Unraveling the structural components of soil humin by use of solution-state nuclear magnetic resonance spectroscopy

Humin is the most recalcitrant and least understood fraction of soil organic matter. By definition, humin is that fraction not extracted by traditional aqueous alkaline soil extractants. Here we show that > or = 70% of the traditional humin fraction is solubilized when 0.1 M NaOH + 6 M urea and dimethyl sulfoxide (DMSO) + 6% H2SO4 are used in series after conventional extraction. Multidimensional solution-state NMR is applied in this study to gain an understanding of the major constituents present in these "solubilized humin fractions". The spectra indicated strong contributions from five main categories of components, namely, peptides, aliphatic species, carbohydrates, peptidoglycan, and lignin. Diffusion edited spectroscopy indicated that all species are present as macromolecules (or stable aggregate species). Although the distribution of the components is generally similar, peptidoglycan is present at significant levels supporting a higher microbial contribution to humin than to humic and fulvic fractions. The abundance of plant- and microbial-derived materials found does not exclude "humic" materials (e.g., oxidized lignin) or the presence of novel compounds at lower concentrations but suggests that a large proportion of humin is formed from classes of known compounds and parent biopolymers.     

Combined chemical and enzymic treatments of corn husk lignocellulosics

The potential for using corn (Zea mays L) husk residues (carbohydrates 827 g kg^?1, lignin 66 g kg^?1 DM) as a carbohydrate source for the production of soluble sugars by combined chemical pretreatment and enzymic hydrolysis was assessed. Comparative investigations of acidic and alkaline pretreatments on corn husk have shown that pentose-containing carbohydrates comprised 86–93% of the solubilised fraction. While pretreatments with 1.25 M NaOH at 25.85° C resulted in preferential extraction of hemicellulose having DP; > 12, acid pretreatments 0.51 M H₂SO₄, 0.51 M H₃PO₄ at (85° C) resulted in extensive depolymerisation of this polysaccharide. Xylose and low molecular weight carbohydrates were identified as the major products. Subsequent hydrolysis of the solubilised fraction with crude hemicellulase preparations yielded 40.90% fermentable sugars. When NaOH (0.02–1.25 M), H₂SO₄ and H₃PO₄ (0.02–0.51 M) were used as pretreatment solvents (25–85° C, 2 h), NaOH was the most effective in increasing the susceptibility of the residual husk towards enzymes, yielding 83–96% reducing sugars. This solvent solubilised up to 60.6% of the lignin and appeared to disintegrate the fibrillar structure of husk. The crystallinity of husk residues increased following the chemical pretreatments and was positively correlated with cellulose content. Enzymic hydrolysis with commercial cellulase preparations proceeded in two stages: a rapid breakdown of amorphous cellulose after which the hydrolysis rate levelled off. Similar biphasic patterns were observed for the pyrolysis temperature of cellulose. Under the most optimal conditions for husk saccharification (pretreatment with 1.25 M NaOH, 25° C, 2 h, followed by enzymic hydrolysis using a mixture of cellulase and cellobiase), 96% of the cellulose-enriched residues was hydrolysed to reducing sugars. A cellulase preparation from Trichoderma reesei exhibited substantial hemicellulolytic activity and could, therefore, be used as the sole saccharifying enzyme preparation for husk lignocellulosics.     

Modelling of the hydrolysis of sorghum straw at atmospheric pressure

Sorghum straw is a renewable, cheap and widespread resource. The acid hydrolysis of sorghum straw to obtain xylose solutions could be a good alternative for this abundant resource. The H₂SO₄ hydrolysis of sorghum straw at two different temperatures (80 and 100 °C) and three H₂SO₄ concentrations (2, 4 and 6%) using a solid/liquid ratio of 1:10 (w/w) was studied. Kinetic parameters of mathematical models for predicting the concentrations of xylose, glucose, acetic acid and furfural were determined. The activation energy of the release reaction was 183.3 kJ mol^?1 for xylose and 185.8 kJ mol^?1 for glucose. The optimal conditions found were 6% H₂SO₄ at 100 °C for 60 min, which allow one to obtain a solution with 18.27 g xylose l^?1, 6.78 g glucose l^?1, 0.7 g furfural l^?1 and 1.35 g acetic acid l^?1. It is concluded that this process has potential for utilisation of this renewable lignocellulosic resource. © 2002 Society of Chemical Industry     

