As NovaCoder mentions, the ACATune line should be at the very top of the startup-sequence, before anything else.
The main purpose of the ACATune tool is to speed up your Amiga, and one of the things it can do is to copy the Kickstart to Fast RAM, which makes things go a bit faster. By using the -maprom * option on the ACAtune line, the Kickstart ROM will be copied to RAM.
You can also use a Kickstart image file instead, but this requires that you get hold of a suitable Kickstart file first. If you do this, you can change the ACATune line to:
C:ACATune -maprom Devs:kick31.rom -cache on -burst on >NIL:
But this assumes that the Kickstart ROM image is called kick31.rom and that it has been copied to the Devs drawer on your boot partition.