The effect of chicken blood and its components on wastewater characteristics and sewage surcharges.

Local wastewater treatment authorities levy surcharges from their non-residential customers that are based, in part, on the concentration of various pollutants in the customer's wastewater. Blood has long been recognized as the most potent contributor to pollutant loads in chicken processing plant wastewater. Quantification of the impact of blood on wastewater characteristics and sewage surcharges is hindered by lack of information on specific characteristics of chicken blood, and by the highly variable methods used by local authorities for calculating surcharges. In this study, the most commonly used wastewater characteristics are determined for whole chicken blood as well as its individual components. The characteristics measured include biochemical oxygen demand, chemical oxygen demand, total suspended solids, fats oil and grease, total Kjeldahl nitrogen, ammonia, and total phosphorus. Sewage surcharge calculation methods were collected from 71 local wastewater authorities. The results show all components of the blood to be extremely high-strength pollutants. The impact of blood on sewage surcharges is shown to be highly variable depending on the rates applied by the local authority.

Flocculation of high purity wheat straw soda lignin.

In industrial process, acidification causes non-sulfonated lignin insolubility. The flocculants poly(diallyldimethylammonium chloride) (pDADMAC) and bovine blood (BB) also caused lignin insolubility while cationic polyacrylamide, chitosan, and soy protein PF 974 were ineffective. Turbidity determined optimal flocculant, but turbidity magnitude with BB was greater than expected. pDADMAC caused negative lignin Zeta potential to became positive, but BB-lignin Zeta potential was always negative. Insoluble lignin did not gravity sediment, and flocculant-lignin mixtures were centrifuged. Pellet and supernatant dry mass and corrected spectroscopic results were in good agreement for optimal pDADMAC and BB. Spectroscopy showed 87-92% loss of supernatant lignin. Nitrogen analysis showed BB concentrated in the pellet until the pellet became saturated with BB. Subtracting ash and BB mass from pellet and supernatant mass confirmed optimal BB. Low levels of alum caused increased lignin flocculation at lower levels of pDADMAC and BB, but alum did not affect optimal flocculant.

Proteins and peptides as renewable flocculants.

Partially hydrolyzed extracts from blood meal, feather meal, and meat and bone meal, as well as a variety of common surplus agricultural proteins were tested for their ability to promote the flocculation of clay. Partial alkaline or enzymatic hydrolyses of blood meal, feather meal, and meat and bone meal were performed to liberate proteins and peptides from their water-insoluble forms. Some of these extracts promoted flocculation. However, if hydrolysis was extensive, low molecular weight peptides were mainly produced, and these extracts did not promote flocculation. Beef skin gelatins and hydrolyzed fish collagen were found to promote flocculation when pH 5.5 buffer was added. Commercial preparations of peptone enzymatic digest and a mixture of keratin and hydrolyzed keratin did not promote flocculation.

Meat & bone meal extract and gelatin as renewable flocculants.

Readily available proteins were tested as renewable flocculants, and their actions were compared to that of anionic PAM, a common, commercial flocculant that requires the coaddition of a calcium ion source. Two soy proteins, a whey fraction, a porcine gelatin, and a meat & bone meal (MBM) extract were used in the flocculation test. It was found that MBM extract and porcine gelatin promoted clay flocculation, and flocculation was complete by 24h with or without the addition of calcium chloride. The other tested proteins did not promote clay flocculation, but all of the proteins were found to be adsorbed to clay. The protein adsorptions were well described by the Langmuir model, and gelatin and MBM extract had higher maximum adsorption capacities than the other proteins. Zwitterionic buffer solutions at pH 5.5, 7.0, and 10.0 were tested in the flocculation experiments. Addition of the pH 5.5 buffer caused the two soy proteins to become clay flocculants and lowered the concentration of gelatin and MBM extract necessary to promote complete flocculation by 24h. Calcium chloride was not required for flocculation. Under optimal testing conditions, the dried weight of gelatin or MBM extract was 2.6 and 17 times higher, respectively, than the weight of anionic PAM required for complete flocculation at 24h.

