MATERIALS AND METHODSMicroalga, oil

MATERIALS AND METHODS
Microalga, oil, lipases and chemicals Wet paste biomass of the marine
microalga N. gaditana was used as an oil-rich substrate. This biomass was facilitated
by the Estación experimental de Las Palmerillas (Cajamar, El Ejido, Spain). Cells were
grown in an outdoor tubular photobioreactor, centrifuged at 7000 rpm for 10 min,
and then stored at 20C until use. This wet biomass contained 24.2% 0.7% w/w of
dry biomass and 29.1 0.0 wt% of total lipids on dry biomass. The total fatty acid
content (or saponifiable lipids as equivalent fatty acids) in the biomass was
12.1 0.2 wt% on dry biomass. Free fatty acids (FFAs) from used vegetable oil (UVO)
were also used. This oil was provides by the biodiesel manufacturer company
Albabío (Nijar, Spain). Table 1 shows the fatty acid composition of both oils. The
esterification reaction was catalysed by the lipase Novozym 435 from Candida
antarctica (Novozymes A/S, Bagsvaerd, Denmark). This lipase is immobilized on a
macroporous acrylic resin and it is positionally non-specific.
Ethanol (96%, analysis quality) and hexane (95% purity, synthesis quality), both
of Panreac (Barcelona, Spain), were used to extract FFAs from the microalga and to
saponify the UVO. HCl (37%, Panreac S.A., Barcelona, Spain), methanol (99.9% purity,
Carlo Erba Reagents, Rodano, Italy), NaOH and KOH (85% purity, J.T. Baker, Deventer,
Holland), all of analytical quality, were also used. All reagents used in the analytical
determinations were also of analytical grade.
Obtaining of FFAs from UVO and microalgal biomass FFAs fromUVO were
obtained by saponification of the oil. The reaction mixture contained 350 mL of oil
and 700 mL of a hydroalcoholic solution of NaOH, which was prepared dissolving
120 g of NaOH in 400 mL of water and 400 mL of ethanol (96% v/v). The saponification
was carried out shaking this mixture at 200 rpm, at 60C for 30 min. The
mixture was then cooled at room temperature, 140 mL of water were added and the
pH was adjusted to values between 1 and 2 using 37% HCl. FFAs were extracted with
300 mL of hexane, shaking at 200 rpm for 30 min. Finally the hydroalcoholic and
hexane phases were separated by decantation and the hexanic phase was washed
twice with water.
FFAs from the microalga N. gaditana were obtained following a modification of
the three-step procedure developed by Ibáñez González et al. (11) (Fig. 1). 0.50 kg of
wet biomass was treated with 3.63 L of ethanol (96% v/v) (30 mL ethanol (96%)/g dry
biomass), containing 24.2 g of KOH (85% purity) in a 10 L reactor, which was jacketed
for temperature control. Direct saponification was carried out at 60C for 1 h with
constant stirring at 200 rpm with a propeller stirrer (Eurostar digital, IKA Staufen,
Germany), in argon atmosphere and covered with aluminium foil to protect from
light. The biomass residue was then separated from the hydroalcoholic solution by
filtration using a porous glass plate (pore diameter 100 mm, Pobel, Madrid, Spain).
This biomass residue was washed with 1.4 L of ethanol (96% v/v) (14 mL ethanol
(96%)/g dry biomass), to increase the fatty acid recovery yield and this washing
solution was added to the hydroalcoholic fatty acid solution from the main extraction.
