was only partially correlated with K'f. Detailed NMR analyses of the sorbents using 1H inversion-recovery experiments showed that 10-nm diameter domains of branched nonpolar aliphatic groups, which account for 20-50% of all nonpolar aliphatic segments and may be associated with the poly(methylene), were responsible for the partial correlation. The correlation between K'f and the amorphous nonpolar aliphatic domains including amorphous poly(methylene) was strong. The rubbery, relatively low-density, and amorphous nonpolar aliphatic domains can be expected to offer an excellent environment for the sorption of phenanthrene by partitioning. These observations suggest that the domains of amorphous poly(methylene) and branched nonpolar aliphatics, which make up 2-9 wt % of the organic fraction in our samples, may serve as good descriptors for the potential of natural organic matter to retain HOCs in the natural environments.

New structural information on a humic acid from two-dimensional 1H-13C correlation solid-state nuclear magnetic resonance.

New information on the chemical structure of a peat humic acid has been obtained using a series of two-dimensional 1H-13C heteronuclear correlation solid-state NMR (HETCOR) experiments with different contact times and with spectral editing by dipolar dephasing and 13C transverse relaxation filtering. Carbon-bonded methyl groups (C-CH3) are found to be near both aliphatic and O-alkyl but not aromatic groups. The spectra prove that most OCH3 groups are connected directly with the aromatic rings, as is typical in lignin. As a result, about one-third of the aromatic C-O groups is not phenolic C-OH but C-OCH3. Both protonated and unprotonated anomeric O-C-O carbons are identified in the one- and two-dimensional spectra. COO groups are found predominantly in OCHn-COO environments, but some are also bonded to aromatic rings and aliphatic groups. All models of humic acids in the literature lack at least some of the features observed here. Compositional heterogeneity was studied by introducing 1H spin diffusion into the HETCOR experiment. Comparison with data for a synthetic polymer, polycarbonate, indicates that the separation between O-alkyl and aromatic groups in the humic acid is less than 1.5 nm. However, transverse 13C relaxation filtering under 1H decoupling reveals heterogeneity on a nanometer scale, with the slow-relaxing component being rich in lignin-like aromatic-C-O-CH3 moieties and poor in COO groups.