Reagent	Concentration
Yeast Extract	5g/L
Tryptone	10g/L
NaCl	10g/L
Agar	2% (eg.100mL medium with 2g agar)

Autoclave at 121°C for 15 min.

One Erlenmeyer flask:

Medium	Reagent
YPD	20 g/L anhydrous glucose	2.5 g/L anhydrous glucose	17.5 g/L Mannitol
YPM	20 g/L mannitol
YP(D+M)	2.5 g/L anhydrous glucose and 17.5 g/L mannitol

Another Erlenmeyer flask:

Reagent	Concentration
Yeast extract	10 g/L
Peptone	20 g/L

Procedure:

1. According to the formula, weigh the required reagent and mix it.
2. Seal with parafilm and rubber band, affix autoclave indicator tape.
3. Autoclave at 115°C for 20 min.
4. Mix the solution from two Erlenmeyer flasks in the laminar flow cabinet.
5. To prepare the solid medium, cool the solution and then add it into the petri dishes in the laminar flow cabinet.

One Erlenmeyer flask:

Medium	Reagent
SD/ΔTrp	20 g/L anhydrous glucose
SM/ΔTrp	20 g/L mannitol

P.S. If a solid medium is made, 20 g/L Agar is added.

Another Erlenmeyer flask:

Reagent	Concentration
YNB	6.7 g/L
Drop-out supplement(ΔLeu/ΔTrp/ΔUra)	0.62 g/L
Leu	0.06 g/L
Ura	0.02 g/L

Procedure:

1. According to the formula, weigh the required material and mix it, and make a mark.
2. Seal with parafilm and rubber band, affix autoclave indicator tape.
3. Autoclave at 115°C for 20 min.
4. Mix the solution from two Erlenmeyer flasks in the laminar flow cabinet.
5. To prepare the solid medium, cooled the solution and then added into the petri dishes in the laminar flow cabinet.

One Erlenmeyer flask: 20 g/L anhydrous glucose.

Medium	Reagent
SD/ΔUra	20 g/L anhydrous glucose
SM/ΔUra	20 g/L mannitol
S(D+M)/ΔUra	2.5 g/L anhydrous glucose and 17.5 g/L mannitol

P.S. If a solid medium is made, 20 g/L Agar is added.

Another Erlenmeyer flask:

Reagent	Concentration
YNB	6.7 g/L
Ura Drop-out supplement	0.62 g/L

Procedure:

1. According to the formula, weigh the required material and mix it, and make a mark.
2. Seal with parafilm and rubber band, affix autoclave indicator tape.
3. Autoclave at 115°C for 20 min.
4. Mix the solution from two Erlenmeyer flasks in the laminar flow cabinet.
5. To prepare the solid medium, cooled the solution and then added into the petri dishes in the laminar flow cabinet.

1. Preparation of kelp hydrolysate

(1) Kelp Pre-treatment
Place the required amount of kelp in a 65°C drying oven for 12 hours to dry out the moisture. Then, use a grinder to crush the dried kelp into a 50-mesh powder. Collect the powder and store it in a refrigerator at 4°C.

(2) Kelp Acid Hydrolysis
First, measure 9.0 mL of concentrated HCl and dilute it in 1 L of distilled water to prepare a 0.1 M dilute hydrochloric acid solution.
Second, weigh 20.0 g of kelp powder into a 500 mL shaker flask and add 200 mL of dilute hydrochloric acid. Sterilize the mixture at 121°C for 20 min under high temperature and pressure. The resulting hydrolysate is the kelp acid hydrolysate.

(3) Enzymatic Hydrolysis of the Acid Hydrolysate
Acidic cellulase and β-glucanase are used for further hydrolysis of the acid hydrolysate to fully release the sugars from the kelp matrix. The optimal reaction conditions for acidic cellulase and β-glucanase are at a pH between 4.5 and 6.0, and a temperature between 65°C and 75°C. Therefore, the pH of the acid hydrolysate is adjusted before enzymatic hydrolysis. The specific steps are as follows:

First, weigh 40.0 g of sodium hydroxide pellets and gradually dissolve them in 100 mL of distilled water to prepare a 10.0 M NaOH solution. After cooling 200 mL of acid hydrolysate to below 70°C, add approximately 2.0 mL of the 10 M sodium hydroxide solution to adjust the pH to 5.0-5.5.
Second, add acidic cellulase and β-glucanase, and mix thoroughly to ensure the enzymes are well incorporated into the acid hydrolysate, avoiding clumping. Perform the enzymatic hydrolysis in a 70°C water bath for three hours to obtain the kelp enzymatic hydrolysate.
Third, filter the kelp enzymatic hydrolysate using a vacuum pump and Buchner funnel to obtain a clear hydrolysate, referred to as kelp hydrolysate, which will be used for subsequent analyses and experiments. The kelp hydrolysate should be freshly prepared and used immediately.