In the following step, water (674 mL) was added to the hydroalcoholic phase to
reach 30% wt in water, to increase the immiscibility of the hydroalcoholic solution
and hexane, and the unsaponifiable contents were extracted with 4.30 L hexane
(hexane/hydroalcoholic solution ratio 1:1 v/v). This extraction was carried out stirring
at 200 rpm for 10 min. Subsequently, the solutionwas decanted for 30 min. The
unsaponifiable hexanic solution was removed, leaving the lower hydroalcoholic
phase in the extractor. Next, concentrated HCl was added to the hydroalcoholic
phase to pH 5, to form the FFAs. These FFAs were extracted with hexane (4.3 L), and
the mixture was stirred at 200 rpm for 10 min. It was then allowed to decant for
30 min and two phases were obtained, a bottom one that was discarded and the
upper hexanic phase containing the FFAs. This FFA solution was evaporated in a
rotary evaporator (Buchi, R210, with vacuum pumpV-700 and controller V-850,
Switzerland) to remove and recover the solvent and the FFAs.
Esterification of FFAs catalysed by lipase Novozym 435 In this reaction
FFAs react with methanol to produce methyl esters (biodiesel) and water in the
presence of the lipase Novozym 435 which acts as catalyst. Experiments were carried
out with 4 g of FFAs, 0.86 mL of methanol (methanol/FFA molar ratio 1.5:1) and
different Novozym 435/FFA ratios. The esterification reaction was carried out in
50 mL Erlenmeyer flasks with silicone-capped stoppers. In a typical experiment the
mixture was incubated at 40C and stirred in an orbital shaking air-bath (Inkubator
1000, Unimax 1010 Heidolph, Klein, Germany) at 200 rpm for 24 h. The reactions
were stopped by separation of Novozym 435 by filtration. The final reaction mixture
was conserved at 4C until analysis. Experiments were carried out modifying the
following variables: reaction time, temperature, stirring velocity, methanol/FFA
molar ratio and Novozym 435 amount. All reactions and their corresponding analyses
were carried out in duplicate, and each value recorded is therefore the arithmetic
mean of four experimental data (data shown as mean value standard
deviation).
Analysis of reaction products The identification of the reaction products
(FFAs, methyl esters and to lesser extent acylglycerols) was carried out by thin layer
chromatography (TLC), followed by gas chromatography (GC), to determine quantitatively
the species present. The fatty acid profile of the oilswas also determined by
GC. TLC was carried out on plates of silica-gel (Precoated TLC plates, SIL G-25;
MachereyeNagel, SigmaeAldrich), activated by heating at 105C for 30 min. The
samples were spotted directly on the plate by adding 0.2 mL of reaction product
mixture. The mobile phase used was chloroform/acetone/methanol (95:4.5:0.5 v/v/
v). Spots of each lipid were visualized by spraying the plate with iodine vapour in a
nitrogen steam. The position of the spot corresponding to each lipidic species is
characterized by the response factor. Each lipid type was scraped from the plates and
methylated according to the method of Rodriguez-Ruiz et al. (12). Fatty acid methyl
esters were analysed by GC with an Agilent Technology 6890 gas chromatograph
(Avondale, PA, USA) using a capillary column of fused silica OmegawaxTM
(0.25 mm 30 mm, 0.25 mm standard film, Supelco, Bellefonte, PA, USA), and a
FID (flame-ionization detector). Nitrogen was the carrier gas. Nonadecanoic acid
(19:0) (SigmaeAldrich) was used as internal standard for quantitative
determination of fatty acids. Matreya (Pleasant Gap, PA, USA) n-3 PUFAs
(polyunsaturated fatty acids) standard (catalogue number 1177) was used for the
quantitative determination of fatty acids.
The esterification degree (ED) represents the percentage of initial FFAs
consumed by methylation. ED was determined by acid-base titration after checking
that the results were similar to those obtained by TLC and GC, which take longer. The
samples were diluted with acetone (ratio 1:1 v/v) and phenolphthalein was used as
indicator. The samples from esterification of microalgal FFAs were washed with
alumina, diluted with acetone (fatty acid/acetone ratio 1:20 v/v) and titrated, using
thymol blue as indicator. Previously the acidity of the initial reaction mixture had
been determined under the same conditions in which the acidity was measured,
once the reaction had finished. ED was calculated by the equation:
where V0 and V are the volumes of 1 or 0.1 N NaOH solution used in the titration of
the initial mixture and esterification product, respectively. To determine the purity
of the FFAs and methyl ester from the FFA microalgal extract and esterification reaction
products, respectively, a sample of known weight was analysed by GC. Purity
(wt%) was determined as the ratio between the methyl ester weight obtained by GC
and the sample weight.

