Reduction of hexavalent chromium using bacterial isolates and a microbial community enriched from tannery effluent.

We investigated microbial growth in increasing concentrations of hexavalent chromium (Cr(VI)) and its reduction by a microbial community enriched from tannery effluent and by the bacterial strains isolated from the enriched community. The bacterial growth was monitored by measuring the optical cell density (OD650), while the Cr(VI) concentration in the samples was determined using spectrophotometry and liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). At a Cr(VI) concentration of 100 mg/L, the isolates affiliated with Pseudomonas aeruginosa (P. aeruginosa) reached higher optical cell densities, but were in general less effective for Cr(VI) reduction than the isolates affiliated with Mammaliicoccus sciuri (M. sciuri). All three M. sciuri isolates and only one of the seven P. aeruginosa isolates were able to reduce 50% of the Cr(VI) with an initial concentration of 100 mg/L within 24 h (pH 7.1), while the six isolates affiliated with P. aeruginosa were less effective. Compared to the isolated, individual bacterial strains, the enriched microbial community was better adapted to the elevated Cr(VI) concentrations, but needed a longer time (48 h) to reduce the Cr(VI) with the same efficacy as the most efficient individual isolates. The ability of the enriched microbial community and the isolated bacterial strains to reduce the Cr(VI) highlights their potential for use in the rapid bioremediation of wastewaters contaminated with Cr(VI).

Kin Discrimination Modifies Strain Distribution, Spatial Segregation, and Incorporation of Extracellular Matrix Polysaccharide Mutants of Bacillus subtilis Strains into Mixed Floating Biofilms.

Microorganisms in nature form multicellular groups called biofilms. In biofilms, bacteria embedded in the extracellular matrix (ECM) interact intensely due to their proximity. Most studies have investigated genetically homogeneous biofilms, leaving a gap in knowledge on genetically heterogeneous biofilms. Recent insights show that a Gram-positive model bacterium, Bacillus subtilis, discriminates between strains of high (kin) and low (nonkin) genetic similarity, reflected in merging (kin) and boundaries (nonkin) between swarms. However, it is unclear how kinship between interacting strains affects their fitness, the genotype assortment, and incorporation of the mutant lacking the main structural ECM polysaccharide (EpsA-O) into floating biofilms (pellicles). We cultivated Bacillus subtilis strains as mixtures of isogenic, kin, and nonkin strain combinations in the biofilm-promoting minimal medium under static conditions, allowing them to form pellicles. We show that in nonkin pellicles, the dominant strain strongly reduced the frequency of the other strain. Segregation of nonkin mixtures in pellicles increased and invasion of nonkin EpsA-O-deficient mutants into pellicles decreased compared to kin and isogenic floating biofilms. Kin and isogenic strains had comparable relative frequencies in pellicles and showed more homogenous cell mixing. Overall, our results emphasize kin discrimination as a social behavior that shapes strain distribution, spatial segregation, and ECM mutant ability to incorporate into genetically heterogenous biofilms of B. subtilis. IMPORTANCE Biofilm communities have beneficial and harmful effects on human societies in natural, medical, and industrial environments. Bacillus subtilis is a biotechnologically important bacterium that serves as a model for studying biofilms. Recent studies have shown that this species engages in kin discriminatory behavior during swarming, which may have implications for community assembly, thus being of fundamental importance. Effects of kin discrimination on fitness, genotype segregation, and success of extracellular matrix (ECM) polysaccharide (EpsA-O) mutant invasion into biofilms are not well understood. We provide evidence that kin discrimination depends on the antagonism of the dominant strain against nonkin by using environmental strains with determined kin types and integrated fluorescent reporters. Moreover, this antagonism has important implications for genotype segregation and for when the bacteria are mixed with ECM producers. The work advances the understanding of kin-discrimination-dependent bacterial sociality in biofilms and its role in the assembly of multicellular groups.

Kin discrimination drives territorial exclusion during Bacillus subtilis swarming and restrains exploitation of surfactin.

