Profiling 5-tolyltriazole biodegrading sludge communities using next-generation sequencing and denaturing gradient gel electrophoresis.

Efficient biodegradation of 5-tolyltriazole (5-TTri) in wastewater treatment would minimize its potential detrimental effects on aquatic systems. Therefore, in order to profile 5-TTri biodegrading activated sludge communities (ASC) by DGGE and NGS, acclimation experiments with (i) easily degradable substrates, and (ii) various complex substrates mimicking wastewater conditions were performed. DGGE revealed four genera: Aminobacter (family Phyllobacteriaceae), Flavobacterium (family Flavobacteriaceae), Pseudomonas (family Pseudomonaceae), and Hydrogenophaga (family Comamonadaceae). Metagenomics (DNA) revealed the dominant families Alcaligenaceae, Pseudomonadaceae and Comamonadaceae that also represented the most active families at the RNA level (metatranscriptomics), which might indicate their importance for 5-TTri biodegradation. ASC acclimation and the composition of the substrate significantly affected 5-TTri biodegradation and the development of biodegrading communities. Using acetate only, a moderate 5-TTri degrading community was detected with a very low biodiversity and Pseudomonas spp. as dominant organisms. In contrast, setups fed 'sludge supernatant' (a complex substrate) efficiently biodegraded 5-TTri and formed a more diverse microbial community but with Hydrogenophaga spp. as the dominant group. Finally, a hypothetical 5-TTri biodegradation pathway was constructed based exclusively on the detected, biodegradation-related, Hydrogenophaga spp. genes.

Characterization of sulfur oxidizing bacteria related to biogenic sulfuric acid corrosion in sludge digesters.

BACKGROUND: Biogenic sulfuric acid (BSA) corrosion damages sewerage and wastewater treatment facilities but is not well investigated in sludge digesters. Sulfur/sulfide oxidizing bacteria (SOB) oxidize sulfur compounds to sulfuric acid, inducing BSA corrosion. To obtain more information on BSA corrosion in sludge digesters, microbial communities from six different, BSA-damaged, digesters were analyzed using culture dependent methods and subsequent polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). BSA production was determined in laboratory scale systems with mixed and pure cultures, and in-situ with concrete specimens from the digester headspace and sludge zones. RESULTS: The SOB Acidithiobacillus thiooxidans, Thiomonas intermedia, and Thiomonas perometabolis were cultivated and compared to PCR-DGGE results, revealing the presence of additional acidophilic and neutrophilic SOB. Sulfate concentrations of 10-87 mmol/L after 6-21 days of incubation (final pH 1.0-2.0) in mixed cultures, and up to 433 mmol/L after 42 days (final pH <1.0) in pure A. thiooxidans cultures showed huge sulfuric acid production potentials. Additionally, elevated sulfate concentrations in the corroded concrete of the digester headspace in contrast to the concrete of the sludge zone indicated biological sulfur/sulfide oxidation. CONCLUSIONS: The presence of SOB and confirmation of their sulfuric acid production under laboratory conditions reveal that these organisms might contribute to BSA corrosion within sludge digesters. Elevated sulfate concentrations on the corroded concrete wall in the digester headspace (compared to the sludge zone) further indicate biological sulfur/sulfide oxidation in-situ. For the first time, SOB presence and activity is directly relatable to BSA corrosion in sludge digesters.

Discovery of keratinases using bacteria isolated from marine environments.

Bacteria are important for the biodegradation of keratin. Thus, a workflow to isolate keratin-degrading bacteria utilizing an optimized azo-keratin assay was established. Deteriorated feather samples, collected in marine shoreline environments from the intertidal zone, yielded 50 unique bacterial isolates exhibiting keratin degradation when feather meal was supplied as keratin substrate. The majority of isolates, identified by 16S sequencing, belonged to genera previously reported to produce keratinases: Bacillus spp. (42%) and Stenotrophomonas spp. (40%). The remaining 18% represented the genera Alcaligenes, Chryseobacterium, Salinivibrio, Delftia, Stappia, and Microbacterium, genera not previously been associated with keratinase production. The workflow, also applied to 21 Bacilli from our in-house culture collection, additionally revealed four Bacilli with remarkable feather degradation potential. The industrial applicability of their associated keratinases was evaluated and the most active keratinase expressed in E. coli to confirm keratinase expression. Enriched keratinase fractions demonstrated activity up to 75°C and retained viability when stored lyophilized at 20°C for up to 200d.