Carbohydrates associated with leaf protein

Heating leaf protein of agricultural crops with 2 N H₂SO₄ removed 5–10% of its dry weight as carbohydrate, of which less than half was reducing sugar; arabinose, galactose, xylose and sometimes a little glucose were found for all the crops studied. Pronase released soluble carbohydrate from red clover and wheat protein as large fragments which gave pentose, mainly arabinose, on heating with 0.2 N H₂SO₄, and galactose and glucose on heating with 2 N H₂SO₄. Similar results on graded acid hydrolysis were shown by a fraction from red clover liquor, the soluble material remaining after heat-precipitation of the protein.     

15N NMR investigation of the reduction and binding of TNT in an aerobic bench scale reactor simulating windrow composting

T15NT was added to a soil of low organic carbon content and composted for 20 days in an aerobic bench scale reactor. The finished whole compost and fulvic acid, humic acid, humin, and lignocellulose fractions extracted from the compost were analyzed by solid-state CP/MAS and DP/MAS 15N NMR. 15N NMR spectra provided direct spectroscopic evidence for reduction of TNT followed by covalent binding of the reduced metabolites to organic matter of the composted soil, with the majority of metabolite found in the lignocellulose fraction, by mass also the major fraction of the compost. In general, the types of bonds formed between soil organic matter and reduced TNT amines in controlled laboratory reactions were observed in the spectra of the whole compost and fractions, confirming that during composting TNT is reduced to amines that form covalent bonds with organic matter through aminohydroquinone, aminoquinone, heterocyclic, and imine linkages, among others. Concentrations of imine nitrogens in the compost spectra suggest that covalent binding by the diamines 2,4DANT and 2,6DANT is a significant process in the transformation of TNT into bound residues. Liquid-phase 15N NMR spectra of the fulvic acid and humin fractions provided possible evidence for involvement of phenoloxidase enzymes in covalent bond formation.     

Analysis of Dietary Fiber in Feces of Rats Fed with Fiber Supplemented Diets

A procedure based on Southgate's fractionation scheme with subsequent colorimetric determination of monomeric constituents in the individual fractions was used for the analysis of feces of rats fed with diets supplemented with cereal bran or its fiber components. Prior to fractionation, dehydrated feces were extracted with chloro-form:methanol (2:1) mixture and hot 80% methanol and then subjected to treatment with both amylolytic (MERCK glucoamylase, 60°C, 2 hr) and proteolytic (CALBIOCHEM pepsin, 40°C, 48 hr) enzymes. Colorimetrically determined values of noncellulosic polysaccharides and cellulose (calculated as sums of hexoses, pentoses, and uronic acids) agreed satisfactorily with those determined by detergent fiber analysis. The sums of neutral sugars determined colorimetrically correlated closely with the values obtained by GLC analysis after Saeman's hydrolysis (2 hr with 72% (w/w) H₂SO₄ at 20°C and 2 hr with 2N H₂SO₄ at 100°C) of defatted samples. The GLC data were, however, consistently higher, especially those for hexoses; the difference was more pronounced with feces than with tested bran material. Confidence in the analysis was strengthened by the observation that the results of proximate and fiber analyses closely approached 100%.     

Isolation of Gellan Gum from Foods by Use of Monovalent Cations

Gellan gum added to foods was recovered by precipitation with 2M NaCl. The precipitate was trapped on glass wool, washed with 1M salt solution and eluted with boiling 40% H₂SO₄ or boiling 2 × 10^?4N NaOH. Elution with dilute NaOH was as effective as use of 40% H₂SO₄, except with dog food, mayonnaise and salad dressing. The hydrocolloid content of the eluate was determined by the carbazole test. Recoveries from milk, beverages, salad dressing and other foods ranged from 75–94% (reproducibility 2–4%). The preferred cations for precipitation were Na⁺ > K⁺ > Li⁺ > NH₄⁺. For the anions, the order was CO₃^? > SO₄^? > Cl^?.     