MATERIALS AND METHODS
Microalga, oil, lipases and chemicals Wet paste biomass of the marine
microalga N. gaditana was used as an oil-rich substrate. This biomass was facilitated
by the Estación experimental de Las Palmerillas (Cajamar, El Ejido, Spain). Cells were
grown in an outdoor tubular photobioreactor, centrifuged at 7000 rpm for 10 min,
and then stored at 20C until use. This wet biomass contained 24.2%  0.7% w/w of
dry biomass and 29.1  0.0 wt% of total lipids on dry biomass. The total fatty acid
content (or saponifiable lipids as equivalent fatty acids) in the biomass was
12.1 0.2 wt% on dry biomass. Free fatty acids (FFAs) from used vegetable oil (UVO)
were also used. This oil was provides by the biodiesel manufacturer company
Albabío (Nijar, Spain). Table 1 shows the fatty acid composition of both oils. The
esterification reaction was catalysed by the lipase Novozym 435 from Candida
antarctica (Novozymes A/S, Bagsvaerd, Denmark). This lipase is immobilized on a
macroporous acrylic resin and it is positionally non-specific.
Ethanol (96%, analysis quality) and hexane (95% purity, synthesis quality), both
of Panreac (Barcelona, Spain), were used to extract FFAs from the microalga and to
saponify the UVO. HCl (37%, Panreac S.A., Barcelona, Spain), methanol (99.9% purity,
Carlo Erba Reagents, Rodano, Italy), NaOH and KOH (85% purity, J.T. Baker, Deventer,
Holland), all of analytical quality, were also used. All reagents used in the analytical
determinations were also of analytical grade.
Obtaining of FFAs from UVO and microalgal biomass FFAs fromUVO were
obtained by saponification of the oil. The reaction mixture contained 350 mL of oil
and 700 mL of a hydroalcoholic solution of NaOH, which was prepared dissolving
120 g of NaOH in 400 mL of water and 400 mL of ethanol (96% v/v). The saponification
was carried out shaking this mixture at 200 rpm, at 60C for 30 min. The
mixture was then cooled at room temperature, 140 mL of water were added and the
pH was adjusted to values between 1 and 2 using 37% HCl. FFAs were extracted with
300 mL of hexane, shaking at 200 rpm for 30 min. Finally the hydroalcoholic and
hexane phases were separated by decantation and the hexanic phase was washed
twice with water.
FFAs from the microalga N. gaditana were obtained following a modification of
the three-step procedure developed by Ibáñez González et al. (11) (Fig. 1). 0.50 kg of
wet biomass was treated with 3.63 L of ethanol (96% v/v) (30 mL ethanol (96%)/g dry
biomass), containing 24.2 g of KOH (85% purity) in a 10 L reactor, which was jacketed
for temperature control. Direct saponification was carried out at 60C for 1 h with
constant stirring at 200 rpm with a propeller stirrer (Eurostar digital, IKA Staufen,
Germany), in argon atmosphere and covered with aluminium foil to protect from
light. The biomass residue was then separated from the hydroalcoholic solution by
filtration using a porous glass plate (pore diameter 100 mm, Pobel, Madrid, Spain).
This biomass residue was washed with 1.4 L of ethanol (96% v/v) (14 mL ethanol
(96%)/g dry biomass), to increase the fatty acid recovery yield and this washing
solution was added to the hydroalcoholic fatty acid solution from the main extraction.
