Thomas A. Foglia

Lipase-catalyzed production of biodiesel

Lipases were screened for their ability to transesterify triglycerides with short-chain alcohols to alkyl esters. The lipase fromMucor miehei was most efficient for converting triglycerides to their alkyl esters with primary alcohols, whereas the lipase fromCandida antarctica was most efficient for transesterifying triglycerides with secondary alcohols to give branched alkyl esters. Conditions were established for converting tallow to short-chain alkyl esters at more than 90% conversion. These same conditions also proved effective for transesterfying vegetable oils and high fatty acid-containing feedstocks to their respective alkyl ester derivatives.

Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates).

Aside from their importance to the survival and general welfare of mankind, agriculture and its related industries produce large quantities of feedstocks and coproducts that can be used as inexpensive substrates for fermentative processes. Successful adoption of these materials into commercial processes could further the realization of a biorefinery industry based on agriculturally derived feedstocks. One potential concept is the production of poly(hydroxyalkanoate) (PHA) polymers, a family of microbial biopolyesters with a myriad of possible monomeric compositions and performance properties. The economics for the fermentative production of PHA could benefit from the use of low-cost agricultural feedstocks and coproducts. This mini-review provides a brief survey of research performed in this area, with specific emphasis on studies describing the utilization of intact triacylglycerols (vegetable oils and animal fats), dairy whey, molasses, and meat-and-bone meal as substrates in the microbial synthesis of PHA polymers.

Immobilized lipase‐catalysed production of alkyl esters of restaurant grease as biodiesel

Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases as biocatalysts. The lipases studied included those of Thermomyces lanuginosa and Candida antarctica supported on granulated silica (gran‐T.l. and gran‐C.a., respectively), C. antarctica supported on a macroporous acrylic resin (SP435) and Pseudomonas cepacia immobilized within a phyllosilicate sol‐gel matrix (IM PS‐30). All alcoholysis reactions were carried out in solvent‐free media employing a one‐step addition of the alcohol to the reaction system. Of the lipases studied, IM PS‐30 was found to be the most effective in catalysing the methanolysis and ethanolysis of grease. The processes catalysed by gran‐T.l. and gran‐C.a. lipases gave poor conversions to esters, and the SP435‐catalysed reactions gave intermediate yields of ethyl and methyl esters. Water activity (aw) was an important factor in the methanolysis reactions; reaction media with aw<0.5 resulted in the highest conversions to methyl esters. Molecular sieves also improved methyl ester yields by as much as 20% in transesterification reactions catalysed by IM PS‐30. The immobilized lipases also were evaluated for their ability to produce alkyl esters of grease with several additional normal and branched‐chain alcohols.

The microbial production of poly(hydroxyalkanoates) from tallow

Abstract The bacteria Pseudomonas oleovorans, P. resinovorans, P. putida, and P. citronellolis were evaluated for their ability to grow and produce poly(hydroxyalkanoates) (PHA) using tallow free fatty acids and tallow triglyceride as carbon substrates. Tallow free fatty acids supported cell growth and PHA production for all four organisms, yielding PHA contents of 18%, 15%, 19% and 3% of their cell dry weights for P. oleovorans, P.␣resinovorans, P. putida, and P. citronellolis respectively. Only P. resinovorans, however, was able to grow and produce PHA polymer, with cells attaining a PHA content of 15% of their cell dry weight, using unhydrolyzed tallow as the substrate. Extracts from 46-h cultures of P. resinovorans were found to have a higher esterase activity (12.80 units μl−1min−1) compared to the activities found for cultures of P. oleovorans, P. citronellolis, and P. putida ( < 0.03 units μl−1min−1). Polymer repeat-unit compositions were determined by GC analysis of the β-hydroxymethyl esters of hydrolyzed PHA, and ranged in carbon-chain lengths from C4 to C14, with some mono-unsaturation in the C12 and C14 side-chains. PHA compositions were similar for the polymers obtained from all four organisms, with repeat units of chain lengths C8 and C10 predominating.

Enzymatic interesterification of tallow-sunflower oil mixtures

In an effort to improve the physical and/or thermal characteristics of solid fats, the enzymatic interesterification of tallow and butterfat with high-oleic sunflower oil and soybean oil was investigated. The two simultaneously occurring reactions, interesterification and hydrolysis, were followed by high-performance liquid chromatography of altered glycerides and by gas-liquid chromatography of liberated free fatty acids. The enzymes used in these studies were immobilized lipases that included either a 1,3-acyl-selective lipase or acis-9-C18-selective lipase. The degree of hydrolysis of the fat/oil mixtures was dependent upon the initial water content of the reaction medium. The extent of the interesterification reaction was dependent on the amount of enzyme employed but not on the reaction temperature over the range of 50–70°C. Changes in melting characteristics of the interesterified glyceride mixtures were followed by differential scanning calorimetry of the residual mixed glycerides after removal of free fatty acids. Interesterification of the glyceride mixes with the two types of enzymes allowed for either a decrease or increase in the solid fat content of the initial glyceride mix.

Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction

Abstract A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding for type II polyhydroxyalkanoate (PHA) synthases. The primer-pair, I-179L and I-179R, was based on the highly conserved sequences found in the coding regions of Pseudomonas phaC1 and phaC2 genes. Purified genomic DNA or lysate of colony suspension can serve equally well as the target sample for the PCR, thus affording a simple and rapid screening of phaC1/C2-containing microorganisms. Positive samples yield a specific 540-bp PCR product representing partial coding sequences of the phaC1/C2 genes. Using the PCR method, P. corrugata 388 was identified for the first time as a medium-chain-length (mcl)-PHA producer. Electron microscopic study and PHA isolation confirmed the production of mcl-PHA in P. corrugata 388. The mcl-PHA of this organism has a higher molecular weight than that of similar polymers produced by other pseudomonads.