2. Preparation of kelp hydrolysate medium A

Weigh 15.0 g of ammonium sulfate, 8.0 g of anhydrous potassium dihydrogen phosphate, 6.15 g of anhydrous magnesium sulfate, and 25.0 g/L of kelp acid hydrolysate. Dilute to a final volume of 1 L. Sterilize at 115°C for 20 min under high temperature and pressure.

3. Preparation of kelp hydrolysate medium B

Weigh 15.0 g of ammonium sulfate, 8.0 g of anhydrous potassium dihydrogen phosphate, 6.15 g of anhydrous magnesium sulfate, and 25.0 g/L of kelp enzymatic hydrolysate. Dilute to a final volume of 1 L. Sterilize at 115°C for 20 min under high temperature and pressure.

Reaction System:

Reagent	Volume
PrimeSTAR Mix	25 μL
Primer-F	1.5 μL
Primer-R	1.5 μL
Template	1 μL
ddH2O	Up to 50 μL

Procedure:

1. Mix reagents according to the table below in the PCR tube.
2. Put the PCR tube in the PCR machine, and set the PCR amplification procedure as follows.

	Temperature	Time
Initial Denaturation	94℃	2 min
Denaturation	98℃	30 s	× 30
Annealing	52℃	5 s
Extension	72℃	1000kb/10s
Final Extension	72℃	10 min
Hold	16℃	∞

Procedure:

Reagent	Volume/Amount	Final Concentration
Agarose	0.5 g	2 %
1× TAE Buffer	25 mL	1

1. Weigh 0.5 g of agarose into a conical flask.
2. Measure 50 mL of 1× TAE Buffer into an Erlenmeyer flask and shake gently.
3. Put the Erlenmeyer flask with agarose into the microwave to heat up（1-1.5min）.
4. When the agarose is completely dissolved, take out the Erlenmeyer flask and place it at room temperature. Cool it until it is slightly hot and then pour the solution into the mold.
5. Immediately add 0.05%-0.1% Nucleic acid dye to the agarose solution in the mold and mix well.
6. Allow the prepared gel to solidify at room temperature (15-20 min).

Sample loading and Electrophoresis：

1. Add 5× loading buffer to the PCR product at a ratio of 1:4.
2. Add a sufficient amount of 1× TAE buffer to the electrophoresis tank (enough to cover the gel block).
3. Remove the solidified gel from the mold and put it into the electrophoresis tank with electrophoresis solution.
4. Make sure the loading pore is close to the negative current flows.
5. Add 5μL DNA marker into the sample wells.
6. Add 4-5μL DNA samples to sample wells. Record the position of the sample added.
7. Cover the electrophoresis tank.
8. Turn on the electrophoresis appliance, set the voltage to 120V, and the time to 20 min.
9. After electrophoresis is completed, take out the gel and place it into a gel image system for development.

Gel Imaging:

1. Take out post-electrophoresis gel with disposable gloves on the hands.
2. Open the gel imager.
3. Put the gel in the imager. Capture the image of the gel.
4. Analyze the bands result.

1. Briefly centrifuge the PCR product.
2. Measure its volume using a pipette and transfer it to a sterile 2mL centrifuge tube.
3. Add the appropriate volume of Buffer DP to the product and vortex for 15 seconds.
   1. To recover > 100bp fragments: add 3 volumes of Buffer DP to the product.
   2. To recover < 100bp fragments: add 3 volumes of Buffer DP and 1 volume of isopropanol to the product.
4. Briefly centrifuge to collect any liquid droplets on the tube walls.
5. Place the HiPure DNA column into a collection tube and transfer the mixture to the column. Centrifuge at 10,000 × g for 15-60 seconds.
6. Discard the filtrate and place the column back into the collection tube. Add 500µl of Buffer DW2 (diluted with anhydrous ethanol) to the column. Centrifuge at 10,000 × g for 15-60 seconds.
7. Discard the filtrate and place the column back into the collection tube. Centrifuge at 10,000 × g for 2 minutes.
8. Place the column in a new 2ml centrifuge tube. Add 7-30µl of Elution Buffer to the center of the membrane. Let it sit for 1 minute. Centrifuge at 10,000 × g for 1 minute.
9. Discard the column and store the DNA at -20°C.