In the following step, water (674 mL) was added to the hydroalcoholic phase to
reach 30% wt in water, to increase the immiscibility of the hydroalcoholic solution
and hexane, and the unsaponifiable contents were extracted with 4.30 L hexane
(hexane/hydroalcoholic solution ratio 1:1 v/v). This extraction was carried out stirring
at 200 rpm for 10 min. Subsequently, the solutionwas decanted for 30 min. The
unsaponifiable hexanic solution was removed, leaving the lower hydroalcoholic
phase in the extractor. Next, concentrated HCl was added to the hydroalcoholic
phase to pH 5, to form the FFAs. These FFAs were extracted with hexane (4.3 L), and
the mixture was stirred at 200 rpm for 10 min. It was then allowed to decant for
30 min and two phases were obtained, a bottom one that was discarded and the
upper hexanic phase containing the FFAs. This FFA solution was evaporated in a
rotary evaporator (Buchi, R210, with vacuum pumpV-700 and controller V-850,
Switzerland) to remove and recover the solvent and the FFAs.
Esterification of FFAs catalysed by lipase Novozym 435 In this reaction
FFAs react with methanol to produce methyl esters (biodiesel) and water in the
presence of the lipase Novozym 435 which acts as catalyst. Experiments were carried
out with 4 g of FFAs, 0.86 mL of methanol (methanol/FFA molar ratio 1.5:1) and
different Novozym 435/FFA ratios. The esterification reaction was carried out in
50 mL Erlenmeyer flasks with silicone-capped stoppers. In a typical experiment the
mixture was incubated at 40C and stirred in an orbital shaking air-bath (Inkubator
1000, Unimax 1010 Heidolph, Klein, Germany) at 200 rpm for 24 h. The reactions
were stopped by separation of Novozym 435 by filtration. The final reaction mixture
was conserved at 4C until analysis. Experiments were carried out modifying the
following variables: reaction time, temperature, stirring velocity, methanol/FFA
molar ratio and Novozym 435 amount. All reactions and their corresponding analyses
were carried out in duplicate, and each value recorded is therefore the arithmetic
mean of four experimental data (data shown as mean value  standard
deviation).
Analysis of reaction products The identification of the reaction products
(FFAs, methyl esters and to lesser extent acylglycerols) was carried out by thin layer
chromatography (TLC), followed by gas chromatography (GC), to determine quantitatively
the species present. The fatty acid profile of the oilswas also determined by
GC. TLC was carried out on plates of silica-gel (Precoated TLC plates, SIL G-25;
MachereyeNagel, SigmaeAldrich), activated by heating at 105C for 30 min. The
samples were spotted directly on the plate by adding 0.2 mL of reaction product
mixture. The mobile phase used was chloroform/acetone/methanol (95:4.5:0.5 v/v/
v). Spots of each lipid were visualized by spraying the plate with iodine vapour in a
nitrogen steam. The position of the spot corresponding to each lipidic species is
characterized by the response factor. Each lipid type was scraped from the plates and
methylated according to the method of Rodriguez-Ruiz et al. (12). Fatty acid methyl
esters were analysed by GC with an Agilent Technology 6890 gas chromatograph
(Avondale, PA, USA) using a capillary column of fused silica OmegawaxTM
(0.25 mm  30 mm, 0.25 mm standard film, Supelco, Bellefonte, PA, USA), and a
FID (flame-ionization detector). Nitrogen was the carrier gas. Nonadecanoic acid
(19:0) (SigmaeAldrich) was used as internal standard for quantitative
determination of fatty acids. Matreya (Pleasant Gap, PA, USA) n-3 PUFAs
(polyunsaturated fatty acids) standard (catalogue number 1177) was used for the
quantitative determination of fatty acids.
