Rafael A. Garcia

Producing Docosahexaenoic Acid (DHA)-Rich Algae from Biodiesel-Derived Crude Glycerol : Effects of Impurities on DHA Production and Algal Biomass Composition

Crude glycerol is the primary byproduct of the biodiesel industry. Producing docosahexaenoic acid (DHA, 22:6 n-3) through fermentation of the alga Schizochytrium limacinum on crude glycerol provides a unique opportunity to utilize a large quantity of this byproduct. The objective of this work is to investigate the effects of impurities contained in the crude glycerol on DHA production and algal biomass composition. Crude glycerol streams were obtained from different biodiesel refineries. All of the glycerol samples contained methanol, soaps, and various elements including calcium, phosphorus, potassium, silicon, sodium, and zinc. Both methanol and soap were found to negatively influence algal DHA production; these two impurities can be removed from culture medium by evaporation through autoclaving (for methanol) and by precipitation through pH adjustment (for soap). The glycerol-derived algal biomass contained 45-50% lipid, 14-20% protein, and 25% carbohydrate, with 8-13% ash content. Palmitic acid (C16:0) and DHA were the two major fatty acids in the algal lipid. The algal biomass was rich in lysine and cysteine, relative to many common feedstuffs. Elemental analysis by inductively coupled plasma showed that boron, calcium, copper, iron, magnesium, phosphorus, potassium, silicon, sodium, and sulfur were present in the biomass, whereas no heavy metals (such as mercury) were detected in the algal biomass. Overall, the results show that crude glycerol was a suitable carbon source for algal fermentation. The crude glycerol-derived algal biomass had a high level of DHA and a nutritional profile similar to that of commercial algal biomass, suggesting a great potential for using crude glycerol-derived algae in omega-3-fortified food or feed.

Use of Biodiesel-Derived Crude Glycerol for Producing Eicosapentaenoic Acid (EPA) by the Fungus Pythium irregulare

Crude glycerol is a major byproduct for the biodiesel industry. Producing value-added products through microbial fermentation on crude glycerol provides opportunities to utilize a large quantity of this byproduct. The objective of this study is to explore the potential of using crude glycerol for producing eicosapentaenoic acid (EPA, 20:5 n-3) by the fungus Pythium irregulare . When P. irregulare was grown in medium containing 30 g/L crude glycerol and 10 g/L yeast extract, EPA yield and productivity reached 90 mg/L and 14.9 mg/L x day, respectively. Adding pure vegetable oils (flaxseed oil and soybean oil) to the culture greatly enhanced the biomass and the EPA production. This enhancement was due to the oil absorption by the fungal cells and elongation of shorter chain fatty acids (e.g., linoleic acid and alpha-linolenic acid) into longer chain fatty acid (e.g., EPA). The major impurities contained in crude glycerol, soap and methanol, were inhibitory to fungal growth. Soap can be precipitated from the liquid medium through pH adjustment, whereas methanol can be evaporated from the medium during autoclaving. The glycerol-derived fungal biomass contained about 15% lipid, 36% protein, and 40% carbohydrate, with 9% ash. In addition to EPA, the fungal biomass was also rich in the essential amino acids lysine, arginine, and leucine, relative to many common feedstuffs. Elemental analysis by inductively coupled plasma showed that aluminum, calcium, copper, iron, magnesium, manganese, phosphorus, potassium, silicon, sodium, sulfur, and zinc were present in the biomass, whereas no heavy metals (such as mercury and lead) were detected. The results show that it is feasible to use crude glycerol for producing fungal biomass that can serve as EPA-fortified food or feed.

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.

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.

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.

Structural and thermal stability of β-lactoglobulin as a result of interacting with sugar beet pectin.

Changes in the structural and thermal stability of β-lactoglobulin (β-LG) induced by interacting with sugar beet pectin (SBP) have been studied by circular dichroism (CD), Fourier transform infrared, and steady-state as well as time-resolved fluorescence spectroscopic techniques. It has been demonstrated that SBP not only is capable of binding to native β-LG but also causes a significant loss in antiparallel β-sheet, ∼10%, accompanied by an increase in either random coil or turn structures. In addition, the interaction also disrupted the environments of all aromatic residues including Trp, Phe, and Tyr of β-LG as evidenced by near-UV CD and fluorescence. When preheated β-LG was combined with SBP, the secondary structure of β-LG was partially recovered, ∼4% gain in antiparallel β-sheet, and Trp19 fluorescence was recovered slightly. Although forming complexes with SBP did not significantly impact the thermal stability of individual secondary structural elements of β-LG, the environment of Trp19 was protected considerably.

Preparation of starch-poly-glutamic acid graft copolymers by microwave irradiation and the characterization of their properties.

Graft copolymers of waxy maize starch and poly-γ-glutamic acid (PGA) were produced in an aqueous solution using microwave irradiation. The microwave reaction conditions were optimized with regard to temperature and pH. The temperature of 180°C and pH7.0 were the best reaction conditions resulting in a PGA graft of 0.45% based on nitrogen analysis. The average graft content and graft efficiency for the starch-PGA graft copolymer prepared at 180°C and pH7.0 were 4.20% and 2.73%, respectively. The starch-PGA graft copolymer produced at 180°C and pH7.0 could absorb more than 20 times its own weight amount of water and form a gel. The preliminary rheology study revealed that the starch-PGA graft copolymer gel exhibited viscoelastic solid behavior while the control sample of waxy starch showed viscoelastic liquid behavior.

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.

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.