Enzymatic characterization of organic phosphorus in animal manure

Information on the forms of P present in animal manure may improve our ability to manage manure P. In most investigations of manure P composition, only inorganic and total P are determined, and the difference between them is assigned as organic P. In this study, we explored the possibility of identifying and quantifying more specific organic P forms in animal manure with orthophosphate-releasing enzymes. Pig (Sus scrofa) manure and cattle (Bos taurus) manure were first sequentially fractionated into water-soluble P, NaHCO3-soluble P, NaOH-soluble P, HCl-soluble P, and residual P. The fractions were separately incubated with wheat phytase, alkaline phosphatase, nuclease P1, nucleotide pyrophosphatase, or their combinations. The released orthophosphate was determined by a molybdate blue method. Part of the organic P in those fractions could be identified by the enzymatic treatments as phytate (i.e., 39% for pig manure and 17% for cattle manure in water-soluble organic P), simple phosphomonoesters (i.e., 43% for pig manure and 15% for cattle manure in NaOH-soluble organic P), nucleotide-like phosphodiesters (2-12%), and nucleotide pyrophosphate (0-4%). Our data indicate that the enzymatic treatment is an effective approach to identify and quantify the organic P forms present in animal manures.     

Comparison of phosphorus forms in wet and dried animal manures by solution phosphorus-31 nuclear magnetic resonance spectroscopy and enzymatic hydrolysis

Both enzymatic hydrolysis and solution (31)P nuclear magnetic resonance (NMR) spectroscopy have been used to characterize P compounds in animal manures. In this study, we comparatively investigated P forms in 0.25 M NaOH/0.05 M EDTA extracts of dairy and poultry manures by the two methods. For the dairy manure, enzymatic hydrolysis revealed that the majority of extracted P was inorganic P (56%), with 10% phytate-like P, 9% simple monoester P, 6% polynucleotide-like P, and 18% non-hydrolyzable P. Similar results were obtained by NMR spectroscopy, which showed that inorganic P was the major P fraction (64-73%), followed by 6% phytic acid, 14 to 22% other monoesters, and 7% phosphodiesters. In the poultry manure, enzymatic hydrolysis showed that inorganic P was the largest fraction (71%), followed by 15% phytate-like P and 1% other monoesters, and 3% polynucleotide-like P. NMR spectroscopy revealed that orthophosphate was 51 to 63% of extracted P, phytic acid 24 to 33%, other phosphomonoesters 6 to 12%, and phospholipids and DNA 2% each. Drying process increased orthophosphate (8.4% of total P) in dairy manure, but decreased orthophosphate (13.3% of total P) in poultry manure, suggesting that drying treatment caused the hydrolysis of some organic P to orthophosphate in dairy manure, but less recovery of orthophosphate in poultry manure. Comparison of these data indicates that the distribution patterns of major P forms in animal manure determined by the two methods were similar. Researchers can utilize the method that best fits their specific research goals or use both methods to obtain a full spectrum of manure P characterization.     

Are naked and common mole-rats eusocial and if so, why?