Characterization of lipoxygenase oxidation products by high-performance liquid chromatography with electron impact-mass spectrometric detection.

Lipoxygenase (LOX) is an enzyme that oxygenates polyunsaturated fatty acids to their corresponding hydroperoxy derivatives. For example, LOX found in plants produce the corresponding 13- and 9-hydroperoxide derivatives of linoleic acid (13-HPOD and 9-HPOD). Identification of the HPOD products is usually accomplished by using gas chromatography with mass spectrometric (MS) detection, which requires extensive derivatization of the thermally unstable hydroperoxy group. Here we report a high-performance liquid chromatographic method in combination with electron impact (EI)-MS detection that separates and characterizes the HPOD isomers generated by soybean LOX type I oxygenation of linoleic (LA) and linolenic acids as well as HPOD products produced by photosensitized oxidation of LA. The method does not required derivatization of the hydroxyperoxide group, and location of its position can be determined by the EI-MS fragmentation pattern. The method has been used for the analysis of HPOD produced by action of partially purified LOX from the micro-alga Chlorella pyrenoidosa on LA. The study suggests the presence of two LOX isozymes in the micro-alga that oxygenate LA to its 13-HPOD and 9-HPOD derivatives. Moreover, the 9-LOX isozyme under anaerobic conditions cleaves 13-HPOD to 13-oxo-tridecadienoic acid and pentane but does not cleave 9-HPOD.

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa.

The micro-alga Chlorella pyrenoidosa expresses an enzymatic activity that cleaves the 13-hydroperoxide derivatives of linoleic acid [13-hydroperoxy-9(Z),11(E)-octadecadienoic acid, 13-HPOD] and linolenic acid [13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid, 13-HPOT] into volatile C(5) and non-volatile C(13) oxo-products. This enzymic activity initially was attributed to a hydroperoxide lyase enzyme; however, subsequent studies showed that this cleavage activity is the result of lipoxygenase activity under anaerobic conditions. Headspace analysis of the volatile products by GC/MS showed the formation of pentane when the substrate was 13-HPOD, whereas a more complex mixture of hydrocarbons was formed when 13-HPOT was the substrate. Analysis of the non-volatile cleavage products from 13-HPOD by liquid chromatography/MS indicated the formation of 13-oxo-9(Z),11(E)-tridecadienoic acid (13-OTA) along with the 13-keto-octadecadienoic acid derivative. When the substrate is 13-HPOT, liquid chromatography/MS analysis indicated the formation of 13-OTA as the major non-volatile product. Aldehyde dehydrogenase (AldDH) oxidizes 13-OTA to an omega-dicarboxylic acid, whereas alcohol dehydrogenase (ADH) reduces 13-OTA to an omega-hydroxy carboxylic acid. AldDH and ADH require the oxidized (NAD(+)) and reduced (NADH) forms of the cofactor NAD, respectively. By combining the action of AldDH and ADH into a continuous cofactor-recycling process, it is possible to simultaneously convert 13-OTA to the corresponding omega-dicarboxylic acid and omega-hydroxy carboxylic acid derivatives.

Cofactor recycling in a coupled enzyme oxidation-reduction reaction: conversion of omega-oxo-fatty acids into omega-hydroxy and dicarboxylic acids.

