Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent

A new enzymatic method of synthesizing methyl esters from plant oil and methanol in a solvent-free reaction system was developed. It is anticipated that such plant oil methyl esters can be used as a biodiesel fuel in the future. Lipase from Rhizopus oryzae efficiently catalyzed the methanolysis of soybean oil in the presence of 4-30 wt% water in the starting materials; however the lipase was nearly inactive in the absence of water. The methyl ester (ME) content in the reaction mixture reached 80-90 wt% by stepwise additions of methanol to the reaction mixture. The kinetics of the reaction appears to be in accordance with the successive reaction mechanism. That is, the oil is first hydrolyzed to free fatty acids and partial glycerides, and the fatty acids produced are then esterified with methanol. Although R. oryzae lipase is considered to exhibit 1(3)-regiospecificity, a certain amount of 1,3-diglyceride was obtained during the methanolysis and hydrolysis of soybean oil by R. oryzae lipase solution. Therefore, the high ME content in the reaction mixture is probably attributable to the acyl migration from the sn-2 position to the sn-1 or sn-3 position in partial glycerides.     

Efficient production of fatty acid methyl ester from waste activated bleaching earth using diesel oil as organic solvent

Fatty acid methyl ester (FAME) production from waste activated bleaching earth (ABE) discarded by the crude oil refining industry was investigated using fossil fuel as a solvent in the esterification of triglycerides. Lipase from Candida cylindracea showed the highest stability in diesel oil. Using diesel oil as a solvent, 3 h was sufficient to obtain a yield of approximately 100% of FAME in the presence of 10% lipase from waste ABE. Kerosene was also a good solvent in the esterification of triglycerides embedded in the waste ABE. Fuel analysis showed that the FAME produced using diesel oil as a solvent complied with the Japanese diesel standard and the 10% residual carbon amount was lower than that of FAME produced using other solvents. Use of diesel oil as solvent in the FAME production from the waste ABE simplified the process, because there was no need to separate the organic solvent from the FAME-solvent mixture. These results demonstrate a promising reutilization method for the production of FAME, for use as a biodiesel, from industrial waste resources containing waste vegetable oils.     

高酸值麻疯树籽油制备生物柴油的杂多酸催化预酯化研究

以探索生物柴油制备中预酯化的绿色技术为目的,研究了杂多酸H3PW12O40催化预酯化高酸值麻疯树籽油。以单因素实验考察了酯化反应中各因素对酯化率的影响,得到H3PW12O40催化预酯化的最佳条件为:反应温度65℃,反应时间3 h,醇油物质的量比9∶1,催化剂H3PW12O40用量1%。在最佳条件下,麻疯树籽油的酯化率为96.1%,酸值(KOH)降至0.61 mg/g。提出了高酸值麻疯树籽油制备生物柴油中杂多酸催化预酯化的工艺路线,分析显示工艺具有一定优势。     

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27°C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.     

Catalytic properties of a lipase from Photobacterium lipolyticum for biodiesel production containing a high methanol concentration

Biodiesel, an alternative fuel, is generated via the transesterification reaction of vegetable oil or animal oil with alcohol. Currently, many reports have noted that microbial lipases might be utilized for the production of biodiesel. Among them, immobilized Candida antarctica lipase B (Novozym435) is frequently utilized for its biocatalytic efficiency and availability. However, as the enzyme is unstable in a medium containing high concentrations of methanol, a multi-stepwise methanol supply is required for the efficient production of biodiesel. Photobacterium lipolyticum lipase (M37) was determined to be quite stable in a medium containing a high concentration of methanol. The enzyme activity was maintained for longer than 48 h without any loss at a methanol concentration of 10%. In an effort to evaluate enzyme performance in the production of biodiesel, we have compared M37 lipase and Novozym435 in the biodiesel production reaction using fresh or waste oil and methanol. In the 3-stepwise methanol feeding method generally conducted for Novozym435 in biodiesel production, the M37 lipase showed a similar or superior conversion yield to Novozym435. However, the M37 lipase evidenced significantly higher conversion yields in the 2 and 1 step methanol feeding reactions. Particularly in the 1 step process using 10% of methanol where almost no conversion was detected by Novozym435, the biodiesel yield achieved with M37 lipase reached a level of up to 70% of the possible maximum yield. Consequently, this methanol-tolerant lipase, M37, has been shown to be a suitable enzyme for use in the biodiesel production process.     

Enhancement of l(+)-lactic acid production using acid-adapted precultures of Rhizopus arrhizus in a bubble column reactor

88 g/L lactic acid was produced from waste potato starch (equivalent to 100 g/L glucose) in a bubble column reactor using appropriate acid-adapted precultures of Rhizopus arrhizus. Further experiment showed that repeated dilution of cultures caused the decrease of lactic acid concentration and productivity due to formation of large fungal pellets.     

