📚 Ecosystems 2.1.2 Anaerobic Respiration Experiment Design | 生态系统 2.1.2 厌氧呼吸实验设计
Anaerobic respiration plays a crucial role in many ecosystems, especially in environments where oxygen is scarce, such as waterlogged soils, sediments, and the digestive tracts of animals. Understanding how organisms like yeast undergo fermentation not only illustrates key biochemical pathways but also provides insight into nutrient cycling and energy flow within ecosystems.
厌氧呼吸在许多生态系统中起着至关重要的作用，尤其是在缺氧环境中，如水涝土壤、沉积物和动物消化道。理解酵母等生物如何进行发酵，不仅能说明关键的生化途径，还能深入了解生态系统中的养分循环和能量流动。
1. The Role of Anaerobic Respiration in Ecosystems | 厌氧呼吸在生态系统中的作用
In the absence of oxygen, certain decomposers and microorganisms switch to anaerobic respiration to break down organic matter, releasing nutrients back into the soil or water. This process is vital for the carbon cycle and can produce methane (CH₄) in wetlands or ethanol (C₂H₅OH) in fermenting fruits.
在无氧条件下，某些分解者和微生物转而进行厌氧呼吸来分解有机物，将养分释放回土壤或水中。这一过程对碳循环至关重要，并可在湿地中产生甲烷 (CH₄) 或在发酵的水果中产生乙醇 (C₂H₅OH)。
Yeast (Saccharomyces cerevisiae) is a classic model organism for studying anaerobic respiration in the laboratory because it can survive with or without oxygen, undergoing alcoholic fermentation when oxygen is lacking.
酵母 (Saccharomyces cerevisiae) 是实验室研究厌氧呼吸的经典模式生物，因为它可以在有氧或无氧条件下生存，在缺氧时进行酒精发酵。
2. Experimental Principle: Yeast Fermentation | 实验原理：酵母发酵
The overall equation for alcoholic fermentation in yeast is:
酵母酒精发酵的总方程式为：
C₆H₁₂O₆ → 2 C₂H₅OH + 2 CO₂ + small amount of ATP
Glucose is broken down into ethanol and carbon dioxide, with only a small yield of ATP compared to aerobic respiration. The production of CO₂ gas can be measured to monitor the rate of fermentation.
葡萄糖被分解为乙醇和二氧化碳，与有氧呼吸相比，仅产生少量 ATP。通过测量 CO₂ 气体的产生量，可以监测发酵速率。
In this experiment, the volume of CO₂ evolved over time will be recorded under strict anaerobic conditions.
本实验将在严格的厌氧条件下，记录随时间推移产生的 CO₂ 体积。
3. Aim and Hypothesis | 实验目的与假设
Aim: To investigate the rate of anaerobic respiration in yeast by measuring carbon dioxide production under oxygen-free conditions.
目的： 通过测量无氧条件下二氧化碳的产量，研究酵母的厌氧呼吸速率。
Hypothesis: If yeast is provided with glucose and placed in an anaerobic environment, it will produce CO₂, and the rate of production will be influenced by factors such as temperature and glucose concentration.
假设： 如果为酵母提供葡萄糖并置于厌氧环境中，它将产生 CO₂，且生成速率会受到温度和葡萄糖浓度等因素的影响。
4. Materials and Apparatus | 材料与设备
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Yeast suspension (10% active dry yeast in warm water, pre-activated)
酵母悬液（10% 活性干酵母溶于温水，预先活化）
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5% glucose solution
5% 葡萄糖溶液
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Liquid paraffin oil
液体石蜡油
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Two boiling tubes with rubber bungs and delivery tubes
两支配有橡胶塞和导气管的沸腾管
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Two test tubes containing limewater (calcium hydroxide solution)
两支装有石灰水（氢氧化钙溶液）的试管
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Water bath set at 30°C
设置为 30°C 的水浴锅
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Stopwatch or timer
秒表或计时器
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Graduated syringes or gas syringe for measuring gas volume
用于测量气体体积的刻度注射器或气体注射器
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Thermometer, pipettes, safety goggles
温度计、移液管、护目镜
5. Procedure for Setting Up Anaerobic Conditions | 设置厌氧条件的步骤
1. Label two boiling tubes as ‘Anaerobic’ and ‘Aerobic’ (control). Add 10 mL of yeast suspension and 10 mL of glucose solution to each tube. Gently mix.
1. 将两支沸腾管分别标记为“厌氧”和“有氧”（对照）。向每支管中加入 10 mL 酵母悬液和 10 mL 葡萄糖溶液，轻轻混匀。
2. In the anaerobic tube, carefully layer liquid paraffin over the mixture to a depth of about 1 cm. This prevents oxygen from entering the solution. Insert the rubber bung with delivery tube, ensuring an airtight seal.
2. 在厌氧管中，在混合液上方小心地加入一层约 1 cm 厚的液体石蜡，这可以防止氧气进入溶液。塞上带有导气管的橡胶塞，确保密封良好。
3. In the aerobic tube, do not add paraffin; simply stopper it loosely to allow air exchange, or leave it open. However, for fair comparison, it’s better to stopper it but without paraffin, so that some oxygen is available initially.