Effect of acclimation and nutrient supply on 5-tolyltriazole biodegradation with activated sludge communities.

The corrosion inhibitor 5-tolyltriazole (5-TTri) can have a detrimental impact on aquatic systems thus implying an acute need to reduce the effluent concentrations of 5-TTri. In this study, 5-TTri biodegradation was enhanced through acclimation and nutrient supply. Activated sludge communities (ASC) were setup in nine subsequent ASC generations. While generation two showed a lag phase of five days without biodegradation, generations four to nine utilized 5-TTri right after inoculation, with biodegradation rates from 3.3 to 5.2 mg L(-1)d(-1). Additionally, centrifuged AS supernatant was used to simulate the nutrient conditions in wastewater. This sludge supernatant (SS) significantly enhanced biodegradation, resulting in removal rates ranging from 3.2 to 5.0 mg L(-1)d(-1) without acclimation while the control groups without SS observed lower rates of ⩽ 2.2 mg L(-1)d(-1).

Screening and monitoring microbial xenobiotics' biodegradation by rapid, inexpensive and easy to perform microplate UV-absorbance measurements.

BACKGROUND: Evaluation of xenobiotics biodegradation potential, shown here for benzotriazoles (corrosion inhibitors) and sulfamethoxazole (sulfonamide antibiotic) by microbial communities and/or pure cultures normally requires time intensive and money consuming LC/GC methods that are, in case of laboratory setups, not always needed. RESULTS: The usage of high concentrations to apply a high selective pressure on the microbial communities/pure cultures in laboratory setups, a simple UV-absorbance measurement (UV-AM) was developed and validated for screening a large number of setups, requiring almost no preparation and significantly less time and money compared to LC/GC methods. This rapid and easy to use method was evaluated by comparing its measured values to LC-UV and GC-MS/MS results. Furthermore, its application for monitoring and screening unknown activated sludge communities (ASC) and mixed pure cultures has been tested and approved to detect biodegradation of benzotriazole (BTri), 4- and 5-tolyltriazole (4-TTri, 5-TTri) as well as SMX. CONCLUSIONS: In laboratory setups, xenobiotics concentrations above 1.0 mg L(-1) without any enrichment or preparation could be detected after optimization of the method. As UV-AM does not require much preparatory work and can be conducted in 96 or even 384 well plate formats, the number of possible parallel setups and screening efficiency was significantly increased while analytic and laboratory costs were reduced to a minimum.

Monitoring benzotriazoles: a 1 year study on concentrations and removal efficiencies in three different wastewater treatment plants.

Benzotriazole (BTri), 4- and 5-tolyltriazole (4-TTri, 5-TTri) were monitored over 1 year in three wastewater treatment plants (WWTPs) with a membrane bioreactor (MBR-MH) and two conventional activated sludge systems (CAS-E, CAS-M). The influent/effluent concentrations and treatment stages removal efficiencies were monitored. 5-TTri was removed best (mean removal 80%) in the WWTP mainly by biodegradation followed by BTri (mean removal 45%) and 4-TTri (mean removal 15%) that showed a significant lower elimination. High removal fluctuations for all three benzotriazoles occurred over the four seasons with lowest removal during winter. All three WWTPs constituted a point source for BTs in the aquatic environment as concentration measurements in the receiving rivers upstream and downstream of the WWTP proved. While MBR-MH and CAS-M significantly increased the downstream concentrations, CAS-E only slightly increased the downstream concentrations as the receiving river was already contaminated with benzotriazoles from hydropower. 5-TTri was detected in lowest concentrations due to its good removal compared to BTri and 4-TTri that contribute to high effluent concentrations with the potential to accumulate due to insufficient self-purification.

