Pullulan is generated by the microbial A. pullulans through the processing mainly of glucose, but can also be produced from maltose, fructose, galactose, sucrose, and mannose. In a commercial setting, pullulan is obtained from a strain of A. pullulans that is non-toxic, non-pathogenic, and unmodified genetically that is given a liquid form of starch in a set environment. The pullulan produced can be modified by different conditions such as the nutrients provided, temperature, pH, oxygen content, and other supplements. The microbial needs to be provided with a source of carbon and nitrogen in order to produce pullulan and the ratio of carbon to nitrogen needs to be precise in order to maximize pullulan production. Higher levels of nitrogen than carbon are required as excess carbon can decrease the efficiency of the enzymes and excess nitrogen can increase the production of biomass, but does not affect the pullulan production. Oxygen is also important for the proliferation of the A. pullulans cells and the production of pullulan. Further supplements can be used in order to increase the level of pullulan production, such as olive oil and tween 80. 
While the manufacturing conditions of pullulan can be altered in order to increase yield, chemical modifications of pullulan can also be used to alter the properties of the pullulan. The unmodified structure of pullulan contains nine hydroxyl groups attached to the backbone of the molecule, and these hydroxyl groups can be replaced with other functional groups. Some examples of processes that can modify the functional groups of pullulan include sulfation, esterification, oxidation, etherification, copolymerization, amidification, and others. Pullulan can be given a negative charge through creating an ester linkage that attaches a carboxylate group to the hydroxyl, which yields a carboxymethyl pullulan. Pullulan is hydrophilic and can be modified to have hydrophobic functionality by adding a cholesterol group. The main benefit of the added hydrophobic functionality is that it makes it so the pullulan can form self assembling micelles. Another notable modification to pullulan is the acetylation of pullulan in order to create pullulan acetate (PA), which also has a hydrophobic functionality. PA has the benefit of forming self-assembled nanoparticles, which can simplify manufacturing of certain pullulan bioconjugates. Pullulan and pullulan derivatives can also be folated in order to improve cancer cell targeting as the nanoparticle can be endocytosed into the cancer cells through folate-mediated endocytosis.