1. Open the spectrophotometer, select the board layout on the computer, select the blank area, standard area, and unknown area.
2. Add 2μL water in the blank area, do not add any substance in the standard area, and add 2μL sample in the unknown area in order.
3. Click the Start button to start the enzyme marker, check the OD260/OD280 in the results, check the plasmid concentration, and make a record.

Reagent	Volume
5×CE II Buffer	2 μL
Exnase II	1 μL
Backbone	X μL
Fragment	Y μL
ddH2O	Up to 10μL

Where X and Y are calculated:

X = 0.01 × Number of backbone’s base ÷ Concentration of backbone

Y = 0.02 × Number of fragment’s base ÷ Concentration of fragment

Then heated in a 37 °C metal bath for 30 min.

1. Inoculate a single colony of Saccharomyces cerevisiae into 5 mL of liquid medium, and incubate overnight in a shaker at 30°C and 220 rpm.
2. Dilute the culture 5 times and measure the OD value, then calculate the required culture volume using the formula below.

   $$V_1 = 5 \times \frac{\text{Required initial OD (0.4)}}{\text{Tested OD}}$$

3. In the laminar flow cabinet, transfer V1 mL of the culture into fresh liquid medium, and incubate in a shaker at 30°C and 220 rpm for about four hours.
4. Collect the cells in the laminar flow cabinet: add 1.2 mL of the culture to a 2 mL centrifuge tube, centrifuge at 7000 rpm for 3 min, and remove the supernatant with a pipette (be careful not to aspirate the pellet).
5. Add 1 mL of solution I, resuspend the pellet, and centrifuge again at 7000 rpm for 3 min. Discard the supernatant in the same manner.
6. Add 100 μL of solution II (lithium acetate solution), aliquot into four 1.5 mL centrifuge tubes (25 μL per tube), and store at -80°C.

1. Take a tube of competent cells (20 μL) from the -80°C freezer, and let it sit on ice for about 5-10 min to thaw the competent cells.
2. Add 10 μL of the overnight ligation or recombinant product to the competent cells, mix well, and let the mixture sit on ice for about 10 min. Preheat the water bath to 42°C.
3. Place the competent cells with the ligation mixture in a 42°C water bath for heat shock for 90 seconds, then immediately place them on ice for 10 min. Add 800 μL of antibiotic-free LB liquid medium to the competent cells, and incubate at 37°C, 180 rpm in a shaker for approximately 1 hour to allow recovery.
   P.S. The heat shock time must be strictly controlled.
4. Centrifuge the competent cells at 5000 rpm for 3 min, then remove 600 μL of the supernatant in the laminar flow cabinet. Resuspend the remaining cells, and spread 200 μL of the suspension evenly on LB solid plates containing kanamycin/ampicillin. Incubate the plates at 37°C in an incubator for 12-16 hours.
5. Patch plating: Sterilize the laminar flow cabinet with UV light in advance, and place kanamycin-resistant LB solid medium plates inside. Observe the transformed E. coli plates, pick 2-3 single colonies from each plate, and incubate them upside down at 37°C for about 6 hours.
6. Perform E. coli colony PCR, and verify the size of the PCR products using agarose gel electrophoresis. Based on the electrophoresis results, select the suspected positive clones, and inoculate them into approximately 5 mL of LB liquid medium with kanamycin/ampicillin in a sterile hood, and incubate overnight at 37°C, 220 rpm. Use a plasmid miniprep kit to extract recombinant plasmids from the culture, and measure the plasmid concentration using a spectrophotometer.
7. Analyze the sequencing results using NCBI BLAST or SnapGene software.

1. In the laminar flow cabinet, mix the following substances, then add 200 μL of Solution III (Transformation solution), followed by vortexing to mix thoroughly.

