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See detailAutomated Microscopic Analysis of Metal Sulfide Colonization by Acidophilic Microorganisms.
Bellenberg, Soren; Buetti-Dinh, Antoine; Galli, Vanni et al

in Applied and environmental microbiology (2018), 84(20),

Industrial biomining processes are currently focused on metal sulfides and their dissolution, which is catalyzed by acidophilic iron(II)- and/or sulfur-oxidizing microorganisms. Cell attachment on metal ... [more ▼]

Industrial biomining processes are currently focused on metal sulfides and their dissolution, which is catalyzed by acidophilic iron(II)- and/or sulfur-oxidizing microorganisms. Cell attachment on metal sulfides is important for this process. Biofilm formation is necessary for seeding and persistence of the active microbial community in industrial biomining heaps and tank reactors, and it enhances metal release. In this study, we used a method for direct quantification of the mineral-attached cell population on pyrite or chalcopyrite particles in bioleaching experiments by coupling high-throughput, automated epifluorescence microscopy imaging of mineral particles with algorithms for image analysis and cell quantification, thus avoiding human bias in cell counting. The method was validated by quantifying cell attachment on pyrite and chalcopyrite surfaces with axenic cultures of Acidithiobacillus caldus, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans. The method confirmed the high affinity of L. ferriphilum cells to colonize pyrite and chalcopyrite surfaces and indicated that biofilm dispersal occurs in mature pyrite batch cultures of this species. Deep neural networks were also applied to analyze biofilms of different microbial consortia. Recent analysis of the L. ferriphilum genome revealed the presence of a diffusible soluble factor (DSF) family quorum sensing system. The respective signal compounds are known as biofilm dispersal agents. Biofilm dispersal was confirmed to occur in batch cultures of L. ferriphilum and S. thermosulfidooxidans upon the addition of DSF family signal compounds.IMPORTANCE The presented method for the assessment of mineral colonization allows accurate relative comparisons of the microbial colonization of metal sulfide concentrate particles in a time-resolved manner. Quantitative assessment of the mineral colonization development is important for the compilation of improved mathematical models for metal sulfide dissolution. In addition, deep-learning algorithms proved that axenic or mixed cultures of the three species exhibited characteristic biofilm patterns and predicted the biofilm species composition. The method may be extended to the assessment of microbial colonization on other solid particles and may serve in the optimization of bioleaching processes in laboratory scale experiments with industrially relevant metal sulfide concentrates. Furthermore, the method was used to demonstrate that DSF quorum sensing signals directly influence colonization and dissolution of metal sulfides by mineral-oxidizing bacteria, such as L. ferriphilum and S. thermosulfidooxidans. [less ▲]

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See detailMulti-omics reveal the lifestyle of the acidophilic, mineral-oxidizing model species Leptospirillum ferriphilum(T).
Christel, Stephan; Herold, Malte UL; Bellenberg, Soren et al

in Applied and environmental microbiology (2017)

Leptospirillum ferriphilum plays a major role in acidic, metal rich environments where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as ... [more ▼]

Leptospirillum ferriphilum plays a major role in acidic, metal rich environments where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of this model species' type strain is available, limiting the possibilities to investigate the strategies and adaptations Leptospirillum ferriphilum(T) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilum(T) DSM 14647 obtained by PacBio SMRT long read sequencing for use as a high quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as substrate and bioleaching cultures containing chalcopyrite (CuFeS2). Leptospirillum ferriphilum(T) adaptations to growth on chalcopyrite included a possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, expression and translation of genes responsible for chemotaxis and motility were enhanced.IMPORTANCELeptospirillum ferriphilum is one of the most important iron-oxidizers in the context of acidic and metal rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilum(T) coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on Leptospirillum ferriphilum(T) adaptation strategies to growth on the copper mineral chalcopyrite. This data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction. [less ▲]

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See detailAnoxic conditions promote species-specific mutualism between gut microbes in silico
Heinken, Almut Katrin UL; Thiele, Ines UL

in Applied and Environmental Microbiology (2015), 81(12), 4049-4061

The human gut is inhabited by thousands of microbial species, most of which are still uncharacterized. Gut microbes have adapted to each other’s presence as well as to the host and engage in complex cross ... [more ▼]

