Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16

Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid–water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg[superscript 2+], Ca[superscript 2+], and Mn[superscript 2+] were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.Malaysia-MIT Biotechnology Partnership Programm

ESolvent-free, enzyme-catalyzed biodiesel production from mango, neem, and shea oils via response surface methodology

Mango, neem and shea kernels produce non-conventional oils whose potentials are not fully exploited. To give an added value to these oils, they were transesterified into biodiesel in a solvent-free system using immobilized enzyme lipozyme from Mucor miehei. The Doehlert experimental design was used to evaluate the methyl ester (ME) yields as influenced by enzyme concentration—EC, temperature—T, added water content—AWC, and reaction time—RT. Biodiesel yields were quantified by (1)H NMR spectroscopy and subsequently modeled by a second order polynomial equation with interactions. Lipozyme enzymes were more tolerant to high temperatures in neem and shea oils reaction media compared to that of mango oil. The optimum reaction conditions EC, T, AWC, and RT assuring near complete conversion were as follows: mango oil 7.25 %, 36.6 °C, 10.9 %, 36.4 h; neem oil EC = 7.19 %, T = 45.7 °C, AWC = 8.43 %, RT = 25.08 h; and shea oil EC = 4.43 %, T = 45.65 °C, AWC = 6.21 % and RT = 25.08 h. Validation experiments of these optimum conditions gave ME yields of 98.1 ± 1.0, 98.5 ± 1.6 and 99.3 ± 0.4 % for mango, neem and shea oils, respectively, which all met ASTM biodiesel standards

Treatment costs of ammonia recovery from biogas digestate by air stripping analyzed by process simulation

Ammonia can be recovered from liquid biogas digestate as ammonium sulfate by air stripping. A full-scale plant treating 30,000 kg h−1 of digestate was simulated in Aspen Plus.The classical sequence stripper–absorber was modified by introducing a flash drum to reduce the buffer capacity. The stripped digestate was mixed with NaOH up to a pH value of 9 and fed to the stripping column using air at 90 °C. The air flow rate was optimized considering the possible recycle from the absorption column. The complete process was modeled using the process simulator Aspen Plus considering a recovery of ammonia equal to 95%. The total capital investment was evaluated by means of Aspen Plus Process Economic Analyzer in about 5.5 million US$, and the costs of energy and chemicals were quantified in less than 1 million US$ year−1. A benefit of 1.6 US$for m3 of digestate was obtained considering an ammonium sulfate price of 522 US$ ton−1.</p