Determination of optimal conditions for 5-methyl-benzotriazole biodegradation with activated sludge communities by dilution of the inoculum.

The aerobic biodegradation of 5-methyl-benzotriazole (5-TTri) was optimized using lab-scale setups and activated sludge communities (ASC) collected from three wastewater treatment plants (WWTP) MBR-MH, CAS-E and CAS-M being different in their treatment technologies. ASC inocula were diluted to rule out non-biodegrading species and incubated under two nutrient conditions: A) mineral salt media (MSM) and B) carbon and nitrogen supplied MSM giving MSM-CN. 5-TTri removal with the ASC ranged from 60% to 100% in only 10 days. 100 µL suspended biomass from the biodegrading setups was subsequently plated on solid media to eliminate possible activated sludge remnants. After growth occurred, mixed colonies were harvested and inoculated in fresh liquid MSM containing 20 mg L(-1) 5-TTri. These bacterial consortia showed good 5-TTri removal in MSM-CN rather than in MSM, indicating nutrient supply being required for efficient biodegradation. In addition, experiments with high 5-TTri concentrations ranging from 20 to 1,000 mg L(-1) were conducted in both, MSM and MSM-CN and the maximal 5-TTri removal capacity of the ASC evaluated. 50 mg L(-1) 5-TTri was still removed in both media whereas 100 mg L(-1) was solely removed in MSM-CN. 5-TTri biodegradation patterns also indicated that 5-TTri might be co-metabolized by microbial consortia. Furthermore, experiments with gradient-solid-media-plates showed 5-TTri to be inhibitory for the ASC in concentrations above 50 mg L(-1) and revealed the optimal conditions regarding carbon and nitrogen concentration and pH value for effective 5-TTri biodegradation by ASC. Nitrogen proved a crucial factor for enhancing organisms' biodegradation capacity with an optimal pH around 7 while carbon showed no such effect.

Xenobiotic benzotriazoles--biodegradation under meso- and oligotrophic conditions as well as denitrifying, sulfate-reducing, and anaerobic conditions.

The intensive use of benzotriazoles as corrosion inhibitors for various applications and their application in dishwasher detergents result in an almost omnipresence of benzotriazole (BTri), 4-methyl- and 5-methyl-benzotriazole (4-TTri and 5-TTri, respectively) in aquatic systems. This study aims on the evaluation of the biodegradation potential of activated sludge communities (ASCs) toward the three benzotriazoles regarding aerobic, anoxic, and anaerobic conditions and different nutrients. ASCs were taken from three wastewater treatment plants with different technologies, namely, a membrane bioreactor (MBR-MH), a conventional activated sludge plant CAS-E (intermittent nitrification/denitrification), and CAS-M (two-stage activated sludge treatment) and used for inoculation of biodegradation setups. All ASCs eliminated up to 30 mg L(-1) 5-TTri and BTri under aerobic conditions within 2-7 and 21-49 days, respectively, but not under anoxic or anaerobic conditions. 4-TTri was refractory at all conditions tested. Significant differences were observed for BTri biodegradation with non-acclimated ASCs from MBR-MH with 21 days, CAS-E with 41 days, and CAS-M with 49 days. Acclimated ASCs removed BTri within 7 days. Furthermore, different carbon and nitrogen concentrations revealed that nitrogen was implicitly required for biodegradation while carbon showed no such effect. The fastest biodegradation occurred for 5-TTri with no need for acclimatization, followed by BTri. BTri showed sludge-specific biodegradation patterns, but, after sludge acclimation, was removed with the same pattern, regardless of the sludge used. Under anaerobic conditions in the presence of different electron acceptors, none of the three compounds showed biological removal. Thus, presumably, aerobic biodegradation is the major removal mechanism in aquatic systems.

