Microorganisms in spent water-miscible metalworking fluids as a resource of strains for their disposal

Metalworking fluids (MWFs) are essential components for modern metalworking technologies. The volume of world consumption of diluted working solutions exceeds 20 million tons annually. Water miscible MWFs accounts to 90% of the global MWFs demand. Spent MWFs contain toxic xenobiotics and residues of biocides. They are hazardous to the environment and require complex processing and detoxification. Microorganisms are able to colonize MWFs and metabolize the majority of available substances (oils and additives). Microbial degradation is one of the most efficient ways to dispose of water-miscible MWFs. The species composition of microorganisms changes during the operation of the fluid. From the point of view of the selection of effective biodegradion agents, the composition of microorganism species at the end of the MWFs service life cycle is of high interest for the industry. The aim of this work is to study the fungal and bacterial diversity of the microbiota of the spent MWF both by the method of isolating axenic cultures of fungi and bacteria, and by NGS-based metagenomic profiling using 16S rRNA for bacteria and ITS for fungi, and evaluate the possibility of their growth in diluted to a working concentration MWFs that differ in composition. Multiplex sequencing revealed (share in the samples more than 0.01%) 18 bacterial genera: Achromobacter, Alcaligenes, Aquamicrobium, Bacillus, Bacteroides, Brevundimonas, Caulobacter, Chryseobacterium, Dechlorobacter, Desulfovibrio, Dysgonomonas, Ochrobactrum, Proteiniphilum, Pseudomonas, Ralstonia, Shewanella, Sphingomonas, Stenotrophomonas and 17 fungal genera: Alternaria, Aspergillus, Bjerkandera, Cadophora, Candida, Cladosporium, Cryptococcus, Cystobasidium, Didymella, Fusarium, Malassezia, Penicillum, Pithomyces, Saccharomyces, Talaromyces, Wickerhamomyces, Yarrowia. Six genera of fungi (Saccharomyces, Fusarium, Malassezia, Bjerkandera, Penicillum, Alternaria) were identified in all tested samples. Yarrowia, Saccharomyces, Cadophora and Fusarium dominated in different samples. The diversity of bacterial biota was significantly higher than that of fungi. There was no clear dominance of any kind of bacteria in any sample. The analysis of isolated axenic cultures revealed that bacteria Shewanella putrefaciens, Proteus sp., Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Aeromonas hydrophila, Pseudomonas pseudoalcaligenes and fungi Fusarium solani, F. oxysporum, Yarrowia lipolytica, Candida metapsilosis were capable of growing in water-miscible MWFs. The data on the microorganism's species composition of the spent MWFs will make it possible to select species-specific DNA regions and design specific and sensitive test systems that can be used for identifying and evaluating the concentration of potential agents for the biodegradation of MWFs intended for disposal.