Reagent	Mass/Volume
gRNA Plasmid	1000 ng
Donor DNA	＞1000 ng
Competent cells containing Cas9 plasmids	20μL

Or

Reagent	Mass/Volume
Plasmid	500 ng
Competent cells	20μL
Competent cells containing Cas9 plasmids	20μL

2. Incubate at 30°C in a shaker for 1 hour, vortex once every 15-30 min for 10 seconds each time, for a total of 4 vortexes.
3. Depending on the liquid volume, centrifuge to remove part of the liquid if necessary, and spread the remaining cells on the corresponding selective YPD/SD plates. Incubate at 30°C for 2-3 days.

1. In the laminar flow cabinet, pick 5-10 patched single colonies and place them in 20 μL of ddH₂O. Heat-lyse the cells in a PCR machine at 98°C for 10 min.
2. Centrifuge the lysed cells at 14,000 rpm for 5 min, and use the supernatant to prepare the following system for PCR amplification.

Reagent	Volume
2 × Hieff Mix	5 μL
Primer-F	0.4 μL
Primer-R	0.4 μL
ddH2O	2.2 μL
Template	2 μL

Procedure:

	Temperature	Time
Initial Denaturation	94℃	2 min
Denaturation	94℃	30 s	× 30
Annealing	55℃	30 s
Extension	72℃	20 （1000 bp/30s）
Final Extension	72℃	10 min
Hold	16℃	∞

1. Pick the patched colony and place it in 20 μL of NaOH solution. Heat-lyse the cells in a PCR machine at 95°C for 20 min.
2. Centrifuge the lysed cells in a tabletop mini-centrifuge for 1 min, and use the supernatant to prepare the following system for PCR amplification.

Reagent	Volume
KOD Buffer	5 μL
Primer-F	0.4 μL
Primer-R	0.4 μL
dNTP	2 μL
ddH2O	1 μL
KOD.FX	0.2 μL
Template	1 μL

Procedure:

	Temperature	Time
Initial Denaturation	94℃	5 min
Denaturation	98℃	10 s	× 33
Annealing	42℃	30 s
Extension	68℃	1 min （1000 bp/60s）
Final Extension	68℃	10 min
Hold	16℃	∞

Add ethanol to Buffer PW before use, check bottle tag for the adding volume.

1. Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μL Buffer BL to CP3. Centrifuge for 1 min at 9,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube.
2. Collect 3-5 mL bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15-30°C), then remove all traces of supernatant until all medium has been drained.
3. Re-suspend the bacterial pellet in 250 μL Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.
4. Add 250 μL Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times.
5. Add 350 μL Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 3 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge.
6. Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 30-60 sec at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.
7. Wash the Spin Column CP3 by adding 600 μL Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 30-60 sec at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Column CP3 back into the Collection Tube.
8. Repeat Step 7.
9. Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to remove residual Buffer PW.
10. Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μL H₂O to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 1 min at 12,000 rpm (~13,400 × g).

Mannitol and glucose can be quantified using High-Performance Liquid Chromatography (HPLC). A Waters 2695 HPLC system was used, with a Milford RI-2414 refractive index detector. The chromatographic column was an Aminex HPX-87H (300 × 7.8 mm) from Bio-Rad. The autosampler was employed, and the mobile phase was 2.5 mM dilute sulfuric acid.

1. HPLC Detection Method:
   The specific method is as follows: set the autosampling program with an injection volume of 10 μL, flow rate of the mobile phase at 0.6 mL/min, and column oven temperature set to 60°C. The analysis time was set to 25 min.
2. Preparation of 2.5 mM Dilute Sulfuric Acid Mobile Phase:
   Measure 2 L of ultrapure water (18.2 MΩ) into a reagent bottle. Take a small amount (about 20 mL) of ultrapure water into a beaker, add 280 μL of concentrated sulfuric acid, and filter it through a 0.22 μm filter membrane for sterilization. Add this to the ultrapure water and degas with ultrasound for 30 min.
3. Preparation of Standard Curves for Samples:
   Prepare standard curves for glucose and mannitol: prepare mixed standard solutions with concentrations of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 g/L using the mobile phase, and filter the solution through a 0.22 μm membrane into chromatographic vials. After HPLC detection, record the peak areas of mannitol and glucose to plot the corresponding standard curves.
4. Sample Preparation:
   Pipette 200 μL of the above kelp hydrolysate into a 1.5 mL centrifuge tube, add 800 μL of the mobile phase, mix thoroughly, and filter it through a 0.22 μm membrane into a chromatographic vial for HPLC detection.
5. Data Analysis:
   After detection, record the peak areas of the samples, and calculate the contents of glucose and mannitol in the samples according to the corresponding standard curves.