Evaluation of Gelation and Solubility of Bovine Plasma Protein Isolates

Gelation and solubility of bovine plasma protein isolates prepared from liquid plasma using different acids (HCl, H₃PO₄, H₂SO₄) and bases (NaOH, Na₂CO₃) were evaluated and compared to commercial plasma. Protein recovery was over 90%. Solubility at 1% (w/v) was low in pH range 4.0 to 5.5 but similar to commerical plasma in pp range 5.5 to 8.0 (p < 0.01). All isolates gelled at a concentration o 6.0%. Only isolate H₃PO₄-Na₂CO₃ and commercial plasma gelled at a minimum concentration of 4.0%. This concentration decreased to 2.4% for the same isolate when 0.025M Ca⁺⁺ ion was added.     

Humin as an electron mediator for microbial reductive dehalogenation

We report that humins extracted as the solid fractions from paddy soils or sediment are involved in extracellular electron transfer, coupled with microbial reductive dehalogenation of pentachlorophenol (PCP), by serving as both electron acceptor and electron donor. In our system, humin is requisite for the dechlorination of PCP, and this activity cannot be maintained when humin is replaced with soluble humic substances or related compounds, including 0.1 M NaOH-extracted humic acid from soil, Aldrich humic acid, and anthraquinone-2,6-disulfonate. The function of humins is stable against treatments with H(2)O(2) (30%, 30 min), HCl (0.1 M, 48 h), NH(2)OH · HCl (0.1 M, 48 h), NaBH(4) (0.1 M, 15 h), and heat (121 °C, 30 min). Cyclic voltammograms indicated that humin harbors redox-active moieties, and electron spin resonance suggested that quinone moieties within humin are the redox-active centers. Fourier-transform infrared and nuclear magnetic resonance analyses verified the presence of the aryl carbonyl carbon group in humin. Although the proportion of redox-active carbon is very small, the potential electron-mediating ability is not negligible. The finding that humin, in solid form, is redox active has important implications for in situ bioremediation, given the wide distribution of humin and the diversity and ubiquity of humic substance-utilizing microorganisms.     

Production of a high‐protein meal and fermentable sugars from defatted soybean meal,a co‐product of the soybean oil industry

The goal of this research was to produce a high‐protein meal by treating defatted soybean meal, a by‐product of soybean oil production, with dilute acid. Treatments were a mild hydrolysis at 80 °C with sulphuric acid at concentrations ranging from 0.5% to 2.0% (w/v) and times varying from 1 to 16 h that were arranged according to a central composite rotatable experimental design. The end products were an enhanced‐protein meal and a carbohydrate concentrate of fermentable and nonfermentable sugars. The highest protein content rise, from 48% to 58%, was for treatments with concentrations of acid ranging between 1.2% and 1.7% and times between 1.0 and 2.6 h. The maximum yield of fermentable sugar was 21.0% d.b. at 2.0% H₂SO₄ and treatments of at least 6 h. The conditions that provide a highest protein and sugar contents were the treatments with concentrations of sulphuric acid ranging from 0.9 to 1.9% H₂SO₄ for 1–4 h.     

HYDROGEN SULFIDE PRODUCTION BY HEATING DIFFERENT PROTEIN FRACTIONS OF TUNA MEAT

Raw albacore tuna was separated into water-soluble and water-insoluble fractions. The water-soluble fraction was separated into two fractions by trichloroacetic acid (2.5%). The water-insoluble fraction was extracted with NaCl (0.45 M), and then the insoluble fraction was further extracted with urea solution (6 M). H₂S production upon heating of each fraction was determined by the reflux-trap technique. Water-soluble proteins produced 71% of the H₂S, while the corresponding contribution from the insoluble fraction was 29%. Non-protein soluble components and salt-soluble proteins produced insignificant amounts of H₂S. Significant increase in the amount of H₂S was observed when water-insoluble proteins were treated with urea and TCA solutions.     

