## 2 Materials and Methods  
Road bitumen 35/50 (according to the European Specification EN) refined from Ural crude oil was used for the research. As a modifier, a bio-agent of fatty acid methyl esters (FAMEs) based on rapeseed oil (RME--rapeseed methyl esters) with a cobalt catalyst in the amount of 0.1% m/m as calculated for metal and with a reaction initiator of cumene hydroperoxide in the amount of 1% m/m was used. Details of the production process, composition, and chemical reaction connected to bio-agent processing are described elsewhere [17]. The bio-modifier was produced by an application of an oxidation reaction promoter to the RME and then by allowing this mixture to be oxidized at a temperature of \(20\pm 5\,^{\circ}\mathrm{C}\). The bio-agent used in this study is a bi-functional material. During the first stage, after being added to the bitumen, it fluidized bitumen by lowering its viscosity. During the second stage, due to the presence of double unsaturated bonds and cobalt catalyzer in the RME, a slow polymerization process started in the bio-agent. Polymerization occurred in the presence of oxygen and partially recovered initial bitumen viscosity. The research presented in this paper was conducted observing the bitumen specimens and the asphalt mixture samples. The following samples were subjected to bitumen tests: clean unmodified bitumen 35/50 and bitumen modified with bio-modifier at the amounts of 1.25%, 2.5%, 3.75% and 5.0% of addition, in terms of bitumen weight. In addition, as reference points, unmodified bitumens 50/70 and 70/100 were also tested. Road bitumen 35/50 was mixed with a bio-modifier for one minute at a temperature of 150 \({}^{\circ}\)C. FAME demonstrated full blendability to the bitumen binder. In order to determine the characteristics of the polymerization process in time, bitumens modified with the bio-agent were conditions in the thin layer of 1 mm for up to 56 days at room temperature. The tests were conducted directly after modification (Day 0) and after 7, 14, 28, and 56 days of conditioning. Bitumens were tested in a dynamic shear rheometer (DSR) for complex modulus G* and for phase angle \(\mid\)\(\delta\)\(\mid\). Tests were accomplished with a constant frequency of 10 rad/s in the temperature range between 0 \({}^{\circ}\)C and 100 \({}^{\circ}\)C, using two parallel plates with a 25 mm diameter and a 1 mm gap. For asphalt mixture tests, asphalt concrete (AC) with a 16 mm maximum aggregate sieve size was used. Two types of asphalt mixtures were tested: a reference AC with 30% RAP with 35/50 bitumen and AC with 30% RAP, 35/50 bitumen, and 2.5% bio-agent (bio-agent content was determined based on the bitumen testing as explained later in this paper). Figure 1 presents an aggregate blend gradation curve, while basic properties of asphalt concrete are shown in Table 1. For air void determination, asphalt mixtures were compacted in a Marshall compact with 2 \(\times\) 75 blows for each sample, according to EN 12697-30 Bituminous mixtures. Test methods for hot mix asphalt. Specim preparation by impact compactor. Compaction level was determined based on the air void determination according to EN 12697-8 Bituminous mixtures. Test methods for hot mix asphalt. Determination of void characteristics of bituminous specimens. During compaction, all samples demonstrated a temperature similar to \(135\pm 5\)\({}^{\circ}\)C. The reference mixture exhibited air void content of 4.6%. The bio-agent application resulted in better mixture compaction (3.2% air voids). The asphalt mixture with bio-agent was not optimized for air void content due to the Polish requirements for asphalt mix design. According to requirements WT-2 [18], the mixture was designed for the required air void range. Once air void content is within the range (3%-6%), further optimization is not required. In addition, in this research, there was an attempt to determine the influence of bio-agent on the compatibility of asphalt mixture and those on its properties.