The esterification degree (ED) represents the percentage of initial FFAs
consumed by methylation. ED was determined by acid-base titration after checking
that the results were similar to those obtained by TLC and GC, which take longer. The
samples were diluted with acetone (ratio 1:1 v/v) and phenolphthalein was used as
indicator. The samples from esterification of microalgal FFAs were washed with
alumina, diluted with acetone (fatty acid/acetone ratio 1:20 v/v) and titrated, using
thymol blue as indicator. Previously the acidity of the initial reaction mixture had
been determined under the same conditions in which the acidity was measured,
once the reaction had finished. ED was calculated by the equation:
where V0 and V are the volumes of 1 or 0.1 N NaOH solution used in the titration of
the initial mixture and esterification product, respectively. To determine the purity
of the FFAs and methyl ester from the FFA microalgal extract and esterification reaction
products, respectively, a sample of known weight was analysed by GC. Purity
(wt%) was determined as the ratio between the methyl ester weight obtained by GC
and the sample weight.

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MATERIALS AND METHODSMicroalga, oil, lipases and chemicals Wet paste biomass of the marinemicroalga N. gaditana was used as an oil-rich substrate. This biomass was facilitatedby the Estación experimental de Las Palmerillas (Cajamar, El Ejido, Spain). Cells weregrown in an outdoor tubular photobioreactor, centrifuged at 7000 rpm for 10 min,and then stored at 20C until use. This wet biomass contained 24.2% 0.7% w/w ofdry biomass and 29.1 0.0 wt% of total lipids on dry biomass. The total fatty acidcontent (or saponifiable lipids as equivalent fatty acids) in the biomass was12.1 0.2 wt% on dry biomass. Free fatty acids (FFAs) from used vegetable oil (UVO)were also used. This oil was provides by the biodiesel manufacturer companyAlbabío (Nijar, Spain). Table 1 shows the fatty acid composition of both oils. Theesterification reaction was catalysed by the lipase Novozym 435 from Candidaantarctica (Novozymes A/S, Bagsvaerd, Denmark). This lipase is immobilized on amacroporous acrylic resin and it is positionally non-specific.Ethanol (96%, analysis quality) and hexane (95% purity, synthesis quality), bothof Panreac (Barcelona, Spain), were used to extract FFAs from the microalga and tosaponify the UVO. HCl (37%, Panreac S.A., Barcelona, Spain), methanol (99.9% purity,Carlo Erba Reagents, Rodano, Italy), NaOH and KOH (85% purity, J.T. Baker, Deventer,Holland), all of analytical quality, were also used. All reagents used in the analyticaldeterminations were also of analytical grade.Obtaining of FFAs from UVO and microalgal biomass FFAs fromUVO wereobtained by saponification of the oil. The reaction mixture contained 350 mL of oiland 700 mL of a hydroalcoholic solution of NaOH, which was prepared dissolving120 g of NaOH in 400 mL of water and 400 mL of ethanol (96% v/v). The saponificationwas carried out shaking this mixture at 200 rpm, at 60C for 30 min. Themixture was then cooled at room temperature, 140 mL of water were added and thepH was adjusted to values between 1 and 2 using 37% HCl. FFAs were extracted with300 mL of hexane, shaking at 200 rpm for 30 min. Finally the hydroalcoholic andhexane phases were separated by decantation and the hexanic phase was washedtwice with water.FFAs from the microalga N. gaditana were obtained following a modification ofthe three-step procedure developed by Ibáñez González et al. (11) (Fig. 1). 0.50 kg ofwet biomass was treated with 3.63 L of ethanol (96% v/v) (30 mL ethanol (96%)/g drybiomass), containing 24.2 g of KOH (85% purity) in a 10 L reactor, which was jacketedfor temperature control. Direct saponification was carried out at 60C for 1 h withconstant stirring at 200 rpm with a propeller stirrer (Eurostar digital, IKA Staufen,Germany), in argon atmosphere and covered with aluminium foil to protect fromlight. The biomass residue was then separated from the hydroalcoholic solution byfiltration using a porous glass plate (pore diameter 100 mm, Pobel, Madrid, Spain).This biomass residue was washed with 1.4 L of ethanol (96% v/v) (14 mL ethanol(96%)/g dry biomass), to increase the fatty acid recovery yield and this washingsolution was added to the hydroalcoholic fatty acid solution from the main extraction.In the following step, water (674 mL) was added to the hydroalcoholic phase toreach 30% wt in water, to increase the immiscibility of the hydroalcoholic solutionand hexane, and the unsaponifiable contents