Eusociality in mammals is defined in the present paper by the following criteria: reproductive altruism (which involves reproductive division of labor and cooperative alloparental brood care), overlap of adult generations, and permanent (lifelong) philopatry. We argue that additional criteria such as the existence of castes, colony size, reproductive skew, and social cohesion are not pertinent to the definition of eusociality in mammals. According to our definition of mammalian eusociality, several rodent species of the African family Bathyergidae can be considered eusocial, including the naked mole-rat (Heterocephalus glaber), Damaraland mole-rat (Cryptomys damarensis), and several additional, if not all, species in the genus Cryptomys. Furthermore, some species of social voles (like Microtus ochrogaster) may also fulfill criteria of mammalian eusociality. Understanding the evolution of eusociality in mole-rats requires answers to two primary questions: (1) What are the preconditions for the development of their eusocial systems? (2) Why do offspring remain in the natal group rather than dispersing and reproducing? Eusociality in mammals is by definition a special case of monogamy (more specifically: monogyny one female breeding), involving prolonged pair bonding for more than one breeding period. We argue that eusociality in mole-rats evolved from a monogamous mating system where cooperative brood care was already established. A tendency for group living is considered to be an ancestral (plesiomorph) trait among African bathyergid mole-rats, linking them to other hystricognath rodents. A solitary lifestyle seen in some genera, such as Bathyergus, Georychus, and Heliophobius, is assumed to be a derived trait that arose independently in different lineages of bathyergids, possibly as a consequence of selective constraints associated with the subterranean environment. In proximate terms, in eusocial mole-rats either puberty is assumed to be developmentally delayed so that under natural conditions most animals die before dispersal is triggered (e.g., in the case of Heterocephalus) or dispersal is induced only by an incidental encounter with an unfamiliar, yet adequate sexual partner (e.g., in the case of Cryptomys). Ultimately, a combination of strategies involving either dispersal and/or philopatry can be beneficial, especially in a highly unpredictable environment. If genetic relatedness among siblings is high (e.g., a coefficient of relatedness of 0.5 or more), then philopatry would not invoke an appreciable loss of fitness, especially if the cost of dispersing is higher than staying within the natal group. High genetic relatedness is more likely in a monogamous mating system or a highly inbred population. In this paper, we argue that the preconditions for eusociality in bathyergid mole-rats were a monogamous mating system and high genetic relatedness among individuals. We argue against the aridity food-distribution hypothesis (AFDH) that suggests a causal relationship between cooperative foraging for patchily distributed resources and the origin of eusociality. The AFDH may explain group size dynamics of social mole-rats as a function of the distribution and availability of resources but it is inadequate to explain the formation of eusocial societies of mole-rats, especially with respect to providing preconditions conducive for the emergence of eusociality.     

Mitochondrial DNA Variability of the Gray Wolf: Genetic Consequences of Population Decline and Habitat Fragmentation

The gray wolf is a large, highly mobile predator whose original geographic range included most of the Northern Hemisphere. High rates of genetic exchange probably characterized even distantly-separated populations in the past, but recent population declines and habitat fragmentation have isolated previously contiguous populations, especially in the Old World. We examine mitochondrial DNA (mtDNA) variability among twenty-six populations of wolves from throughout their geographic range. We find eighteen mtDNA genotypes in gray wolves, seven of these are derived from hybridization with coyotes, four are confined to the New World, six are confined to the Old World and one is shared by both areas. Genetic differentiation among wolf populations is significant but small in magnitude. In the Old World, most localities have a single unique genotype, whereas in the New World several genotypes occur at most localities and three of the five genotypes are nearly ubiquitous. The pattern of genetic differentiation in the gray wolf contrasts with that of another large, highly vagile canid, the coyote, in which genetic differentiation among populations is not significant even among widely separated localities. We suggest that the difference between these two species reflects the rapid, recent increase in coyote numbers and expansion of their geographic range, and the coincident decline in gray wolf populations. Apparent genetic differences among extant wolf populations may be a recent phenomenon reflecting population declines and habitat fragmentation rather than a long history of genetic isolation.     

Links among nitrification, nitrifier communities, and edaphic properties in contrasting soils receiving dairy slurry

Soil biotic and abiotic factors strongly influence nitrogen (N) availability and increases in nitrification rates associated with the application of manure. In this study, we examine the effects of edaphic properties and a dairy (Bos taurus) slurry amendment on N availability, nitrification rates and nitrifier communities. Soils of variable texture and clay mineralogy were collected from six USDA-ARS research sites and incubated for 28 d with and without dairy slurry applied at a rate of ~300 kg N ha(-1). Periodically, subsamples were removed for analyses of 2 M KCl extractable N and nitrification potential, as well as gene copy numbers of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Spearman coefficients for nitrification potentials and AOB copy number were positively correlated with total soil C, total soil N, cation exchange capacity, and clay mineralogy in treatments with and without slurry application. Our data show that the quantity and type of clay minerals present in a soil affect nitrifier populations, nitrification rates, and the release of inorganic N. Nitrogen mineralization, nitrification potentials, and edaphic properties were positively correlated with AOB gene copy numbers. On average, AOA gene copy numbers were an order of magnitude lower than those of AOB across the six soils and did not increase with slurry application. Our research suggests that the two nitrifier communities overlap but have different optimum environmental conditions for growth and activity that are partly determined by the interaction of manure-derived ammonium with soil properties.     

