Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with <5% of the original TNT recovered as diaminonitrotoluene metabolites. Eubacterium ruminantium, Lactobacillus ruminis, Ruminobacter amylophilus, Streptococcus bovis, and Wolinella succinogenes were able to completely degrade 100 mg/L TNT, with 23-60% of the TNT recovered as aminodinitrotoluene and/or diaminonitrotoluene metabolites. Clostridium polysaccharolyticum, Megasphaera elsdenii, Prevotella bryantii, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens were able to degrade 80-90% of 100 mg/L TNT. Desulfovibrio desulfuricans subsp. desulfuricans, Prevotella albensis, and Treponema bryantii degraded 50-80% of the TNT. Anaerovibrio lipolytica was completely inhibited by 100 mg/L TNT. These results indicate that a variety of rumen bacteria is capable of transforming TNT.

Bioremediation is of great interest in the detoxification of soil contaminated with residues from explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although there are numerous forms of in situ and ex situ bioremediation, ruminants would provide the option of an in situ bioreactor that could be transported to the site of contamination. Bovine rumen fluid has been previously shown to transform 2,4,6-trinitrotoluene (TNT), a similar compound, in 4 h. In this study, RDX incubated in whole ovine rumen fluid was nearly eliminated within 4 h. Whole ovine rumen fluid was then inoculated into five different types of media to select for archaeal and bacterial organisms capable of RDX biotransformation. Cultures containing 30 μg mL(-1) RDX were transferred each time the RDX concentration decreased to 5 μg mL(-1) or less. Time point samples were analyzed for RDX biotransformation by HPLC. The two fastest transforming enrichments were in methanogenic and low nitrogen basal media. After 21 days, DNA was extracted from all enrichments able to partially or completely transform RDX in 7 days or less. To understand microbial diversity, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted. Cloning and sequencing of partial 16S rRNA fragments were performed on both low nitrogen basal and methanogenic media enrichments. Phylogenetic analysis revealed similar homologies to eight different bacterial and one archaeal genera classified under the phyla Firmicutes, Actinobacteria, and Euryarchaeota. After continuing enrichment for RDX degraders for 1 year, two consortia remained: one that transformed RDX in 4 days and one which had slowed after 2 months of transfers without RDX. DGGE comparison of the slower transforming consortium to the faster one showed identical banding patterns except one band. Homology matches to clones from the two consortia identified the same uncultured Clostridia genus in both; Sporanaerobacter acetigenes was identified only in the consortia able to completely transform RDX. This is the first study to examine the rumen as a potential bioremediation tool for soils contaminated with RDX, as well as to discover S. acetigenes in the rumen and its potential ability to metabolize this energetic compound.

Bacterial endospores are highly resistant structures and dipicolinic acid is a key component of their resilience and stability. Due to the difficulty in controlling endospore contaminants, they are of interest in clean rooms, food processing, and production industries, while benefical endospore-formers are sought for potential utility. Dipicolinic acid production has traditionally been recognized in Bacilli, Clostridia, and Paenibacilli. Here, sixty-seven strains of aerobic and anaerobic endospore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Clostridium, Fontibacillus, Lysinibacillus, Paenibacillus, Rummeliibacillus, and Terribacillus were grown axenically and sporulated biomasses were assayed for dipicolinic acid production using fluorimetric detection. Strains testing positive were sequenced and the genomes analyzed to identify dipicolinic acid biosynthesis genes. The well-characterized biosynthesis pathway was conserved in 59 strains of Bacilli and Paenibacilli as well as two strains of Clostridia; six strains of Clostridia lacked homologs to genes recognized as involved in dipicolinic acid biosynthesis. Our results confirm dipicolinic acid production across different classes and families of Firmicutes. We find that members of Clostridium (cluster I) lack recognized dipicolinic acid biosynthesis genes and propose an alternate genetic pathway in these strains. Finally, we explore why the extent and mechanism of dipicolinic acid production in endospore-forming bacteria should be fully understood. We believe that understanding the mechanism by which dipicolinic acid is produced can expand the methods to utilize endospore-forming bacteria, such as novel bacterial strains added to products, for genes to create inputs for the polymer industry and to be better equipped to control contaminating spores in industrial processes.