Lipid metabolism in Fusarium oxysporum

Certain aspects of lipid metabolism in Fusarium oxysporum were studied using acetate-1-C¹⁴ and H₃P³²O₄ for incorporation of radioactivity into the fungal mycelium. During initial stages of growth of the organism (up to 1 h of incubation) the phospholipid fraction exhibited relatively higher specific activity values indicating its rapid rate of synthesis. Higher turnover rates were observed for phosphatidic acid (PA) and polyglycerophosphatides (PGP). It is postulated that PGP represents a labile pool where PA synthesised in excess of the metabolic needs of the cell is stored to be subsequently used during periods of intense phospholipid biosynthesis. In pulse and chase experiments, higher specific activities were observed for PA, PGP and cardiolipin (CL) fractions after 1-day chase on unlabelled media. Rapid turnover rates were observed for PA, PGP and cardiolipin (CL) fractions. It is suggested that these are involved in the growth of new cells and (or) in the cell repair processes. In cells grown both under normal and carbon-starvation conditions, the specific activity pattern of phosphatidyl choline (PC), and phosphatidyl ethanolamine and phosphatidyl glycerol (PE + PG measured collectively) was very similar indicating similarity in the metabolism of these two classes of phosphatides. Addition of sodium arsenite, alloxan, p. benzoquinone and citric acid in the incubation media was found to affect the synthesis of lipid classes in different ways. For example, among the polar lipids, PA, PC, PE + PG, CL and SGP + FFA fractions were inhibited by p.benzoquinone, while the incorporation of label from C¹⁴ was found to be stimulated into the phosphatidyl inositol fraction.     

Dietary fibre of coconuts from a pacific atoll: Soluble and insoluble components in relation to maturity

Polysaccharide fractions were measured in coconuts at three stages of maturity, which corresponded to dietary usage in the Tokelau Islands of the South Pacific. Kernel was sequentially extracted with cold water (CW), hot water (HW) hot 0.5% ammonium oxalate (HOX), 1M H² SO⁴ and 72% H² SO⁴, and the monosaccharide compositions of the fractions determined. Total readily soluble fractions (CW+HW+HOX) were predominantly galactomannan, and decreased from 77% of the polysaccharide in the immature kernel to 8.8% of that in the mature kernel. Insoluble mannan increased during maturation to be the major polysaccharide component in mature kernel. This indicates that marked differences exist between the three stages of maturity examined, in the properties of their dietary fibre. The results suggest that in studies of diets in which coconut is a staple part, there is a need for careful definition of the coconut component with respect to maturity. Furthermore, they show the importance of using dietary fibre methods which are appropriate to the solubility characteristics of the indigestible polysaccharide.     

Distribution of aged 14C-PCB and 14C-PAH residues in particle-size and humic fractions of an agricultural soil

Organic matter is considered to be the single most importantfactor limiting availability and mobility of persistent organic pollutants (POPs) in soil. This study aimed to characterize the distribution of 14C-PCB (congeners 28 and 52) and 14C-PAH (fluoranthene and benzo[a]pyrene) residues in an Orthic Luvisol soil obtained from two lysimeter studies initiated in 1990 at the Agrosphere Institute (Forschungszentrum Jülich GmbH, Germany). The lysimeter soils contained a low-density OM fraction, isolated during soil washing, which contained a significant fraction (3-12%) of the total 14C-activity. Soils were also fractionated according to three particle sizes: >20, 20-2, and <2 microm. Relative affinity values of 14C-activity for the different particle sizes varied in the order 20-2 microm > (<2 microm) approximately (>20 microm) for the PCBs. Relative affinity values of 14C-activity for the different particle sizes varied in the order 20-2 microm > (<2 microm) > (>20 microm) for the PAHs. The distribution of 14C-PCB or 14C-PAH residues in the organic and inorganic matrixes of the particle-size fractions was determined using methyl isobutyl ketone (MIBK). 14C-PCB and 14C-PAH-associated activities were primarily located in the humin fraction of the 20-2 and <2 microm particle-size fractions of the soil. A small fraction was associated with the fulvic and humic acid fractions; these were quantitatively more important for the PAHs than the PCBs. There appeared to be a high degree of association of 14C-activity with the mineral fraction following MIBK separation of the humic fractions, ranging between 8 and 52% for 14C-PCBs and 57-80% for 14C-PAHs. The mineral (inorganic) component of the soils apparently played a significant (previously unreported) role in the sequestration of both PCBs 28 and 52 and the PAHs fluoranthene and benzo[a]pyrene.     