3. 在有氧管中，不添加液体石蜡；只需松散地塞住管口以允许空气交换，或直接敞口。但为了公平比较，最好同样塞上塞子但不加石蜡，这样初始时有氧气可用。
4. Connect the delivery tube from each boiling tube to a test tube containing limewater. Alternatively, attach a gas syringe to measure the volume of gas produced directly.
4. 将每支沸腾管的导气管连接到装有石灰水的试管中。或者，连接一支气体注射器以直接测量产生的气体体积。
5. Place both setups in a water bath at 30°C to maintain constant temperature.
5. 将两套装置放入 30°C 水浴中，保持恒温。
6. Measuring Carbon Dioxide Production | 测量二氧化碳产量
CO₂ can be detected by passing the gas through limewater, which turns from clear to milky (forms calcium carbonate precipitate). For quantitative data, a gas syringe is preferred to record the volume of CO₂ collected at regular intervals, e.g., every 2 minutes for 20 minutes.
CO₂ 可通过将气体通入石灰水来检测，石灰水会从澄清变浑浊（生成碳酸钙沉淀）。为获得定量数据，最好使用气体注射器，每隔一定时间记录收集到的 CO₂ 体积，例如每 2 分钟记录一次，持续 20 分钟。
If limewater is used, note the time taken for the limewater to turn milky; a shorter time indicates a faster rate of CO₂ production.
如果使用石灰水，记录石灰水变浑浊所需的时间；时间越短，表示 CO₂ 生成速率越快。
| Time (min) | Volume of CO₂ (cm³) – Anaerobic | Volume of CO₂ (cm³) – Aerobic |
|---|---|---|
| 0 | 0 | 0 |
| 2 | ||
| 4 |
Record your results in a table like the one above for clear data presentation.
将结果记录在如上所示的表格中，以便清晰呈现数据。
7. Variables and Controls | 变量与控制
Independent variable: Presence or absence of oxygen (anaerobic vs. aerobic conditions). In a more advanced design, glucose concentration or temperature can be the independent variable, keeping oxygen absent.
自变量： 氧气的有无（厌氧条件 vs. 有氧条件）。在更进阶的设计中，可将葡萄糖浓度或温度作为自变量，同时保持无氧条件。
Dependent variable: Rate of carbon dioxide production (measured as cm³ of CO₂ per minute).
因变量： 二氧化碳生成速率（以每分钟产生的 CO₂ 立方厘米数测量）。
Control variables: Volume and concentration of yeast suspension, volume and concentration of glucose solution, temperature (use water bath), pH (buffered if necessary), and incubation time.
控制变量： 酵母悬液的体积和浓度、葡萄糖溶液的体积和浓度、温度（使用水浴）、pH（必要时使用缓冲液）以及孵育时间。
A control tube containing boiled (dead) yeast may also be set up to confirm that CO₂ production is due to living organisms.
还可以设置一支包含煮沸（灭活）酵母的对照管，以确认 CO₂ 的产生是由活细胞引起的。
8. Data Collection and Presentation | 数据收集与呈现
Plot a graph of volume of CO₂ produced (y-axis) against time (x-axis) for both conditions. The slope of the line indicates the rate of fermentation.
绘制两种条件下 CO₂ 产生体积（y 轴）随时间（x 轴）变化的图线。线的斜率表示发酵速率。
Calculate the rate of CO₂ production by dividing the volume of gas by the time interval. Compare the rates between the anaerobic and aerobic setups. Note that in aerobic conditions, yeast will initially respire aerobically, producing more CO₂ per glucose molecule, but the rate may differ.
通过将气体体积除以时间间隔来计算 CO₂ 生成速率。比较厌氧和有氧装置之间的速率。注意，在有氧条件下，酵母最初进行有氧呼吸，每个葡萄糖分子产生更多的 CO₂，但速率可能不同。
Use statistical tests if multiple trials are performed, such as calculating the mean and standard deviation.
如果进行多次试验，可使用统计检验，例如计算平均值和标准差。
9. Safety Considerations | 安全注意事项
Wear safety goggles to protect eyes from splashes. Liquid paraffin is flammable—keep away from open flames. Yeast and glucose cultures may cause allergies; wash hands after handling. Be careful when using glassware to avoid breakage. Ethanol produced is in small quantities but still flammable.
戴上护目镜防止液体飞溅伤害眼睛。液体石蜡易燃——远离明火。酵母和葡萄糖培养物可能引起过敏；操作后洗手。使用玻璃器皿时小心避免破碎。产生的乙醇量虽小但仍可燃。
10. Expected Results and Ecological Implications | 预期结果与生态学意义
Under anaerobic conditions, CO₂ will be steadily produced, turning limewater milky more slowly or rapidly depending on conditions. In the aerobic setup, respiration may initially be aerobic, but as oxygen depletes, it may switch to fermentation, yielding a mixed result. Typically, the rate of CO₂ production per unit glucose is lower in anaerobic respiration than in aerobic respiration because of incomplete oxidation.
在厌氧条件下，CO₂ 会稳定产生，使石灰水慢慢或快速变浑浊，这取决于条件。在有氧装置中，呼吸最初可能为有氧呼吸，但随着氧气耗尽，可能转而进行发酵，导致结果混合。通常，单位葡萄糖的 CO₂ 生成速率在厌氧呼吸中低于有氧呼吸，因为氧化不完全。
Ecologically, anaerobic respiration allows organisms to thrive in oxygen-depleted niches, driving the decomposition of organic matter and the recycling of nutrients such as
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