Aldehydes are reduced to alcohols by the enzyme alcohol dehydrogenase (ADH), whereas the enzyme aldehyde dehydrogenase (AldDH) oxidizes aldehydes to carboxylic acids. ADH and AldDH require, respectively, the reduced and oxidized forms of the cofactor NAD (NAD+/NADH). By combining both oxidation and reduction reactions into one process, it is possible to produce alcohols and carboxylic acids simultaneously from aldehydes by continuous recycling of the NAD+/NADH cofactor. However, both enzymes need to be active within the same pH region and buffer system. To test this hypothesis, the pH profile (Vmax and Vmax/Km) as well as the pKa of the prototropic groups involved in catalysis for both dehydrogenases were determined using (Z,Z)-nona-2,4-dienal as a model substrate. The pH profile (Vmax and Vmax/Km) of both enzymes overlapped in the pH range of 6-8 in potassium phosphate buffer. When the coupled enzyme system was used at pH 7 with 10% NAD+ cofactor, over 90% of the starting aldehyde was converted to its corresponding acid and alcohol derivatives in a 1:1 ratio. The sequential action of the enzymes lipoxygenase and hydroperoxide lyase converts polyunsaturated fatty acids to aldehydic fatty acids. The products arising from the oxidation or reduction of the aldehydic functionality are of industrial interest. It was found that 13-oxo-9-(Z),11-(E)-tridecadienoic acid, the product of the sequential reaction of soya bean lipoxygenase and hydroperoxide lyase from Chlorella pyrenoidosa on linoleic acid, is also a substrate in this coupled enzyme system.

A gas chromatographic-mass spectrometric method using a PoraPLOT column for the detection of hydroperoxide lyase in Chlorella pyrenoidosa.

A gas chromatographic-mass spectrometric (GC-MS) method using a PoraPLOT Q column was developed for the analysis and identification of the volatile products produced by the action of hydroperoxide lyase (HPLS) upon 13-hydroperoxylinoleic or 13-hydroperoxylinolenic acids. The developed procedure required no derivatization, was not affected by the presence of water, did not require cryogenic conditions to be maintained during injection, and allowed for the quantitation of most products. An acetone powder preparation of Chlorella pyrenoidosa cells was triturated with borate buffer pH = 8.0, and the mixture centrifuged at 12,000 x g. The supernatant and pellet were assayed for HPLS activity by GC-MS analysis of the volatile products given by linoleic acid hydroperoxide. The data showed that the majority of HPLS activity resides in the pellet fraction, and that the primary volatile component was pentane, with smaller amounts of 2-(Z)-pentene and 1-pentene being produced. The fact that HPLS activity resides in the water-insoluble fraction of the acetone powder suggests that HPLS from Chlorella is a membrane-associated enzyme. This investigation also determined that a spectrophotometric assay using alcohol dehydrogenase for measuring HPLS activity was not specific, but measured enzymatic activity other than HPLS.

Analysis of lipoxygenase kinetics by high-performance liquid chromatography with a polymer column.

Soybean lipoxygenase (LOX; EC 1.12.11.12) catalyzes the oxygenation of polyunsaturated fatty acids, acylglycerols and phosphoglycerols, producing a regio- and enantiospecific hydroperoxide product. The goal of this work was to measure the relative rate of LOX-catalyzed oxidation of mixtures of lipids containing linoleate, using high-performance liquid chromatography (HPLC) and a light-scattering detector (LSD). Previous literature suggested that reversed-phase HPLC with silica-based columns could be used for the separation of individual fatty acids, acylglycerols, phosphoglycerides and their oxidation products. However, these columns produced ineffective separations of phosphoglycerides unless choline chloride and a strong base, such as KOH, are present in the mobile phase. Such modifiers precluded the use of the LSD. It was found that a reversed-phase column based upon an organic polymer support, rather than on silica, was able to separate these mixtures with a ternary solvent gradient of methanol/water/acetonitrile without the need for the addition of modifiers. The oxidation time course of a mixture of linoleic acid, trilinolein and 1-linoleoyl-2-stearoyl-sn-glycero-3-phosphocholine was followed using the developed HPLC method. The results showed that trilinolein and phosphatidylcholine reacted at one-tenth the rate of linoleic acid. The diacylglycerol, 1,3-dilinolein, was oxidized at a rate that was approximately 40% that of linoleic acid, with the formation of mono- and dihydroperoxides as well as other unidentified products.