The human gut is inhabited by thousands of microbial species, most of which are still uncharacterized. Gut microbes have adapted to each other’s presence as well as to the host and engage in complex cross-feeding. Constraint-based modeling has been successfully applied to predicting microbe-microbe interactions, such as commensalism, mutualism, and competition. Here, we apply a constraint-based approach to model pairwise interactions between 11 representative gut microbes. Microbe-microbe interactions were computationally modeled in conjunction with human small intestinal enterocytes and subjected to three diets with varying levels of carbohydrate, fat, and protein in normoxic or anoxic environments. Each microbe engaged in species-specific commensal, parasitic, mutualistic, or competitive interactions. For instance, Streptococcus thermophilus efficiently outcompeted paired microbes in agreement with the domination of streptococci in the small intestinal microbiota. Under anoxic conditions, the probiotic Lactobacillus plantarum displayed mutualistic behavior towards six other species, which, surprisingly, were almost entirely abolished under normoxic conditions. This finding suggests that the anoxic conditions in the large intestine drive mutualistic cross-feeding, leading to the evolvement of a more complex ecosystem than the small intestinal microbiota. Moreover, we predict that the presence of the small intestinal enterocyte induces competition over host-derived nutrients. The presented framework can readily be expanded to a larger gut microbial community. This modeling approach will be of great value for subsequent studies aiming to predict conditions favoring desirable microbes or suppressing pathogens. [less ▲]

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See detailGenes of the N-methylglutamate pathway are essential for growth of Methylobacterium extorquens DM4 with monomethylamine
Gruffaz; Muller, Emilie UL; Yousra, Louhichi-Jelail et al

in Applied and Environmental Microbiology (2014), 80(11),

Monomethylamine (MMA, CH3NH2) can be used as carbon and nitrogen source by many methylotrophic bacteria including Methylobacterium extorquens DM4 which lacks the MMA dehydrogenase-encoded by mau genes ... [more ▼]

Monomethylamine (MMA, CH3NH2) can be used as carbon and nitrogen source by many methylotrophic bacteria including Methylobacterium extorquens DM4 which lacks the MMA dehydrogenase-encoded by mau genes, which in M. extorquens AM1 is essential for growth on MMA. Identification and characterization of minitransposon mutants with MMA-dependent phenotype showed that strain DM4 grows with MMA as the sole source of carbon, energy and nitrogen by the N-methylglutamate pathway. Independent mutations were found in a chromosomal region containing genes gmas, mgdABCD, and mgsABC for the three enzymes of the pathway, gamma-glutamylmethylamide (GMA) synthetase, N-methylglutamate (NMG) synthase and NMG dehydrogenase, respectively. RT-PCR confirmed the operonic structure of the two divergent gene clusters mgsABC-gmas and mgdABCD, and their induction during growth with MMA. Genes mgdABCD and mgsABC were found to be essential for utilization of MMA as the carbon and nitrogen source. Gene gmas was essential for MMA utilization as the carbon source, but residual growth was observed for mutant DM4gmas growing with succinate and MMA as the nitrogen source. Plasmid copies of gmas and of the gmas homolog METDI4690 encoding a protein 39% identical to GMAS fully restored the ability of mutants DM4gmas and DM4gmasΔmetdi4690 to use MMA as the carbon and nitrogen source. Similarly, chemically synthesized GMA, the product of gmas, could be used as a nitrogen source for growth in the wild-type strain as well as in DM4gmas and DM4gmasΔmetdi4690 mutants. [less ▲]

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See detailCharacterization of Extracellular Polymeric Substances from Acidophilic Microbial Biofilms
Jiao, Yongqin; Cody, George D.; Harding, Anna K. et al

in Applied and Environmental Microbiology (2010), 76(9), 2916-2922

We examined the chemical composition of extracellular polymeric substances (EPS) extracted from two natural microbial pellicle biofilms growing on acid mine drainage (AMD) solutions. The EPS obtained from ... [more ▼]

We examined the chemical composition of extracellular polymeric substances (EPS) extracted from two natural microbial pellicle biofilms growing on acid mine drainage (AMD) solutions. The EPS obtained from a mid-developmental-stage biofilm (DS1) and a mature biofilm (DS2) were qualitatively and quantitatively compared. More than twice as much EPS was derived from DS2 as from DS1 (approximately 340 and 150 mg of EPS per g [dry weight] for DS2 and DS1, respectively). Composition analyses indicated the presence of carbohydrates, metals, proteins, and minor quantities of DNA and lipids, although the relative concentrations of these components were different for the two EPS samples. EPS from DS2 contained higher concentrations of metals and carbohydrates than EPS from DS1. Fe was the most abundant metal in both samples, accounting for about 73% of the total metal content, followed by Al, Mg, and Zn. The relative concentration profile for these metals resembled that for the AMD solution in which the biofilms grew, except for Si, Mn, and Co. Glycosyl composition analysis indicated that both EPS samples were composed primarily of galactose, glucose, heptose, rhamnose, and mannose, while the relative amounts of individual sugars were substantially different in DS1 and DS2. Additionally, carbohydrate linkage analysis revealed multiply linked heptose, galactose, glucose, mannose, and rhamnose, with some of the glucose in a 4-linked form. These results indicate that the biochemical composition of the EPS from these acidic biofilms is dependent on maturity and is controlled by the microbial communities, as well as the local geochemical environment. [less ▲]

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