Multiple generations of biofuel manufacturing technologies have been proposed and developed, among which first generation technology is the most mature. It typically utilizes corn grain as feedstock in bio-refineries in many countries such as the United States. However, the use of edible matter of crops like corn may lead to a food-versus-fuel competition. To address such an issue, second generation biofuel manufacturing technology using the non-edible matter of crops has been developed. In this paper, the economic viability and environmental sustainability are analyzed and quantified, when restructuring the biofuel supply chain from employing first generation manufacturing technology to the one utilizing second generation technology considering the existing supply chain infrastructure. Two supply chain restructuring strategies, i.e., distributed and centralized preprocessing deployment, are modeled for implementing biomass preprocessing for second generation biofuel manufacturing. Bi-objective optimization formulations for the corn stover-sourced biofuel (a typical second generation biofuel) supply chain considering both economic and environmental aspects are proposed under both strategies. Different decision variables such as the locations selected for constructing preprocessing centers and the corresponding corn stover handling capacities of such centers, as well as the material flows between farms, preprocessing centers, and bio-refinery plants are identified. A case study based on the state of Missouri in the United States is implemented to illustrate the effectiveness of the proposed model and analyze the performance of different strategies. The results of the case study show that when corn stover is used as feedstock in bio-refineries, the unit cost of bioethanol production can be reduced, while the unit emission is increased compared to the corn-sourced supply chain under both supply chain restructuring strategies. Specifically, in Missouri where the daily stover handling amount per bio-refinery plant is relatively low and the stover collection radius is relatively small, the centralized strategy outperforms the distributed one with a higher cost reduction and a smaller increase in emissions. It reduces the unit cost by 27.39%, while increasing the unit emission by 24.42%, compared to the corn-sourced supply chain.