Preparation and FT-IR characterization of metal phytate compounds

Phytic acid (inositol hexaphosphoric acid, IP6) has long been recognized as the predominant organic P form in soil and animal manure. Whereas many studies have investigated the wet chemistry of IP6, there is little information on the characterization of solid metal IP6 compounds. This information is essential for further understanding and assessing the chemical behavior of IP6 in diverse soil-plant-water ecosystems. As the first step in full characterization, we synthesized eight metal phytate compounds and investigated their structural features using Fourier transform infrared spectroscopy (FT-IR). The absorption features from 900 to 1200 cm(-1) in FT-IR could be used to identify these phytates as: (i) light divalent metal (Ca and Mg) compounds with a sharp band and a broad band, (ii) heavy divalent metal (Cu and Mn) compounds with splitting broad bands, and (iii) trivalent metal (Al and Fe) compounds with a broad band and a shoulder band. Three different types of chemical structures of metal-phytate compounds were presented based on the FT-IR information. We further demonstrated that metal orthophosphates possessed different FT-IR spectral characteristics from their IP6 counterparts. The unique spectral features of metal phytates from 1000 to 700 cm(-1) could be used to distinguish phytate compounds from metal phosphate compounds. Thus, FT-IR analysis after fine tuning could provide an analytical tool to investigate the basic metal phytate chemistry in molecular levels, such as the competitive interactions between phosphate and phytate with a specific metal ion, and the conversion (or hydrolysis) of metal phytate to metal phosphate under various conditions.     

Impact of three interactive Texas state regulatory programs to decrease ambient air toxic levels

The Federal Clean Air Act (FCAA) framework envisions a federal-state partnership whereby the development of regulations may be at the federal level or state level with federal oversight. The U.S. Environmental Protection Agency (EPA) establishes National Ambient Air Quality Standards to describe “safe” ambient levels of criteria pollutants. For air toxics, the EPA establishes control technology standards for the 187 listed hazardous air pollutants (HAPs) but does not establish ambient standards for HAPs or other air toxics. Thus, states must ensure that ambient concentrations are not at harmful levels. The Texas Clean Air Act authorizes the Texas Commission on Environmental Quality (TCEQ), the Texas state environmental agency, to control air pollution and protect public health and welfare. The TCEQ employs three interactive programs to ensure that concentrations of air toxics do not exceed levels of potential health concern (LOCs): air permitting, ambient air monitoring, and the Air Pollutant Watch List (APWL). Comprehensive air permit reviews involve the application of best available control technology for new and modified equipment and ensure that permits protect public health and welfare. Protectiveness may be demonstrated by a number of means, including a demonstration that the predicted ground-level concentrations for the permitted emissions, evaluated on a case-by-case and chemical-by-chemical basis, do not cause or contribute to a LOC. The TCEQ's ambient air monitoring program is extensive and provides data to help assess the potential for adverse effects from all operational equipment in an area. If air toxics are persistently monitored at a LOC, an APWL area is established. The purpose of the APWL is to reduce ambient air toxic concentrations below LOCs by focusing TCEQ resources and heightening awareness. This paper will discuss examples of decreases in air toxic levels in Houston and Corpus Christi, Texas, resulting from the interactive nature of these programs.

Implications: Texas recognized through the collection of ambient monitoring data that additional measures beyond federal regulations must be taken to ensure that public health is protected. Texas integrates comprehensive air permitting, extensive ambient air monitoring, and the Air Pollutant Watch List (APWL) to protect the public from hazardous air toxics. Texas issues air permits that are protective of public health and also assesses ambient air to verify that concentrations remain below levels of concern in heavily industrialized areas. Texas developed the APWL to improve air quality in those areas where monitoring indicates a potential concern. This paper illustrates how Texas engaged its three interactive programs to successfully address elevated air toxic levels in Houston and Corpus Christi.     

