The acetylene reduction assay (ARA) was used to measure nitrogen fixation by quantifying nitrogenase activity through the conversion of acetylene to ethylene. Ethylene production was measured using gas chromatography corrected for blanks, and normalized to biomass. This assay enabled comparison of nitrogen fixation efficiency across strains and conditions, helping identify high and low performing nitrogen-fixing cultures for further analysis.
| Item | Quantity | Supplier | Notes |
|---|---|---|---|
| Mastigocladus laminosus cultures | — | — | Cultures grown in nitrogen-free conditions prior to assay |
| ND medium (nitrogen-free growth medium) | approximately 10 ml per incubation vial | — | — |
| 20 mL crimp-sealed serum vials | 1 vial per light and dark replicate | — | — |
| rubber septa and aluminum crimp caps | 1 set per vial | — | Ensure airtight sealing |
| Calcium carbide (CaC₂) | 5g | — | Used for acetylene gas generation |
| acetylene gas generation | 100ml | — | acetylene gas generation |
| Acetylene gas (C₂H₂) | 5ml per vial | — | Substrate for nitrogenase |
| Gas-tight syringes | 5-20 ml capacity | — | For acetylene injection and headspace sampling |
| Ethylene gas standards | — | — | For standard curve preparation |
1. Prepare cultures (normalized biomass): Homogenize culture gently (break large clumps but keep chains intact) and normalize to OD at 750nm = 0.050+ or - 0.003
2. Set up incubations: Add 10 mL ND medium into 20 mL crimp-sealed vials (gas-tight).
3. Inoculate and replicate: For each sub-line, prepare one light and one dark replicate vial.
4. Generate acetylene gas: Produce acetylene by reacting 5 g calcium carbide with 100 mL deionized water (collect the acetylene gas produced).
5. Start the assay: At the beginning of the light cycle, inject 5 mL acetylene gas into each incubation vial.
6. Incubate: Incubate for 4 hours under the assigned condition (light or dark).
7. Collect headspace: At the end, withdraw ~15 mL headspace gas from each incubation vial and inject it into a pre-evacuated 5 mL crimp vial.
8. Measure ethylene: Analyze headspace on an FID gas chromatograph (Shimadzu GC-2014 or equivalent) to quantify ethylene produced.
9. Quantify + normalize: Use an ethylene standard curve, blank-correct using vials containing only ND medium, and normalize results to OD = 0.050.
1. You should detect an ethylene (C₂H₄) peak in GC-FID chromatograms from active samples, while blank ND medium vials should be near zero after correction.
2. Higher ethylene production = higher nitrogenase activity, allowing quantitative comparison across strains/conditions once blank-corrected and normalized to OD = 0.050.
3. Light vs dark incubations typically show whether nitrogenase activity is light-supported (higher in light) or maintained in the dark under your conditions (often lower).
1. No/very low ethylene peak
-Culture not actively fixing N₂ (not well acclimated to ND medium, wrong timing vs light cycle)
-OD normalization incorrect (too little biomass)
-Acetylene not injected properly / vial leaks (check septa, crimps, and syringe tightness)
2. Inconsistent replicates
-Unequal OD across vials (recheck OD₇₅₀ and mixing before dispensing)
-Unequal incubation conditions (light intensity differences, temperature fluctuations)
-Variable headspace sampling volume (use consistent syringe volume and technique)
3. Broad/unstable GC peaks
-Contaminated syringe/lines (flush syringes; ensure clean sampling practice)
-Poor standard curve or drift (run standards and blanks routinely; ensure consistent GC conditions)
4. High background in blanks
-Contamination of ND medium or vials with ethylene/organics
-Carryover in GC system (run blank injections between samples)
1. Acetylene is highly flammable/explosive: Keep away from flames/sparks/heat; work in a well-ventilated area and use gas-tight vials/syringes to prevent leaks.
2. Calcium carbide reaction is hazardous: Reaction with water releases acetylene and can be vigorous; add water carefully, avoid pressure buildup, and wear lab coat, gloves, safety goggles.
3. Crimp-sealed vials are pressurized: Use proper crimping technique; do not over-pressurize; handle syringes carefully to prevent needle-stick injuries.
4. GC/FID safety: Follow instrument safety rules (hot zones, flammable gases used by FID, and cylinder handling if applicable).