The relationship between extent of hemoglobin purification and the performance characteristics of a blood-based flocculant.

BACKGROUND: Whole blood is a highly complex substance. Hemoglobin, the most abundant blood protein, can function as a flocculant; most of the other blood components exhibit poor flocculant activity. For the purpose of processing raw whole blood into a flocculant product, the practical value of hemoglobin purification is uncertain. RESULTS: This study compares the flocculant performance of whole blood to that of three different semi-purified hemoglobin preparations. The whole blood is processed to remove the plasma proteins, the solid cell components, or both. The flocculant performance of whole blood and each hemoglobin preparation is compared over wide ranges of flocculant dose and suspension pH. The clarified liquids are examined for increases in chemical oxygen demand and Kjeldahl nitrogen. Hemoglobin preparations that excluded plasma gave peak flocculation performance at approximately 30 mg solids per gram of suspended kaolin, and gave greatly reduced performance at higher doses; preparations that included plasma gave very similar peak performance, but also maintained relatively high performance at doses up to at least 200 mg g-1 . CONCLUSION: It is shown that removal of the plasma and the cell solids does not improve the flocculant performance or lessen the residual pollutants in the treated water. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Identification of highly active flocculant proteins in bovine blood.

Synthetic polymeric flocculants are used extensively for wastewater remediation, soil stabilization, and reduction in water leakage from unlined canals. Sources of highly active, inexpensive, renewable flocculants are needed to replace synthetic flocculants. High kaolin flocculant activity was documented for bovine blood (BB) and blood plasma with several anticoagulant treatments. BB serum also had high flocculant activity. To address the hypothesis that some blood proteins have strong flocculating activity, the BB proteins were separated by SEC. Then, the major proteins of the flocculant-active fractions were separated by SDS-PAGE. Identity of the major protein components was determined by tryptic digestion and peptide analysis by MALDI TOF MS. The sequence of selected peptides was confirmed using TOF/TOF-MS/MS fragmentation. Hemoglobin dimer (subunits α and ß) was identified as the major protein component of the active fraction in BB; its high flocculation activity was confirmed by testing a commercial sample of hemoglobin. In the same manner, three proteins from blood plasma (fibrinogen, γ-globulin, α-2-macroglobulin) were found to be highly active flocculants, but bovine serum albumin, α-globulin, and ß-globulin were not flocculants. On a mass basis, hemoglobin, γ-globulin, α-2-macroglobulin were as effective as anionic polyacrylamide (PAM), a widely used synthetic flocculant. The blood proteins acted faster than PAM, and unlike PAM, the blood proteins flocculants did not require calcium salts for their activity.

Nonfeed application of rendered animal proteins for microbial production of eicosapentaenoic acid by the fungus Pythium irregulare.

Rendered animal proteins are well suited for animal nutrition applications, but the market is maturing, and there is a need to develop new uses for these products. The objective of this study is to explore the possibility of using animal proteins as a nutrient source for microbial production of omega-3 polyunsaturated fatty acids by the microalga Schizochytrium limacinum and the fungus Pythium irregulare. To be absorbed by the microorganisms, the proteins needed to be hydrolyzed into small peptides and free amino acids. The utility of the protein hydrolysates for microorganisms depended on the hydrolysis method used and the type of microorganism. The enzymatic hydrolysates supported better cell growth performance than the alkali hydrolysates did. P. irregulare displayed better overall growth performance on the experimental hydrolysates compared to S. limacinum. When P. irregulare was grown in medium containing 10 g/L enzymatic hydrolysate derived from meat and bone meal or feather meal, the performance of cell growth, lipid synthesis, and omega-3 fatty acid production was comparable to the that of culture using commercial yeast extract. The fungal biomass derived from the animal proteins had 26-29% lipid, 32-34% protein, 34-39% carbohydrate, and <2% ash content. The results show that it is possible to develop a nonfeed application for rendered animal protein by hydrolysis of the protein and feeding to industrial microorganisms which can produce omega-3 fatty acids for making omega-3-fortified foods or feeds.

Rendered-protein hydrolysates for microbial synthesis of cyanophycin biopolymer.

Cyanophycin is a poly(arginyl-aspartate) biopolymer produced and stored intracellularly by bacteria. Cyanophycin has been proposed as a renewable replacement for petrochemical-based industrial products. An abundant source of amino acids and nitrogen such as in the form of protein hydrolysates is needed for the biosynthesis of cyanophycin. Rendered proteins are largely used as a feed supplement in animal husbandry and aquaculture. New uses would expand the market size of this class of protein coproducts. We prepared and thoroughly characterized the hydrolysates of meat and bone meal, and proceeded to demonstrate for the first time that these hydrolysates could be used in the fermentative production of cyanophycin. Using the enzyme-hydrolyzed meat and bone meal preparation, we obtained crude cyanophycin product at 33-35% level of that produced using the reference casamino acids in both shake-flask and 10-L bioreactor fermentation studies. Polyacrylamide-gel electrophoresis of the cyanophycin under denaturing conditions showed the molecular weight of the isolated polyamide at 24kDa. Our results open a new avenue for the utilization of rendered protein coproducts to produce the cyanophycin biopolymer.