Swarming is the collective movement of bacteria across a surface. It requires the production of surfactants (public goods) to overcome surface tension and provides an excellent model to investigate bacterial cooperation. Previously, we correlated swarm interaction phenotypes with kin discrimination between B. subtilis soil isolates, by showing that less related strains form boundaries between swarms and highly related strains merge. However, how kin discrimination affects cooperation and territoriality in swarming bacteria remains little explored. Here we show that the pattern of surface colonization by swarming mixtures is influenced by kin types. Closely related strain mixtures colonize the surface in a mixed swarm, while mixtures of less related strains show competitive exclusion as only one strain colonizes the surface. The outcome of nonkin swarm expansion depends on the initial ratio of the competing strains, indicating positive frequency-dependent competition. We find that addition of surfactin (a public good excreted from cells) can complement the swarming defect of nonkin mutants, whereas close encounters in nonkin mixtures lead to territorial exclusion, which limits the exploitation of surfactin by nonkin nonproducers. The work suggests that kin discrimination driven competitive territorial exclusion may be an important determinant for the success of cooperative surface colonization.

Kin discrimination promotes horizontal gene transfer between unrelated strains in Bacillus subtilis.

Bacillus subtilis is a soil bacterium that is competent for natural transformation. Genetically distinct B. subtilis swarms form a boundary upon encounter, resulting in killing of one of the strains. This process is mediated by a fast-evolving kin discrimination (KD) system consisting of cellular attack and defence mechanisms. Here, we show that these swarm antagonisms promote transformation-mediated horizontal gene transfer between strains of low relatedness. Gene transfer between interacting non-kin strains is largely unidirectional, from killed cells of the donor strain to surviving cells of the recipient strain. It is associated with activation of a stress response mediated by sigma factor SigW in the donor cells, and induction of competence in the recipient strain. More closely related strains, which in theory would experience more efficient recombination due to increased sequence homology, do not upregulate transformation upon encounter. This result indicates that social interactions can override mechanistic barriers to horizontal gene transfer. We hypothesize that KD-mediated competence in response to the encounter of distinct neighbouring strains could maximize the probability of efficient incorporation of novel alleles and genes that have proved to function in a genomically and ecologically similar context.

Influence of Diclofenac on Activated Sludge Bacterial Communities in Fed-Batch Reactors.

RESEARCH BACKGROUND: The occurrence and environmental toxicity of pharmaceuticals have recently attracted increasing attention. Diclofenac is a highly consumed non-steroidal anti-inflammatory drug, which is often detected in wastewaters, but investigations of its influence on bacteria are scarce. EXPERIMENTAL APPROACH: We investigated the influence of this pharmaceutical on bacterial community in activated sludge exposed to increasing concentrations of diclofenac in fed-batch reactors over 41 days. Nitrification activity of the activated sludge was measured and changes in bacterial community structure were followed using culture-independent molecular method (terminal restriction fragment length polymorphism, T-RFLP) and by the cultivation approach. RESULTS AND CONCLUSIONS: Nitrification activity was not detectably influenced by the addition of diclofenac, while the main change of the bacterial community structure was detected only at the end of incubation (after 41 days) when diclofenac was added to artificial wastewater as the only carbon source. Changes in community composition due to enrichment were observed using cultivation approach. However, taxonomic affiliation of isolates did not match taxons identified by T-RFLP community profiling. Isolates obtained from activated sludge used as inoculum belonged to five genera: Comamonas, Arthrobacter, Acinetobacter, Citrobacter and Aeromonas, known for their potential to degrade aromatic compounds. However, only Pseudomonas species were isolated after the last enrichment step on minimal agar plates with diclofenac added as the sole carbon source. NOVELTY AND SCIENTIFIC CONTRIBUTION: Our results suggest that the selected recalcitrant and commonly detected pharmaceutical does not strongly influence the sensitive and important nitrification process of wastewater treatment. Moreover, the isolated strains obtained after enrichment procedure that were able to grow on minimal agar plates with diclofenac added as the only carbon source could serve as potential model bacteria to study bacterial diclofenac degradation.

A Combinatorial Kin Discrimination System in Bacillus subtilis.