Quantitative analysis of limonene in the product was conducted using a Shimadzu GC-2014C Gas Chromatograph, equipped with a flame ionization detector (FID). The chromatographic column used was an Agilent HP-5 capillary column (5% Ph-Me silicone, 30 m × 0.320 mm × 0.25 μm), with nitrogen as the carrier gas.

1. GC-FID Detection Method:
   The program settings were as follows: set the autosampler for automatic injection with an injection volume of 1 μL, using the split mode with a split ratio of 15:1. The detector temperature was set to 300°C, and the injection port temperature was set to 250°C. The column temperature was raised to 200°C at a rate of 30°C/min and held for 3 min, with a total run time of 20.91 min.
2. Preparation of the Limonene Standard Curve:
   Take 40 μL of 100 mM limonene stock solution into a 2 mL centrifuge tube, add 1960 μL of ethyl acetate to dilute it to 2 mM, and mix thoroughly. Prepare a series of standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mM from the 2 mM limonene stock solution. Filter the solutions through a 0.22 μm filter membrane into chromatographic vials. After gas chromatography detection, record the peak areas of limonene and plot the standard curve.
3. Detection of Fermentation Samples:
   Take the recovered organic phase sample from the fermentation broth into a 1.5 mL centrifuge tube, centrifuge at 10,000 rpm for 1 min, and transfer 300 μL of the supernatant into a clean EP tube. Add 300 μL of ethyl acetate (EtAc), mix thoroughly, and filter through a 0.22 μm membrane into a GC vial. After GC detection, record the peak area and calculate the yield of limonene based on the standard curve.

Prepare in advance:

• 0.100 g/L sodium alginate standard solution (dissolve 0.0100 g sodium alginate in 100 mL water) — wash a 250 mL bottle and seven test tubes with rubber stoppers, then dry them.
• 5 g/L sodium hydroxide standard solution (dissolve 0.5 g sodium hydroxide in 100 mL water) — wash a 250 mL bottle and dry it.
• 0.0125 mol/L sodium tetraborate sulfuric acid solution (dissolve 0.478 g sodium tetraborate decahydrate in 100 mL concentrated sulfuric acid) — wash a brown bottle capable of holding 100 mL of solution and dry it.

1. Sample Preparation
   Solid samples are divided to approximately 100 g and then quickly ground to pass through a 0.50 mm sieve (if the sample is wet, use a 1.00 mm sieve). Mix thoroughly and store in a clean, dry container. For liquid samples, shake well and quickly take out approximately 100 mL, and place in a clean, dry container.
2. Preparation of Sample Solution
   Weigh 0.5g to 2g of the well-mixed sample, and place it in a 250 mL volumetric flask. For liquid samples, add water directly to the volume mark. For solid samples, add about 150 mL of water and place in a constant-temperature shaker at (25 ± 5)°C, shaking at a frequency of (180 ± 20) r/min for 30 min. Afterward, top up with water to the volume mark and filter (or let it settle until clear).
3. Preparation of Standard Curve
   Pipette 0 mL, 0.10 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, and 1.00 mL of sodium alginate standard solution (0.100 g/L) into seven stoppered test tubes. Add water to each to make a total volume of 1.00 mL and mix well. The concentrations of sodium alginate in the standard series solutions are 0 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, and 100 μg/mL, respectively. Place the test tubes in an ice water bath. Slowly add 6.00 mL of Na2B4O7-H2SO4 (c(sodium tetraborate decahydrate) = 0.0125 mol/L, which is prepared by dissolving 0.478g Na2B4O7 in 100 mL of concentrated H2SO4) to each test tube, stopper, and mix well. Heat the test tubes in a boiling water bath for 5 min, then cool to room temperature. Add 100 μL of m-hydroxybiphenyl solution (1.5 g/L, prepared by dissolving 0.15g of m-hydroxybiphenyl in 100 mL of 5 g/L NaOH solution, stored in a brown bottle) as a color developer, and mix well. After standing for 10 min at room temperature, measure the absorbance at 520 nm with a colorimetric cell, setting the 0 μg/mL solution to zero. Plot the standard curve using the concentration of sodium alginate (μg/mL) on the horizontal axis and the corresponding absorbance on the vertical axis.
   P.S. If the standard solution has color before adding the developer, prepare a separate set of test tubes and add the same volume of standard solution. During the color development step, use 100 μL of 5 g/L sodium hydroxide solution instead of m-hydroxybiphenyl solution, then perform the same procedure to measure the absorbance, and subtract this from the final result. The intensity of the color from uronic acids is affected by the concentration and purity of the sulfuric acid. Therefore, when preparing the sample and the standard curve, ensure the sulfuric acid used is of the same batch and specification to maintain consistency.
4. Sample Solution Measurement
   Accurately pipette 1.00 mL of the sample solution or diluted sample solution (containing 10 μg~100 μg of sodium alginate) into a test tube. Perform colorimetric analysis under the same conditions as the standard series solutions, setting the blank test solution as the zero reference and reading the absorbance. Use the standard curve to find the corresponding concentration (μg/mL).
   P.S. If the sample solution shows color before the addition of the color reagent, prepare another test tube with the same volume of sample solution. Replace the color development step with 100 μL of 5 g/L sodium hydroxide solution instead of resorcinol, follow the same procedure, measure the absorbance, and subtract the result during calculation.
5. Blank Test
   Follow the same procedure as for the sample solution, except without the addition of the sample.
   P.S. To eliminate nitrate interference, you may attempt to use Sephadex G10 gel chromatography. The reference method is as follows: take 1 mL of sample solution (containing 10 mg~20 mg of alginate) and perform Sephadex G10 gel filtration chromatography with a gel height of 75 cm and a diameter of 1.6 cm at a flow rate of 1 mL/min. Collect between 55 and 95 min, then make up the volume to 100 mL to obtain the sample solution.