Indicator assay for amino acid decarboxylases.

Indicator assays have been devised for glutamate decarboxylase and arginine decarboxylase as models for a general procedure for amino acid decarboxylases. Since the decarboxylation results in the absorption of a proton in the pH range of maximum enzymatic activity, the change in absorbance of an acid-base indicator can be used to follow the progress of the reaction. For glutamate decarboxylase, the substrate was used as the buffer, and 1-1'-diethyl-2-2'-cyanine iodide was used as the indicator. For arginine decarboxylase, acetate was used as the buffer, and bromcresol green was used as the indicator. The change in absorption with extent of reaction is linear if the indicator pK is equal to the buffer pK.

Purification and Characterization of Calmodulins from Papaver somniferum and Euphorbia lathyris.

Calmodulins (CaM) were isolated and characterized from two well-known latex producing plants, Papaver somniferum and Euphorbia lathyris. The molecular weights of both were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 17,000 comparable to that of bovine brain CaM. Amino acid compositions also compared similarly with those of known CaMs, with regard to the presence of trimethyllysine and the ratio of phenylalanine to tyrosine. The Cornish-Bowden equation (SDeltan) revealed strong statistical correlations of P. somniferum and E. lathyris CaM with those of other plants and animals, although their amino acid compositions were not identical. Both plant CaM stimulated CaM dependent cAMP phosphodiesterase: for Papaver somniferum the K(a) was found to be 1.09 nanomolar and for Euphorbia lathyris, 2.01 nanomolar.

Enhancement of Terpenoid Biosynthesis from Mevalonate in a Fraction of the Latex from Euphorbia lathyris.

A latex pellet fraction from Euphorbia lathyris incorporates mevalonate into triterpenols and their fatty acid esters. Conditions for improved incorporation were determined. CaCl(2) or CaCl(2) plus MnCl(2) stimulated biosynthesis, and the metal ion chelator, ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) enhanced stimulation. Ethylenediaminetetraacetic acid was almost as effective as EGTA, but phthalic acid and citric acid were relatively poor stimulators. The concentration of the Ca(2+)-EGTA complex was directly measured, and the incorporation data are best fitted by a curve that shows that the receptor for the complex is saturable. In the presence of the metal-chelate complex, the addition of fructose, 1,6-bisphosphate plus aldolase (triose-P) or malate provided additional stimulation. Incorporation was maximum at 40 micromolar R-mevalonate, and inhibition occurred at higher concentrations. The apparent K(m) for R-mevalonate was 15 micromolar. Under improved reaction conditions, the rate of triterpenoid biosynthesis from mevalonate is 25 times faster than was previously observed (GJ Piazza, EJ Saggese, KM Spletzer [1987] Plant Physiol 83: 177-180).

Isolation and characterization of bovine mammary calmodulin.

Bovine mammary gland calmodulin, purified by conventional fractionation procedures, was compared with similarly purified bovine brain calmodulin. Affinity chromatography on W-7 agarose of the crude fractions from mammary gland and brain yielded pure proteins containing one trimethyllysine residue per 16,800 daltons with essentially identical amino acid compositions. Kinetic parameters of these two proteins with respect to their ability to activate phosphodiesterase were determined. The constants for half maximum activation were .39 and .44 nM for bovine brain and bovine mammary gland calmodulins, respectively; both proteins gave similar maximum velocities. Based on the amino acid composition and kinetic data, it is concluded that the two proteins are essentially identical.

Triterpene Biosynthesis in the Latex of Euphorbia lathyris: Effect of Calmodulin Antagonists and Chlorinated Phenoxy Compounds.