Production of fermentable media from vine‐trimming wastes and bioconversion into lactic acid by Lactobacillus pentosus

Trimmings of vineshoots, an agricultural waste with little use, were hydrolysed with dilute sulphuric acid (1–5%) in order to obtain sugar solutions suitable as fermentation media. The operational conditions for hydrolysis were selected on the basis of both the generation of hemicellulosic sugars (mainly xylose) and glucose and the concentrations of reaction byproducts affecting fermentation (furfural, hydroxymethylfurfural and acetic acid). Hemicellulosic hydrolysates were supplemented with nutrients and fermented with Lactobacillus pentosus, without any previous detoxification stage, to produce lactic acid. Under the best operational conditions assayed (3% H₂SO₄ and 15 min), 21.8 g lactic acid l^?1 was produced (Q_P = 0.844 g l^?1 h^?1, Y_P/S = 0.77 g g^?1), which represents a theoretical yield of 99.6%. Acetic acid was the primary byproduct formed from xylose, at about 25% of the lactic acid level. Copyright © 2004 Society of Chemical Industry     

Distribution of Proteolytic Activity in the Different Protein Fractions of Tropical Shrimp Head Waste

Ammonium sulfate fractionated protein fractions at levels of 25%, 30%, 35%, 40%, 42.5% and 45% ammonium sulfate were recovered from the head waste of tropical marine tiger (MTS), culture tiger (CTS), white (WS) and brown shrimp (BS) and then characterized for protease activity. Distribution of buffer (pH 7.1) extracted protein among ammonium sulfate fractions showed that total protein in 25% fraction of MTS, CTS, WS and BS was 48, 54, 34 and 24 times more than that in the respective 42.5% fraction of the head waste of these shrimps. The highest proteolytic activity was observed in 42.5% (NH₄)₂SO₄ protein fraction of the head waste of MTS, CTS, WS and BS, the values were 19, 1.7, 11.6 and 2 times, respectively, more than that of the corresponding 25% (NH₄)₂SO₄ protein fractions. Highest caseinolytic activity (pH 8.5) and gelatinolytic activity (pH 7.1) was observed in the 42.5% fraction of the head waste of CTS and WS, respectively; but the highest albuminolytic activity (pH 8) was observed in the same fraction of the head waste of both MTS and BS. The optimum pH for highest gelatinolytic and albuminolytic activity of the 42.5% (NH₄)₂SO₄ protein fraction of the head waste of MTS and BS was 4; the same for highest gelatinolytic activity of the same protein fraction of the head waste of WS, and these fractions included acid proteases such as pepsin, the optimum pH for the above activity of the same fraction of CTS was 6–8.5, and the fraction was an alkaline protease such as chymotrypsin. The SDS-PAGE pattern of 42.5% (NH₄)₂SO₄ protein fraction of the head waste of BS, CTS, WS and MTS was almost similar with a dark band close to the marker band 20kDa. Proteases make up to 48% of industrial enzymes and are mostly used in detergents, leather production and food industry.     

Comparison of acid and amyloglucosidase hydrolysis for estimation of non‐structural polysaccharides in feed samples

Enzymatic methods (amyloglucosidase) and methods based on acid solutions (0.1, 0.2 and 0.3 M H₂SO₄ for 1, 2 and 3 h at 100 °C) for the hydrolysis of non‐structural carbohydrates from different feed samples were compared. The monomeric units resulting from the enzymatic and acid hydrolysis were determined by the glucose oxidase and reducing sugar methods. There was a significant effect of acid concentration and of time of hydrolysis on the glucose and reducing sugar values in the hydrolysate. Glucose values were similar for both the amyloglucosidase method and the most intense conditions of hydrolysis (0.3 M for 3 h) for some samples. Under these conditions, however, the reducing sugar values were higher. No acid hydrolysis method was found to estimate correctly the total non‐structural carbohydrates, but α‐linked glucose polymers in biological samples may be determined by sample hydrolysis with a 0.3 M H₂SO₄ solution for 3 h at 100 °C since the glucose in the hydrolysate is determined by the glucose oxidase method and the sucrose content of the sample is negligible. © 1999 Society of Chemical Industry     