1. Setup of the Apparatus:
   Set up a 1000 mL distillation flask and pour the organic phase sample containing limonene into the flask. Ensure the liquid volume does not exceed 1/3 of the flask’s capacity. Wrap Teflon tape around the ground joint as shown in the diagram, and assemble the apparatus from bottom to top, right to left.
   
   First, connect the condenser to water, with water entering from the bottom and exiting from the top, then connect it to the apparatus. Secure the condenser with a clamp, placing the clamp screw upwards, and position it at the lower middle section of the condenser. Use a round-bottom flask as the receiving flask, and connect the vacuum outlet at the top of the condenser to the vacuum pump via a rubber tube.
   
   Once the apparatus is assembled, it should be observed from the front or side to ensure all components are on the same plane. This system is a vacuum setup, so all glassware must be thick-walled to prevent accidents.
2. Start Vacuum Rotary Evaporation:
   After confirming the setup is correct, start the rotary evaporator and gradually increase the rotation rate until a liquid film forms on the inner wall of the distillation flask. Turn on the vacuum pump, close the back-pressure valve, and once the liquid inside the distillation flask stabilizes, begin heating. Increase the temperature gradually and continue heating until boiling gas appears at the mouth of the distillation flask. Slow the rate of temperature increase and maintain the temperature when liquid droplets start appearing at the condenser outlet. Continue distillation at this temperature. When boiling in the distillation flask ceases, or no more droplets appear at the condenser, raise the temperature by 0.5°C, repeating this process. Continue raising the temperature until it reaches the upper limit of the water bath (~100°C), then stop the distillation.
3. End of Vacuum Rotary Evaporation:
   After distillation is complete, release the back-pressure valve, turn off the vacuum pump, and disassemble the rotary evaporation apparatus. Collect the product from the round-bottom flask (the product will be a layered liquid, with the lower layer primarily water and the upper layer primarily limonene).
4. Calculation:
   Afterward, separate the residue in the distillation flask and the product in the round-bottom flask. Dry a clean glass graduated cylinder and tare it on a balance. Then, pour the separated liquid into the graduated cylinder, weigh it, and measure its volume. Finally, take 500 μL samples from the organic phase of the residue and product, dilute them by 10⁴, and perform gas chromatography detection.
   
   Calculate the relative purity and recovery rate of the purified limonene sample using the following formulas:
   
   

$$\text{\% Relative purity}=\left(\frac{\text{Peak area of limonene in the product}}{\text{Total peak area in the product}}\times\frac{\text{Peak area of limonene in primary standard}}{\text{Total peak area of primary standard}}\right)\times 100\%$$

Use 95% limonene (CAS: 7705-14-8, Macklin) as the reference standard.

The formula to calculate limonene recovery is as follows:

$$\text{\% Recovery} = \left(\frac{\text{The mass of limonene in the product (g)}}{\text{The mass of limonene in raw materials (g)}}\right) \times 100\%$$