were extracted with 4.30 L hexane(hexane/hydroalcoholic solution ratio 1:1 v/v). This extraction was carried out stirringat 200 rpm for 10 min. Subsequently, the solutionwas decanted for 30 min. Theunsaponifiable hexanic solution was removed, leaving the lower hydroalcoholicphase in the extractor. Next, concentrated HCl was added to the hydroalcoholicphase to pH 5, to form the FFAs. These FFAs were extracted with hexane (4.3 L), andthe mixture was stirred at 200 rpm for 10 min. It was then allowed to decant for30 min and two phases were obtained, a bottom one that was discarded and theupper hexanic phase containing the FFAs. This FFA solution was evaporated in arotary evaporator (Buchi, R210, with vacuum pumpV-700 and controller V-850,Switzerland) to remove and recover the solvent and the FFAs.Esterification of FFAs catalysed by lipase Novozym 435 In this reactionFFAs react with methanol to produce methyl esters (biodiesel) and water in thepresence of the lipase Novozym 435 which acts as catalyst. Experiments were carriedout with 4 g of FFAs, 0.86 mL of methanol (methanol/FFA molar ratio 1.5:1) anddifferent Novozym 435/FFA ratios. The esterification reaction was carried out in50 mL Erlenmeyer flasks with silicone-capped stoppers. In a typical experiment themixture was incubated at 40C and stirred in an orbital shaking air-bath (Inkubator1000, Unimax 1010 Heidolph, Klein, Germany) at 200 rpm for 24 h. The reactionswere stopped by separation of Novozym 435 by filtration. The final reaction mixturewas conserved at 4C until analysis. Experiments were carried out modifying thefollowing variables: reaction time, temperature, stirring velocity, methanol/FFAmolar ratio and Novozym 435 amount. All reactions and their corresponding analyseswere carried out in duplicate, and each value recorded is therefore the arithmeticmean of four experimental data (data shown as mean value standarddeviation).Analysis of reaction products The identification of the reaction products(FFAs, methyl esters and to lesser extent acylglycerols) was carried out by thin layerchromatography (TLC), followed by gas chromatography (GC), to determine quantitativelythe species present. The fatty acid profile of the oilswas also determined byGC. TLC was carried out on plates of silica-gel (Precoated TLC plates, SIL G-25;MachereyeNagel, SigmaeAldrich), activated by heating at 105C for 30 min. Thesamples were spotted directly on the plate by adding 0.2 mL of reaction productmixture. The mobile phase used was chloroform/acetone/methanol (95:4.5:0.5 v/v/v). Spots of each lipid were visualized by spraying the plate with iodine vapour in anitrogen steam. The position of the spot corresponding to each lipidic species ischaracterized by the response factor. Each lipid type was scraped from the plates andmethylated according to the method of Rodriguez-Ruiz et al. (12). Fatty acid methylesters were analysed by GC with an Agilent Technology 6890 gas chromatograph(Avondale, PA, USA) using a capillary column of fused silica OmegawaxTM(0.25 mm 30 mm, 0.25 mm standard film, Supelco, Bellefonte, PA, USA), and aFID (flame-ionization detector). Nitrogen was the carrier gas. Nonadecanoic acid(19:0) (SigmaeAldrich) was used as internal standard for quantitativedetermination of fatty acids. Matreya (Pleasant Gap, PA, USA) n-3 PUFAs(polyunsaturated fatty acids) standard (catalogue number 1177) was used for thequantitative determination of fatty acids.The esterification degree (ED) represents the percentage of initial FFAsconsumed by methylation. ED was determined by acid-base titration after checkingthat the results were similar to those obtained by TLC and GC, which take longer. Thesamples were diluted with acetone (ratio 1:1 v/v) and phenolphthalein was used asindicator. The samples from esterification of microalgal FFAs were washed with
alumina, diluted with acetone (fatty acid/acetone ratio 1:20 v/v) and titrated, using
thymol blue as indicator. Previously the acidity of the initial reaction mixture had
been determined under the same conditions in which the acidity was measured,
once the reaction had finished. ED was calculated by the equation:
where V0 and V are the volumes of 1 or 0.1 N NaOH solution used in the titration of
the initial mixture and esterification product, respectively. To determine the purity
of the FFAs and methyl ester from the FFA microalgal extract and esterification reaction
products, respectively, a sample of known weight was analysed by GC. Purity
(wt%) was determined as the ratio between the methyl ester weight obtained by GC
and the sample weight.