植物生长调节剂对城市污水中小球藻生长和油脂积累的影响

研究了不同质量浓度植物生长调节剂(IAA、TRIA、6-BA、2,4-D)对蛋白核小球藻(Chlorella pyrenoidosa)生长和油脂积累的影响,并从藻细胞形态,脂肪酸组成及元素含量等方面对机理进行了探讨。结果表明:2,4-D对城市污水中小球藻生长的促进作用最为显著,当2,4-D的质量浓度为1.5 mg/L时小球藻干重和油脂产量分别达到最大值,比空白对照组分别提高了30.77%和51.43%。利用扫描电镜观察可以发现处理组小球藻个体大小略大于空白对照组。通过对生成的脂肪酸甲酯进行气相色谱分析,结果显示植物生长调节剂的投加对小球藻脂肪酸组成影响不大,但提升了单不饱和脂肪酸含量,有利于提高所得生物柴油的品质。     

Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium and Staphylococcus aureus in a food model system and on the bacterial cell membranes

The effects of different concentrations of Zataria multiflora Boiss. essential oil (EO: 0, 5, 15 and 30 μl 100 ml⁻¹) and nisin (N: 0, 0.25 and 0.5 μg ml⁻¹), temperatures (T: 25 and 8 °C), and storage times (up to 21 days) on growth of Salmonella typhimurium and Staphylococcus aureus in a commercial barley soup were evaluated in a factorial design study. The growth of S. typhimurium was significantly (P < 0.05) decreased by EO concentrations and their combinations with N concentrations at 8 °C. For S. aureus, the viable count was significantly (P < 0.05) inhibited by EO and N concentrations and their combinations, incubated at both storage temperatures. The mechanism of the antimicrobial action of EO, N, and their combinations against cell membranes of the tested organisms were also studied by measurement of the release of cell constituents and by the electronic microscopy observations of the cells. The significant increase of the cell constituents’ release of both organisms was observed as a result of treatments with EO and EO in combination with N. Electronic microscopy observations revealed that the cell membranes of S. typhimurium treated by EO and EO in combination with N were significantly damaged, while cells treated with only N looked similar to untreated cells. The electron micrographs of treated cells of S. aureus with EO, N, and their combination also showed important morphological damages and disrupted membranes.     

Effect of an additionally introduced degQ gene on di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) production by recombinant Bacillus subtilis in a single culture production system

Di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) was produced directly from sucrose using a single culture of recombinant Bacillus subtilis 168 carrying the levan fructotransferase (lft) gene. In this study, three plasmids carrying the degQ36 gene, which is a degQ allele of B. subtilis (degQ36) with a degQ36 mutation on its promoter, were constructed to overproduce intact DegQ in B. subtilis 168. The transformant B. subtilis/pHT-D36 (with the degQ36 gene) consumed sucrose and produced levan at a higher rate than B. subtilis/pHT43 (without the degQ36 gene). The transformant B. subtilis/pLFT-GD36, carrying the lft and degQ36 genes, also consumed sucrose at a higher rate and produced more DFA IV than B. subtilis/pLFT-G, carrying the lft but without the degQ36 gene. B. subtilis/pLFT-GD36 produced 43.5 g/l of DFA IV and consumed 240 g/l of sucrose (96% of added sucrose) by 72 h of cultivation, whereas B. subtilis/pLFT-G produced 23.4 g/l of DFA IV with 76.9 g/l of sucrose still remaining in the system. Sucrose-inducible expression vectors were also constructed, which made it possible to produce DFA IV without IPTG induction. Using these vectors, sucrose consumption rates were enhanced and DFA IV production was increased upon introduction of the degQ36 gene. From these results, it can be concluded that the additionally introduced regulatory gene, degQ, was able to stimulate sucrose conversion to levan, and therefore increased DFA IV production in this system.     

Inactivation kinetics of a piezotolerant Staphylococcus aureus isolated from high-pressure-treated sliced ham by high pressure in buffer and in a ham model system: Evaluation in selective and non-selective medium

The kinetics of inactivation by high pressure of a pressure-resistant strain of Staphylococcus aureus isolated from pressure-treated packaged sliced ham, in buffer and in a ham model system was studied. Selective (BP agar) and enrichment media (BHI agar) were used for enumeration in order to count healthy and sublethally injured cells of the pathogen. A first-order kinetic inactivation was observed in both suspension media, and a very significant increase in D values was apparent when the microorganism was suspended and pressurized in the model food system compared to buffer. In the case of phosphate buffer as suspension medium, the z_p values obtained were 107.5 and 113.6 MPa for the two recovery media, i.e. BP and BHI agars, respectively. In contrast, in the case of the food model system, a two-phase linear relation was apparent and the PDT (Pressure Death Time) curve can be divided into two linear sections, so that two z_p values could be defined, one for each section. Z_p values of 100 and 79.4 MPa correspond to pressures < 500 MPa for the BP and BHI counts, respectively, while z_p values of 416.7 and 333.3 MPa correspond to higher pressures > 500 MPa for the selective and non-selective medium, respectively. When S. aureus had been pressurized in phosphate buffer, the BHI agar was slightly better in cell recovery, while in the case of the ham model system, the BP agar proved superior and gave significantly higher colony counts.

Industrial relevance

The paper provides significant information for the food processing industry as it deals with the effect of high-pressure technology on a piezotolerant pathogen that may survive in sliced ham. This technology is already applied in ham products and this paper supports the need for the use of real food in pressure studies in order to avoid underestimation of the effect and hence the processing times. It is also shown that different recovery media, i.e. selective and non-selective, should be used to avoid underestimation of the surviving cells.