Multicellularity inherently involves a number of cooperative behaviors that are potentially susceptible to exploitation but can be protected by mechanisms such as kin discrimination. Discrimination of kin from non-kin has been observed in swarms of the bacterium Bacillus subtilis, but the underlying molecular mechanism has been unknown. We used genetic, transcriptomic, and bioinformatic analyses to uncover kin recognition factors in this organism. Our results identified many molecules involved in cell-surface modification and antimicrobial production and response. These genes varied significantly in expression level and mutation phenotype among B. subtilis strains, suggesting interstrain variation in the exact kin discrimination mechanism used. Genome analyses revealed a substantial diversity of antimicrobial genes present in unique combinations in different strains, with many likely acquired by horizontal gene transfer. The dynamic combinatorial effect derived from this plethora of kin discrimination genes creates a tight relatedness cutoff for cooperation that has likely led to rapid diversification within the species. Our data suggest that genes likely originally selected for competitive purposes also generate preferential interactions among kin, thus stabilizing multicellular lifestyles.

Kin discrimination between sympatric Bacillus subtilis isolates.

Kin discrimination, broadly defined as differential treatment of conspecifics according to their relatedness, could help biological systems direct cooperative behavior toward their relatives. Here we investigated the ability of the soil bacterium Bacillus subtilis to discriminate kin from nonkin in the context of swarming, a cooperative multicellular behavior. We tested a collection of sympatric conspecifics from soil in pairwise combinations and found that despite their history of coexistence, the vast majority formed distinct boundaries when the swarms met. Some swarms did merge, and most interestingly, this behavior was only seen in the most highly related strain pairs. Overall the swarm interaction phenotype strongly correlated with phylogenetic relatedness, indicative of kin discrimination. Using a subset of strains, we examined cocolonization patterns on plant roots. Pairs of kin strains were able to cocolonize roots and formed a mixed-strain biofilm. In contrast, inoculating roots with pairs of nonkin strains resulted in biofilms consisting primarily of one strain, suggestive of an antagonistic interaction among nonkin strains. This study firmly establishes kin discrimination in a bacterial multicellular setting and suggests its potential effect on ecological interactions.

Effect of Low-Density Static Magnetic Field on the Oxidation of Ammonium by Nitrosomonas europaea and by Activated Sludge in Municipal Wastewater.

Ammonium removal is a key step in biological wastewater treatment and novel approaches that improve this process are in great demand. The aim of this study is to test the hypothesis that ammonium removal from wastewater can be stimulated by static magnetic fields. This was achieved by analysis of the effects of static magnetic field (SMF) on the growth and activity of Nitrosomonas europaea, a key ammonia-oxidising bacterium, where increased growth and increased ammonia oxidation rate were detected when bacteria were exposed to SMF at 17 mT. Additionally, the effect of SMF on mixed cultures of ammonia oxidisers in activated sludge, incubated in sequencing batch bioreactors simulating wastewater treatment process, was assessed. SMFs of 30 and 50 mT, but not of 10 mT, increased ammonium oxidation rate in municipal wastewater by up to 77% and stimulated ammonia oxidiser growth. The results demonstrate the potential for use of static magnetic fields in increasing ammonium removal rates in biological wastewater treatment plants.

Effects of low-density static magnetic fields on the growth and activities of wastewater bacteria Escherichia coli and Pseudomonas putida.

The aim of this study was to explore the influence of a moderate static magnetic field (SMF) of different densities on Escherichia coli and Pseudomonas putida that are commonly found in wastewater treatment plants. In line with literature reports that SMF increases the efficiency of wastewater treatment the findings of this study indicated that SMF negatively influenced the growth but positively influenced the enzymatic activities and ATP levels of the two model bacteria. The inhibitory effect of SMF on growth of E. coli and P. putida was most pronounced at their optimal growth temperature (37°C and 28°C respectively) and was reversible shortly after the SMF had been terminated. Finally, the results suggested that the induced energy metabolism reflected in higher dehydrogenase activities and ATP levels may be more important for survival, and adaptation to SMF induced stress than the increase in the expression of the rpoS gene.

Total bacterial and ammonia-oxidizer community structure in moving bed biofilm reactors treating municipal wastewater and inorganic synthetic wastewater.