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วัสดุและวิธีการ
สาหร่าย , น้ำมัน , ไลเปสและสารเคมีชีวมวลวางเปียกของทะเล
สาหร่าย . gaditana ถูกใช้เป็นริชพื้นผิว ระบบนี้สะดวก
โดย estaci เลอองทดลอง de las palmerillas ( Cajamar เอลจิโด้ , สเปน ) เซลล์มีการปลูกในท่อ
photobioreactor กลางแจ้งที่ระดับ 7 , 000 รอบต่อนาที เป็นเวลา 10 นาที แล้วเก็บรักษา
ที่ 20 C จนใช้ชีวมวลเปียกนี้ประกอบด้วย 40 % 0.7 % w / w ของ
ชีวมวลแห้งและไม่ 0.0 เปอร์เซ็นต์โดยน้ำหนักของไขมันทั้งหมดในมวลชีวภาพแห้ง เนื้อหาทั้งหมดกรด
ไขมัน ( หรือ saponifiable ไขมันเทียบเท่ากับกรดไขมันในชีวมวลเป็น 0.2 เปอร์เซ็นต์โดยน้ำหนักใน
12.1 มวลชีวภาพแห้ง กรดไขมันอิสระ ( ffas ) จากน้ำมันพืชใช้แล้ว ( uvo )
ยังใช้ น้ำมันนี้มีให้โดยผู้ผลิตไบโอดีเซล บริษัท albab í o
( ไนเจอร์ , สเปน )ตารางที่ 1 แสดงองค์ประกอบของกรดไขมันของน้ำมัน ปฏิกิริยาเอสเทอริฟิเคชัน
catalysed โดยไลเปสจาก Candida โนโวไซม์ 435
แอนตาร์กติกา ( กุ้ง / S , bagsvaerd , เดนมาร์ก ) เมื่อถูกตรึงบน
ยางอะคริลิก macroporous และเป็น positionally เฉพาะ .
