Oof, this one is going to be tricky since you almost certainly have mixed samples. You could amplify, clone, streak, and sequence.

Or just use a next-gen sequencing system.

Sanger sequencing requires that you have only 1 type of molecule to give any useful data.

And, 16S primers are sensitive enough that they will amplify from pretty much anything - human, fish, mushrooms, bacteria, etc.

Wrong approach. And if you continue with that, you will just waste time and resources.

You need to first decide what you want to see and how you want to proceed. Made a strategy for that.

Or ask for a strategy if you dont know waht and how. If you are just asking question related to what you are already doing, the answer would be stop that, since it is not the correct approach to undertake.

It sounds like you have attempted direct colony sequencing using universal 16S primers but the results were not successful, even for known microbes. In this case, it may be worth considering DNA extraction and purification as an alternative method. Although it may be time-consuming, it can often provide higher quality DNA for downstream sequencing.

If you prefer not to do DNA extraction, colony PCR can be an alternative approach. You can use universal 16S primers to amplify the 16S rRNA gene from the colonies and perform PCR cleanup to remove any residual PCR components. However, producing clean bands from colony PCR can sometimes be difficult due to potential inhibitors or low template DNA. Optimizing PCR conditions and using appropriate positive and negative controls can help with this issue.

Regarding the possibility of some of the unknown microbes being fungal, it would be more appropriate to use fungal-specific primers if you suspect that some of them are fungal. Using universal 16S primers may not amplify fungal 16S rRNA genes. Alternatively, you can use ITS (Internal Transcribed Spacer) primers, which are commonly used for fungal identification.

If direct colony sequencing using universal 16S primers has failed, DNA extraction and purification or colony PCR with appropriate controls can be alternative approaches to obtain DNA for sequencing. If you suspect that some of the microbes are fungal, using fungal-specific primers or ITS primers may be more appropriate.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU