Microalgae cultivation using organic carbon waste has been proved as a promising way to enhance filamentous algal biomass bio-protection. However, the carbon source in anaerobic digestate (AD; the effluent of biogas plants) contains various organic carbon source and other components, with a complex metabolic process in algae. The utilization of organic carbon including volatile fatty acids (VFAs) and glycerin by Tribonema minus were investigated. The results showed that the utilization of VFAs as the carbon source by T. minus with the addition of 0.3% CO2 decreased pH value below 6.0 and accumulated the undissociated VFAs (UVFAs) (UVFAs contents: 99.29 ± 8.34 mg L−1, 176.52 ± 4.05 mg L−1, 115.02 ± 5.86 mg L−1, and 127.52 ± 7.24 mg L−1 for acetic acid, propanoic acid, butyric acid, and valeric acid, respectively after 1st day cultivation), which inhibited T. minus growth. However, this inhibition effect was not observed when using glycerin as a carbon source (Biomass: 2.95 g L−1). To study the inhibition mechanism of UVFAs on the algae, the growth and metabolism of algae using acetic acid and butyric acid as carbon source was studied under different pH (4.5, 5.5, 6.5). The study found that the UVFAs content at pH 4.5–5.5 was higher than that at pH 6.5 with the same carbon concentrations. UVFAs inhibited the biomass accumulation of T. minus and decreased photosynthetic and enzymatic activities. This study concluded that T. minus can be cultivated with a mixotrophic mode using AD that contains VFAs with controlled pH value around neutral.