Bio-succinic acid production using microorganisms has been interesting as an environmentally friendly process. Palm oil mill effluent (POME) was considered as a cheap substrate to lower the cost of production. It was revealed that 2-fold diluted POME produced more succinic acid than undiluted and 5-fold diluted POME. In addition, the effects of various neutralizing agents on succinic acid production utilized to manage pH and CO2 supply indicated that the utilization of MgCO3 as a neutralizing agent produced succinic acid of 11.5 g/L with a small amount of by-product synthesis. Plackett–Burman Design (PBD) was used to screen the most significant nutrients for bio-succinic acid production from 2-fold diluted POME using E. gallinarum. From the Pareto chart, MgCO3 and peptone presented the highest positive effect on the production of succinic acid. In addition, Box–Behnken Design (BBD) was conducted to increase bio-succinic acid production. Experiments showed the highest production of succinic acid of 23.7 g/L with the addition of 22.5 g/L MgCO3 and 12.0 g/L peptone in 2-fold diluted POME. Moreover, the experiment of replacing MgCO3 with CO2 from biogas resulted in 19.1 g/L of succinic acid, simultaneously creating the high purity of biogas and a higher CH4 content.