เอทานอล ( การวิเคราะห์คุณภาพ 96% ) เฮกเซน ( คุณภาพและความบริสุทธิ์ 95% ) ทั้ง
ของ panreac ( บาร์เซโลนา ) ,ใช้สารสกัดจากสาหร่าย และ ffas

ทำให้เป็นสบู่ที่ uvo . กรดไฮโดรคลอริก ( 37% panreac S.A . , บาร์เซโลนา , สเปน ) , เมทานอล ( 99.9% บริสุทธิ์
คาร์โลมา rodano reagents , อิตาลี ) , NaOH และเกาะ ( 85% ความบริสุทธิ์ , J.T . Baker , Deventer
, ฮอลแลนด์ ) , คุณภาพวิเคราะห์ ยังใช้ ทั้งหมดเพื่อใช้ในการวิเคราะห์ข้างต้นยังวิเคราะห์

เกรดได้รับของ ffas จาก uvo สาหร่ายชีวมวลและ ffas fromuvo ถูก
ได้โดยการออกซิเดชันทางเคมีของน้ำมัน ปฏิกิริยาผสมที่มีอยู่ 350 มล. ของน้ำมัน
และ 700 มิลลิลิตรของสารละลาย hydroalcoholic NaOH ที่เตรียมละลาย
120 กรัม 400 มิลลิลิตร และมีน้ำ 400 มิลลิลิตรเอทานอล ( 96 % v / v ) การออกซิเดชันทางเคมี
ทำการเขย่าส่วนผสมนี้ที่ 200 รอบต่อนาทีที่อุณหภูมิ 60
C เป็นเวลา 30 นาทีส่วนผสมก็เย็นที่อุณหภูมิห้อง , 140 มล. ของน้ำเพิ่ม
pH ปรับค่าระหว่าง 1 และ 2 ใช้ 37 % HCL . ffas ถูกสกัดด้วยเฮกเซน
300 มิลลิลิตร เขย่าที่ความเร็วรอบ 200 รอบต่อนาทีเป็นเวลา 30 นาที ในที่สุด hydroalcoholic
เฟสและเฮกเซนแยกได้จาก decantation และเฟส hexanic ล้าง

ffas สองครั้งด้วยน้ำ จากสาหร่าย )

การแปล กรุณารอสักครู่..

ภาษาอื่น ๆ

การสนับสนุนเครื่องมือแปลภาษา: กรีก, กันนาดา, กาลิเชียน, คลิงออน, คอร์สิกา, คาซัค, คาตาลัน, คินยารวันดา, คีร์กิซ, คุชราต, จอร์เจีย, จีน, จีนดั้งเดิม, ชวา, ชิเชวา, ซามัว, ซีบัวโน, ซุนดา, ซูลู, ญี่ปุ่น, ดัตช์, ตรวจหาภาษา, ตุรกี, ทมิฬ, ทาจิก, ทาทาร์, นอร์เวย์, บอสเนีย, บัลแกเรีย, บาสก์, ปัญจาป, ฝรั่งเศส, พาชตู, ฟริเชียน, ฟินแลนด์, ฟิลิปปินส์, ภาษาอินโดนีเซี, มองโกเลีย, มัลทีส, มาซีโดเนีย, มาราฐี, มาลากาซี, มาลายาลัม, มาเลย์, ม้ง, ยิดดิช, ยูเครน, รัสเซีย, ละติน, ลักเซมเบิร์ก, ลัตเวีย, ลาว, ลิทัวเนีย, สวาฮิลี, สวีเดน, สิงหล, สินธี, สเปน, สโลวัก, สโลวีเนีย, อังกฤษ, อัมฮาริก, อาร์เซอร์ไบจัน, อาร์เมเนีย, อาหรับ, อิกโบ, อิตาลี, อุยกูร์, อุสเบกิสถาน, อูรดู, ฮังการี, ฮัวซา, ฮาวาย, ฮินดี, ฮีบรู, เกลิกสกอต, เกาหลี, เขมร, เคิร์ด, เช็ก, เซอร์เบียน, เซโซโท, เดนมาร์ก, เตลูกู, เติร์กเมน, เนปาล, เบงกอล, เบลารุส, เปอร์เซีย, เมารี, เมียนมา (พม่า), เยอรมัน, เวลส์, เวียดนาม, เอสเปอแรนโต, เอสโทเนีย, เฮติครีโอล, แอฟริกา, แอลเบเนีย, โคซา, โครเอเชีย, โชนา, โซมาลี, โปรตุเกส, โปแลนด์, โยรูบา, โรมาเนีย, โอเดีย (โอริยา), ไทย, ไอซ์แลนด์, ไอร์แลนด์, การแปลภาษา.