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Biodiesel is a widely used biofuel in diesel engines, which is of particular interest as a renewable fuel because it possesses the similar properties as the diesel fuel. The pure soybean biodiesel was tested in an optical constant volume combustion chamber using natural flame luminosity and forward illumination light extinction (FILE) methods to explore the combustion process and soot distribution at various ambient temperatures (800 K and 1000 K) and oxygen concentrations (21%, 16%, 10.5%). Results indicated that, with a lower ambient temperature, the autoignition delay became longer for all three oxygen concentrations and more ambient air was entrained by spray jet and more fuel was burnt by premixed combustion. With less ambient oxygen concentration, the heat release rate showed not only a longer ignition delay but also longer combustion duration.