Cultivar and Maturity Effects on Muskmelon ( Cucumis melo) Colour,Texture and Cell Wall Polysaccharide Composition

Cell wall polysaccharides (CWP) of two types of melons were isolated and purified. Fractionations were performed using cyclohexanetrans-1,2-diamine tetraacetate (CDTA), Na₂CO₃, guanidinium thiocyanate (GTC) and KOH. Alditol acetate derivatives of neutral sugars from each CWP fraction were prepared and analysed by gas chromatography. Trifluoro-acetic acid insoluble fractions were analysed colorimetrically and uronic acid was determined. The CDTA and Na₂CO₃ fractions were found to be composed of typical pectic materialscontaining primarily galacturonic acid with the neutral sugars arabinose, galactose, rhamnose and a smaller amount of xylose. As maturity increased, CDTA fraction yields increased, though total neutral sugar CWP compositions decreased. GTC and KOH fractions were typical of hemicellulose, and contained principally xylose, glucose, galactose, mannose and fucose, with very small amounts of uronic acid, arabinose and rhamnose. The residues contained principally glucose and galactose, with smaller amounts of mannose, xylose, arabinose and fucose. With the exception of xylose and glucose, all neutral sugars decreased significantly during ripening in both the Cantaloupe and Honey Dew melons. Total uronic acid did not change as maturity increased, except for Cantaloupe, where total uronic acid decreased from the ripe to overripe stages. Relationships between firmness, drip loss and other composition measurements, as well as the total CWP sugar composition, were also determined. Only the CDTA fraction yields were negatively correlated with the changes in firmness of both melons and positively correlated with changes in drip loss as maturity increased.     

Acid hydrolysis and fermentation of brewer's spent grain to produce xylitol

The hemicellulosic fraction of brewer's spent grain (BSG) was hydrolysed with diluted acid under different conditions of liquid/solid ratio (8–12 g g⁻¹), sulfuric acid concentration (100–140 mg g⁻¹ dry matter) and reaction time (17–37 min) in order to produce a liquor with a large amount of xylose and good fermentability to produce xylitol. Results showed that all the evaluated reaction conditions were able to hydrolyse xylan and arabinan with efficiencies higher than 85.8 and 95.7% respectively, and even under the mildest reaction condition a considerable amount (92.7%) of the hemicellulosic fraction could be extracted. The hydrolysates presented different fermentabilities when used as fermentation media for xylitol production by Candida guilliermondii yeast, owing to the differences in their composition. Based on statistical analysis, the best condition for BSG acid hydrolysis was the use of a liquid/solid ratio of 8 g g⁻¹, 100 mg H₂SO₄ g⁻¹ dry matter and a reaction time of 17 min. Under this condition a high extraction efficiency of hemicellulosic sugars (92.7%) and good fermentation results (Y_P/S = 0.70 g g⁻¹ and Q_P = 0.45 g dm⁻³ h⁻¹) were attained. Copyright © 2005 Society of Chemical Industry     

Neutral Salt Effects on Stability of Whey Protein Isolate Foams

Addition of 0.1 M Na₂SO₄, NaCl, or NaSCN reduced maximum overruns of whey protein isolate (WPI) by 33%, 27%, and 38%. This was attributable to an ionic effect, i.e., the ions interacted with countercharges on the proteins thereby reducing electrostatic interactions. In contrast, at high concentrations (1M), Na₂SO₄ improved foam stability by 76% compared to WPI without salt. Chloride had an intermediate effect, whereas NaSCN did not improve foam stability. Increasing Na₂SO₄ concentration (2M) improved foam stability by 127% compared to control. The relative effectiveness of salts at improving foam stability and heat stability followed the Hofmeister series (SO₄^2-??).