Poly(hydroxyalkanoate) biosynthesis from triglyceride substrates

Abstract The biosynthesis of poly(hydroxyalkanoates) (PHA) by Pseudomonas resinovorans from triglyceride substrates was investigated. Each triglyceride, whether animal fat or vegetable oil, supported cellular growth to relatively high average cell yields (3.3 ± 0.2 g/l). PHA yields ranged from 1.1 g/l to 2.1 g/l, representing approximately 45% of the bacterial cell dry weight. The repeat-unit composition of the polymers was determined by gas chromatography (GC) and GC/mass spectrometry of the β-hydroxyalkanoate methyl esters from the hydrolyzed polymers. With the exception of PHA from soybean oil (PHA-soy), each polyester was composed of β-hydroxyacyl moieties with chain lengths ranging from C4 to C14, with C8 and C10 being the predominant species. PHA-soy contained an additional fraction (2%) of C16 monomers. The alkyl side-chains of the PHA contained varying degrees of unsaturation. PHA from coconut oil was composed entirely of saturated side-chains, whereas PHA-soy contained 4.2 mol% olefinic groups in its side-chains. The increase in the degree of side-chain unsaturation caused decreased melting temperatures, enthalpies of fusion, and glass transition temperatures. The molar masses of the polymers were relatively constant and ranged from 6.5 × 104 to 10.1 × 104 g/mol.

Heat Resistance and Fatty Acid Composition of Listeria monocytogenes: Effect of pH, Acidulant, and Growth Temperature

The objective of this study was to determine the influence of pH, acidulant, and growth temperature history on the heat resistance and fatty acid composition of Listeria monocytogenes Scott A. Cells were grown to late exponential phase (OD600 = 0.6) at 10, 19, or 37 degrees C in brain heart infusion broth acidified to pH 5.4 or 7 with either acetic or lactic acid. Thermal death times at 60 degrees C subsequently were determined by using a submerged-coil heating apparatus. The surviving cell population was enumerated by spiral-plating heated samples onto tryptic soy agar supplemented with 0.6% yeast extract and 1% sodium pyruvate. The thermal resistance of cells cultured at a particular temperature was significantly lower (P < 0.05) when lactic acid was used to acidify the medium of pH 5.4. Regardless of acid identity, D values significantly decreased (P < 0.05) with increased growth temperature when the pH of the growth medium was 5.4, whereas D values significantly increased (P < 0.05) with increased temperature at pH 7. At pH 5.4 adjusted with lactic acid, D values were 1.30, 1.22, and 1.14 min for cells grown at 10, 19, and 37 degrees C, respectively. At pH 5.4 adjusted with acetic acid, L. monocytogenes failed to grow at 10 degrees C; the D values were 1.32 and 1.22 min when the cells were grown at 19 and 37 degrees C, respectively. At pH 7, the D values were 0.95, 1.12, and 1.28 min with lactic acid and 0.83, 0.93, and 1.11 min with acetic acid at 10, 19, and 37 degrees C, respectively. The most abundant fatty acids (44 to 82%) were branched-chain saturated fatty acids (anteiso-and iso-C15:0 and iso-C17:0) regardless of pH, acidulant, or growth temperature. However, there was an increase in C15:0 isomers at the expense of iso-C17:0 when the growth temperature was lowered from 37 to 10 degrees C. While variable changes in longer-chain fatty acids were found, the percentage of longer-chain (C16 and C18) fatty acids was greatest when L. monocytogenes was grown at 37 degrees C regardless of pH or acidulant. This study demonstrates that the heat resistance of L. monocytogenes depends upon its growth conditions.

Analysis and Characterization of Sophorolipids by Liquid Chromatography with Atmospheric Pressure Chemical Ionization

SummaryA reversed phase high performance liquid chromatographic method combined with atmospheric pressure chemical ionization mass detection (LC/APCI-MS) has been developed for the separation and analysis of sophorolipids produced byC. bombicola when grown on fatty acid mixtures. Using this method it was found that the incorporation of palmitic, linoleic, and linolenic acids into the sophorolipid structure was dependent on the initial fatty acid, content of these acids, whereas the incorporation of oleic acid, was independent of its initial content in the mixture. Also observed was the incorporation of esterified glycerol into the sophorolipid structure, which has not been reported previously.

Decarbonylation dehydration of fatty acids to alkenes in the presence of transition metal complexes

The catalytic decarbonylation of stearic acid into a mixture of heptadecenes has been accomplished by use of a catalyst system comprised of rhodium trichloride (RhCl3) and triphenylphosphine. The isomeric composition of the heptadecenes formed was found, by spectroscopic and chemical methods, to be dependent upon the type of rhodium catalyst employed. Anhydrous RhCl3 produced about the same amount of 2-heptadecene, twice the amount of 3-heptadecene, and about half the amount of 1-heptadecene as hydrated RhCl3. The active catalytic species formed in situ has been identified as the rhodium I complex [(C6H5)3P]2Rh(CO)Cl. A mechanistic pathway to account for the observed decarbonylation products employing the above catalyst is presented.