Industrial-scale bioreactors treat wastewater of temporally variable composition under different weather conditions, while the microbial populations of wastewater treatment plants are often studied in controlled laboratory-scale systems with defined influent at a constant temperature. 16S rRNA- and ammonia oxidising amoA-gene-defined bacterial community structure was investigated in industrial and laboratory-scale moving bed biofilm bioreactors (MBBRs) treating municipal wastewater (WW) or synthetic ammonium solution (AS). Nitrification activity, 16S rRNA and amoA gene T-RFLP profiles were comparable between industrial and laboratory scale WW bioreactors. AS bioreactors exhibited higher nitrification and higher relative abundances of Nitrosomonadaceae and Nitrospiraceae families but only small changes in the general bacterial community structure was detected compared to WW MBBRs. Nitrosomonas europaea lineage dominated WW, while uncultivated Nitrosomonas-like sequences prevailed in AS bioreactors. These results suggest that influent type has a stronger influence on community structure than operational conditions, such as temperature or bioreactor size.

Nitrification activity and community structure of nitrite-oxidizing bacteria in the bioreactors operated with addition of pharmaceuticals.

Pharmaceuticals represent a group of the new emerging contaminants, which might influence microbial communities in the activated sludge. Nitrification activity and Nitrospira community structure in the small-scale reactors supplied with different concentrations (0, 50, 200, 500 µg L(-1)) of the selected pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid) were evaluated. Ammonia removal was not influenced by selected pharmaceuticals. However, in the two reactors operated with 50 µg L(-1) of pharmaceuticals (R50 and R50P), the effluent concentration of N-(NO(2)(-)+NO(3)(-)) was significantly higher than in the other reactors. Nitrospira community structure was assessed by terminal restriction fragment length polymorphism (T-RFLP) and by cloning and sequencing of the partial genes for 16S rRNA. Nitrospira spp. were detected in all reactors. The two dominant T-RFs represented the sublineages I and II of the genus Nitrospira. Main shifts were observed in the reactors R50 and R50P, where the T-RF representing sublineage II was much higher as compared to the other reactors. Consistent with this, the Nitrospira sublineage II was detected only in the clone libraries from the reactors R50 and R50P. Our results suggest that the relative abundance of Nitrospira sublineage II could be related to the effluent N-(NO(2)(-)+NO(3)(-)) concentration.

Influence of pharmaceutical residues on the structure of activated sludge bacterial communities in wastewater treatment bioreactors.

Concern is growing over contamination of the environment with pharmaceuticals because of their widespread use and incomplete removal during wastewater treatment, where microorganisms drive the key processes. The influence of pharmaceuticals on bacterial community structure in activated sludge was assessed in small-scale wastewater treatment bioreactors containing different concentrations (5, 50, 200 and 500microgL(-1)) of several commonly used pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid). T-RFLP analyses of the bacterial 16S rRNA genes indicated a minor but consistent shift in the bacterial community structure in the bioreactor R50 supplied with pharmaceuticals at a concentration of 50microgL(-1), compared to the control reactor R0, which was operated without addition of pharmaceuticals. In the reactors operated with higher concentrations of pharmaceuticals, a greater structural divergence was observed. Bacterial community composition was further investigated by preparation of two clone libraries of bacterial 16S rRNA genes from reactors R0 and R50. Most clones in both libraries belonged to the Betaproteobacteria, among which Thauera, Sphaerotilus, Ideonella and Acidovorax-related spp. dominated. Nitrite-oxidizing bacteria of the genus Nitrospira sp., which are key organisms for the second stage of nitrification in wastewater treatment plants, were found only in the clone library of the reactor without pharmaceuticals. In addition, diversity indices were calculated for the two clone libraries, indicating a reduced diversity of activated sludge bacterial community in the reactor supplied with 50microgL(-1) of each of selected pharmaceuticals.

The Rok protein of Bacillus subtilis represses genes for cell surface and extracellular functions.

Rok is a repressor of the transcriptional activator ComK and is therefore an important regulator of competence in Bacillus subtilis (T. T. Hoa, P. Tortosa, M. Albano, and D. Dubnau, Mol. Microbiol. 43:15-26, 2002). To address the wider role of Rok in the physiology of B. subtilis, we have used a combination of transcriptional profiling, gel shift experiments, and the analysis of lacZ fusions. We demonstrate that Rok is a repressor of a family of genes that specify membrane-localized and secreted proteins, including a number of genes that encode products with antibiotic activity. We present evidence for the recent introduction of rok into the B. subtilis-Bacillus licheniformis-Bacilllus amyloliquefaciens group by horizontal transmission.