This study focused on the cultivation of pure culture SMX biodegrading organisms to perform specific biodegradation experiments. It is known that cultivation, especially on solid media, is affected with the problem described as “viable but non cultivable” (VBNC) [30, 31]. Solid media being implicitly required for the isolation of pure cultures is for sure limited in its cultivation efficiency mainly due to reduced water content and different or inappropriate nutrient conditions. Thus only a low percentage of around 1% of the active organisms in environmental samples  and around 15% from activated sludge can be cultivated [33, 34]. In this study 9 different isolates out of 110 pure cultures were obtained that showed SMX biodegradation. This quite high percentage of almost 10% was only possible with a two-step SMX-acclimation experiment that was conducted to increase the chance to cultivate SMX biodegrading organisms by applying a strong selective pressure using 10 mg L-1 SMX in the media. Furthermore, R2A medium that is known to work well for isolation of aquatic organisms  was applied for the cultivation of bacteria being assumed to be at least SMX-resistant when growth was observed on SMX-reinforced R2A. However, a lot more organisms compared to those cultivated in this study might be present in activated sludge capable of SMX biodegradation. These VBNCs might be taxonomically characterized by culture-independent methods, e.g. restriction fragment length polymorphism screening [36, 37]. However, for our focus on linking biodegradation patterns, rates and nutrient utilization to specific species these methods were not feasible. Only with actively biodegrading pure cultures a clear and precise coherence between SMX biodegradation and taxonomically identified species is possible. As a final goal, pure cultures would allow to analyze species-specific biodegradation products and thus determine potential SMX biodegradation pathways. Applying that knowledge to WWTP techniques would provide a strategy to selectively enhance biodegrading species in activated sludge systems improving and stabilizing SMX removal efficiency.
Therefore phylogenetic identification of potential SMX biodegrading species is implicitly required. As shown in this study five of the nine SMX biodegrading species found belonged to the genus Pseudomonas confirming this group to play an important role for the biodegradation of micropollutants. This was proved for e.g. acetaminophen or chlorinated compounds by many other studies [38–40]. Additionally, two isolates SMXB24 and SMX348 were identified as Microbacterium sp.. It was shown that Microbacterium sp. SMXB24 is closely related to Microbacterium sp. 7 1 K, an organism that was found to be related with phytoremediation. The second Microbacterium sp. SMX348 is closely related to Microbacterium sp. BR1 which was isolated from an acclimated SMX biodegrading membrane bioreactor, proving this species’ crucial role for the biodegradation of SMX . In addition the general potential of different Microbacteria species for the biodegradation of xenobiotic compounds has been highlighted in the literature [41, 42]. Also Variovorax paradoxus, closely related to the isolated Variovorax sp. SMX332, is known from literature to be capable of biodegrading a large variety of pollutants including sulfolene and other heterocyclic compounds . Therefore it seems likely that the isolated Variovorax sp. SMX332 might also be able to biodegrade SMX. Finally, also for the group Brevundimonas spp. some literature data exist proving that these organisms might play a role in the removal of antibiotics .
Taxonomic identification was followed by observing influences on biodegradation rate and efficiency due to the availability of nutrients. Biodegradation rates decreased with reduced nutrient content from the complex R2A-UV over nutrient-poor MSM-CN and MSM media and more time was needed to remove SMX. MSM media contained SMX as sole carbon and nitrogen source at a concentration of 10 mg L-1 and thus provided just around 4.8 mg L-1 carbon and 1.7 mg L-1 nitrogen. These conditions, with SMX being the only nutrient in MSM, showed an effect on biodegradation and reduced removal efficiency but proved the organisms’ ability to utilize SMX as sole nutrient and/or energy source. However, this indicates that complex nutrients and higher nutrient concentrations seem to have a positive effect on biodegradation due to co-metabolic  or diauxic effects  as the very high SMX removal rates of 2.5 mg L-1 d-1 confirmed that they were significantly higher than the one of 0.0079 mg L-1 d-1 found in a previous study .
In general, SMX biodegradation might be based more on a diauxic process, i.e. readily degradable nutrients are used up first followed by SMX utilization, rather than real co-metabolism, i.e. two substrates are used up in parallel when provided together, as experiments with R2A-UV media showed. A strong increase in UV-AM, attributed to biomass growth due to a fast nutrient consumption provided by the complex R2A-UV media, was followed by a rapid SMX elimination. In MSM-CN or MSM, as the nutrients concentrations were too low to foster excessive biomass growth, such an increase was not observed . Even at low cell densities SMX was rapidly removed proving that biomass concentration is not as important as cellular activity. Therefore, the higher removal rates in presence of sufficient nutrients also showed that SMX biodegradation was a rapid and complex metabolic process.
Therefore, information about the biodegradation potential of the isolated bacterial strains with respect to the availability of nutrients might increase the elimination efficiency in WWTPs as the treatment process could be specifically adapted to the needs of the biodegrading species.
For future research, the availability of isolated species will allow screening for biodegradation intermediates and/or stable metabolites and determination of species-specific biodegradation pathways. To date only few data on SMX metabolites such as 3-amino-5-methyl-isoxazole found in SMX degrading activated sludge communities  and hydroxy-N-(5-methyl-1,2-oxazol-3-yl)benzene-1-sulfonamide detected in an SMX degrading consortium of fungi and Rhodococcus rhodochrous exists . Further research is also needed to screen for the nutrient influence on metabolite formation, i.e. if the isolated pure cultures produce different metabolites due to changing nutrient conditions.