Characterization of pure cultures isolated from sulfamethoxazole-acclimated activated sludge with respect to taxonomic identification and sulfamethoxazole biodegradation potential.

BACKGROUND: Sulfamethoxazole (SMX, sulfonamide antibiotic) biodegradation by activated sludge communities (ASC) is still only partly understood. The present work is focusing on nine different bacteria species capable of SMX biodegradation that were isolated from SMX-acclimated ASC. RESULTS: Initially 110 pure cultures, isolated from activated sludge, were screened by UV-absorbance measurements (UV-AM) for their SMX biodegradation potential. Identification via almost complete 16S rRNA gene sequencing revealed five Pseudomonas spp., one Brevundimonas sp., one Variovorax sp. and two Microbacterium spp.. Thus seven species belonged to the phylum Proteobacteria and two to Actinobacteria. These cultures were subsequently incubated in media containing 10 mg L(-1) SMX and different concentrations of carbon (sodium-acetate) and nitrogen (ammonium-nitrate). Different biodegradation patterns were revealed with respect to media composition and bacterial species. Biodegradation, validated by LC-UV measurements to verify UV-AM, occurred very fast with 2.5 mg L(-1) d(-1) SMX being biodegraded in all pure cultures in, for UV-AM modified, R2A-UV medium under aerobic conditions and room temperature. However, reduced and different biodegradation rates were observed for setups with SMX provided as co-substrate together with a carbon/nitrogen source at a ratio of DOC:N - 33:1 with rates ranging from 1.25 to 2.5 mg L(-1) d(-1). CONCLUSIONS: Media containing only SMX as carbon and nitrogen source proved the organisms' ability to use SMX as sole nutrient source where biodegradation rates decreased to 1.0 - 1.7 mg L(-1) d-(1). The different taxonomically identified species showed specific biodegradation rates and behaviours at various nutrient conditions. Readily degradable energy sources seem to be crucial for efficient SMX biodegradation.

Swimming behavior of selected species of Archaea.

The swimming behavior of Bacteria has been studied extensively, at least for some species like Escherichia coli. In contrast, almost no data have been published for Archaea on this topic. In a systematic study we asked how the archaeal model organisms Halobacterium salinarum, Methanococcus voltae, Methanococcus maripaludis, Methanocaldococcus jannaschii, Methanocaldococcus villosus, Pyrococcus furiosus, and Sulfolobus acidocaldarius swim and which swimming behavior they exhibit. The two Euryarchaeota M. jannaschii and M. villosus were found to be, by far, the fastest organisms reported up to now, if speed is measured in bodies per second (bps). Their swimming speeds, at close to 400 and 500 bps, are much higher than the speed of the bacterium E. coli or of a very fast animal, like the cheetah, each with a speed of ca. 20 bps. In addition, we observed that two different swimming modes are used by some Archaea. They either swim very rapidly, in a more or less straight line, or they exhibit a slower kind of zigzag swimming behavior if cells are in close proximity to the surface of the glass capillary used for observation. We argue that such a "relocate-and-seek" behavior enables the organisms to stay in their natural habitat.

The mode of cell wall growth in selected archaea is similar to the general mode of cell wall growth in bacteria as revealed by fluorescent dye analysis.

The surfaces of 8 bacterial and 23 archaeal species, including many hyperthermophilic Archaea, could be stained using succinimidyl esters of fluorescent dyes. This allowed us for the first time to analyze the mode of cell wall growth in Archaea by subculturing stained cells. The data obtained show that incorporation of new cell wall material in Archaea follows the pattern observed for Bacteria: in the coccoid species Pyrococcus furiosus incorporation was in the region of septum formation while for the rod-shaped species Methanopyrus kandleri and Methanothermus sociabilis, a diffuse incorporation of cell wall material over the cell length was observed. Cell surface appendages like fimbriae/pili, fibers, or flagella were detectable by fluorescence staining only in a very few cases although their presence was proven by electron microscopy. Our data in addition prove that Alexa Fluor dyes can be used for in situ analyses at temperatures up to 100°C.