Recognized calmodulin antagonists and chlorinated phenoxyalkylamines were tested as inhibitors of mevalonate incorporation into triterpenols and their fatty acid esters in a centrifuged pellet from the latex of Euphorbia lathyris. The calmodulin antagonists, chlorpromazine (II), fluphenzine, and trifluoperazine were good inhibitors; I(50) values for II and trifluoperazine were 150 and 55 micromolar, respectively. Inhibition by the phenoxyalkylamines increased with increasing chlorine substitution, and I(50) for 2-(pentachlorophenoxy)ethyl N,N-diethylamine (IX) was 35 micromolar. The calmodulin-stimulated phosphodiesterase catalyzed hydrolysis of cAMP was used as an assay to quantitate the calmodulin antagonism of the tested compounds. Compounds II and IX were calmodulin antagonists over a concentration range similar to their effective range in the biosynthesis of triterpenes. The antagonism of the chlorinated phenoxy compounds increased in parallel to their inhibitory effect upon triterpene biosynthesis.

Triterpene Biosynthesis in the Latex of Euphorbia lathyris: Calmodulin Antagonists Are Ineffective in Whole Latex.

The calmodulin antagonists chlorpromazine, fluphenazine, trifluoperazine, and 2-(pentachlorophenoxy)ethyl N,N-diethylamine are not inhibitors of acetate incorporation into triterpenols (TOH) and their fatty acid esters (TE) in whole tapped latex from Euphorbia lathyris, although prior work demonstrated that these antagonists are good inhibitors of mevalonate incorporation into TOH and TE in a centrifuged pellet from the latex. Antagonist absorption into the endogenous terpene pool is the primary reason for antagonist ineffectiveness in whole latex; changes in the utilized substrate or chemical deactivation of the antagonists were ruled out as factors. A biosynthetically inactive, latex supernatant fraction containing the endogenous terpene pool was prepared. This fraction blocks antagonist action when added to the latex pellet, and proved to be a useful tool for demonstrating that inhibition of triterpene biosynthesis by a calmodulin antagonist is partially reversible.

Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach.

(14)CO(2) photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C(3) plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg(2+) were present in the assays, their influence upon CO(2) assimilation was also examined. Free Mg(2+) was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO(2) assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).PGA-supported O(2) evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O(2) evolution slowed as time passed, corresponding to the effect of ADP on CO(2) assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O(2) evolution became more severe with time. This did not mirror CO(2) assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O(2) evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O(2) evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO(2) assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.The appearance of label in the medium was measured when [U-(14)C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg(2+) complexes. The rate of back exchange was stimulated by the presence of Mg(2+). This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg(2+) complexes. A portion of the CO(2) assimilation and O(2) evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations.

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts.

Photoassimilation of (14)CO(2) by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO(2) assimilation occurred.The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.

Medium effects in enzyme-catalyzed decarboxylations.

Carbon isotope effects and steady-state kinetic parameters have been measured for the decarboxylation of arginine and homoarginine by the pyridoxal 5'-phosphate dependent arginine decarboxylase from Escherichia coli. In water at pH 5.25, 5 degrees C, homoarginine shows an isotope effect k12/k13 = 1.601, indicating that the decarboxylation step is entirely rate determining. In the presence of 16 mol % ethylene glycol under otherwise identical conditions, the decarboxylation rate is increased 3-fold, and the carbon isotope effect is 1.044, indicating that the rate of the decarboxylation step is increased by the presence of the less polar solvent. The decarboxylation or arginine under the same conditions shows a similar trend: in water, the isotope effect is 1.027, decreasing to 1.003 in 16% ethylene glycol, with little change in the steady-state rate. Again, the rate of the decarboxylation step is substantially increased by the presence of the nonpolar solvent. Thus, pyridoxal phosphate dependent enzymatic decarboxylations show a medium effect similar to that observed in a number of nonenzymatic decarboxylations. This suggests that these enzymes may accelerate the decarboxylation step by providing a nonpolar environment. Evidence is also presented that desolvation of the substrate carboxyl group may contribute to catalysis.