The non-nutritional performance characteristics of peptones made from rendered protein.

Economic considerations require the use of inexpensive feedstocks for the fermentative production of moderate-value products. Our previous work has shown that peptones capable of supporting the growth of various microorganisms can be produced from inexpensive animal proteins, including meat and bone meal, feather meal, and blood meal, through alkaline or enzymatic hydrolysis. In this work, we explore how these experimental peptones compare to commercial peptones in terms of performance characteristics other than chemical make-up; these characteristics can impact fermentation operating cost. It is shown that experimental peptone powders produced through enzymatic hydrolysis are highly hygroscopic and that their physical form is not stable to humid storage conditions; those produced through alkaline hydrolysis and commercial peptones are less hygroscopic. When used in growth medium, all peptones contribute haze to the solution; experiments show that the source of haze is different when using enzyme- versus alkali-hydrolyzed peptones. Alkali-hydrolyzed peptones and all peptones made from blood meal are stronger promoters of media foaming than the commercial peptones; some enzyme-hydrolyzed peptones support very little foam formation and are superior to the commercial peptones in this sense. Alkali-hydrolyzed peptones are roughly equivalent to commercial peptones in the coloration they contribute to media, while enzyme-hydrolyzed peptones contribute intense coloration to media. No peptone caused a significant change in the viscosity of media. The experimental peptones studied here may be acceptable low-cost substitutes for commercial peptones, but none is equivalent to the commercial products in all respects.

Isolation of unsaturated diols after oxidation of conjugated linoleic acid with peroxygenase.

Oat seeds are a rich source of peroxygenase, an iron heme enzyme that participates in oxylipin metabolism in plants. An isomer of CLA, 9(Z),11 (E)-octadecadienoic acid (1), believed to have anticarcinogenic activity, was used as a substrate for peroxygenase in an aqueous medium using t-butyl hydroperoxide as the oxidant. After acidification of the reaction medium, the products were extracted with ethyl ether, converted to their methyl esters, and characterized using HPLC. Major products after reaction for 24 h showed resonances from 1H NMR spectroscopy that were further downfield than the expected epoxides and were thought to be diol hydrolysis products. However, analyses by HPLC with atmospheric pressure chemical ionization MS (APCI-MS) of the putative allylic diols or their bis-trimethylsilyl ether derivatives gave incorrect M.W. The M.W. of the diols could be obtained by APCI-MS after removal of unsaturation by hydrogenation or by EI-MS after conversion of the allylic 1,2-diols to cyclic methyl boronic esters. Data from MS in conjunction with analyses using 1H and 13C NMR showed that the methylated products from 1 were methyl 9,10(threo)-dihydroxy-11 (E)-octadecenoate, methyl 9,10(erythro)-dihydroxy-11(E)-octadecenoate, methyl 9,12(erythro)-dihydroxy-10(E)-octadecenoate, and methyl 9,12(threo)-dihydroxy-10(E)-octadecenoate. Solid-phase extraction without prior acidification and conversion of the products to methyl esters allowed identification of the following epoxides: methyl 9,10(Z)-epoxy-11 (E)-octadecenoate (6M), methyl 9,10(E)-epoxy-11 (E)-octadecenoate, and methyl 11,12(E)-epoxy-9(Z)-octadecenoate. At times of up to at least 6 h, 6M accounted for approximately 90% of the epoxide product. Product analysis after the hydrolysis of isolated epoxide 6M showed that hydrolysis of epoxide 6 could largely account for the diol products obtained from the acidified reaction mixtures.

Purification of lipoxygenase from Chlorella: production of 9- and 13-hydroperoxide derivatives of linoleic acid.

Oxygenation of linoleic acid by the enzyme lipoxygenase (LOX) that is present in the microalga Chlorella pyrenoidosa is known to produce the corresponding 9- and 13-hydroperoxide derivatives of linoleic acid (9- and 13-HPOD, respectively). Previous work with this microalga indicated that partially purified LOX, present in the 30-45 and 45-80% saturated (NH4)2SO4 precipitate fractions, produced both HPOD isomers but in different ratios. It was not clear, however, if the observed activity in the two isolates represented the presence of one or more isozymes. In the present work, LOX isolated from the intracellular fraction of Chlorella by (NH4)2SO4 precipitation (35-80% saturated) was purified by ion exchange and hydrophobic interaction chromatography to apparent homogeneity. Analysis of the purified protein by SDS-PAGE and subsequent native size exclusion chromatography demonstrated that LOX in Chlorella is a single monomeric protein with a molecular mass of approximately 47 kDa. The purified LOX produced both the 9-HPOD and 13-HPOD isomers from linoleic acid in equal amounts, and the isomer ratio was not altered over the pH range of 6 to 9. Optimal activity of LOX was at pH 7.5.