Enzymatic hydrolysis of organic phosphorus in swine manure and soil

Organic phosphorus (Po) exists in many chemical forms that differ in their susceptibility to hydrolysis and, therefore, bioavailability to plants and microorganisms. Identification and quantification of these forms may significantly contribute to effective agricultural P management. Phosphatases catalyze reactions that release orthophosphate (Pi) from Po compounds. Alkaline phosphatase in tris-HCl buffer (pH 9.0), wheat (Triticum aestivum L.) phytase in potassium acetate buffer (pH 5.0), and nuclease P1 in potassium acetate buffer (pH 5.0) can be used to classify and quantify Po in animal manure. Background error associated with different pH and buffer systems is observed. In this study, we improved the enzymatic hydrolysis approach and tested its applicability for investigating Po in soils, recognizing that soil and manure differ in numerous physicochemical properties. We applied (i) acid phosphatase from potato (Solanum tuberosum L.), (ii) acid phosphatases from both potato and wheat germ, and (iii) both enzymes plus nuclease P1 to identify and quantify simple labile monoester P, phytate (myo-inositol hexakis phosphate)-like P, and DNA-like P, respectively, in a single pH/buffer system (100 mM sodium acetate, pH 5.0). This hydrolysis procedure released Po in sequentially extracted H2O, NaHCO3, and NaOH fractions of swine (Sus scrofa) manure, and of three sandy loam soils. Further refinement of the approach may provide a universal tool for evaluating hydrolyzable Po from a wide range of sources.     

Phosphorus distribution in dairy manures

The chemical composition of manure P is a key factor determining its potential bioavailability and susceptibility to runoff. The distribution of P forms in 13 dairy manures was investigated with sequential fractionation coupled with orthophosphate-releasing enzymatic hydrolysis. Among the 13 dairy manures, manure total P varied between 4100 and 18,300 mg kg(-1) dry matter (DM). Water-extractable P was the largest fraction, with inorganic phosphorus (P(i)) accounting for 12 to 44% of manure total P (1400-6800 mg kg(-1)) and organic phosphorus (P(o)) for 2 to 23% (130-1660 mg kg(-1)), respectively. In the NaHCO(3) fraction, P(i) varied between 740 and 4200 mg P kg(-1) DM (4-44% of total manure P), and P(o) varied between 340 and 1550 mg P kg(-1) DM (2-27% of total manure P). In the NaOH fraction, P(i) fluctuated around 200 mg P kg(-1) DM, and P(o) ranged from 130 to 630 mg P kg(-1) DM. Of the enzymatically hydrolyzable P(o) in the three fractions, phytate-like P dominated, measuring 26 to 605 mg kg(-1) DM, whereas monoester P and DNA-like P were relatively low and less variable. Although concentrations of various P forms varied considerably, significant correlations between manure total P and certain P forms were observed. For example, H(2)O-extracted P(i) was correlated with total manure P (R(2) = 0.62), and so was NaOH-extracted P(o) (R(2) = 0.81). Data also show that the amount of P released by a single extraction with sodium acetate (100 mM, pH 5.0) was equivalent to the sum of P in all three fractions (H(2)O-, NaHCO(3)-, and NaOH-extractable P). Thus, a single extraction by sodium acetate buffer could provide an efficient evaluation of plant-available P in animal manure, while the sequential fractionation approach provides more detailed characterization of manure P.     

Influence of decomposition on chemical properties of plant- and manure-derived dissolved organic matter and sorption to goethite

Sorption of dissolved organic matter (DOM) plays an important role in maintaining the fertility and quality of soils in agricultural ecosystems. Few studies have examined the effects of decomposition on DOM sorption and chemical characteristics. This study investigated the sorption to goethite (alpha-FeOOH) of fresh and decomposed hydrophilic (HPL) and hydrophobic (HPB) DOM fractions extracted from the shoots and roots of crimson clover (Trifolium incarnatum L.), corn (Zea mays L.), soybean [Glycine max (L.) Merr.], hairy vetch (Vicia villosa L.), and dairy and poultry manures. Sorption was positively related to apparent molecular weight (MWAP), aromaticity as measured by absorptivity at 280 nm, and phenolic acid content. A 10-d laboratory microbial decomposition of the source organic matter generally increased the sorption of the extracted DOM onto goethite. The decomposition effect on sorption was greater for the HPL fractions than for the HPB fractions. There was a decrease in the MWAP values of the DOM samples following sorption to goethite. In many cases the reduction in MWAP was large, indicating a strong preference by goethite for the higher MWAP DOM fractions. The results of this laboratory-based research demonstrate that microbial processes affect the chemical characteristics of DOM which may affect the distribution of soil organic C pools.