The genus Rhodococcus comprises numerous strains recognized for degradation of pollutants and production of secondary metabolites, including biosurfactants, lignin breakdown, and utilization of volatile organic compounds. Often, gene redundancies and the evolution of alternative pathways are attributed to such characteristics. Rhodococcus opacus 1CP, initially isolated as a chlorophenol-degrading strain, was found to be a model organism comprising several such features. In this study, we analyzed the genome and transcriptome and provided evidence that the strain 1CP uses three different pathways of ortho-, meta-, and side-chain attack for the degradation of aromatic compounds. The wild-type strain and the single and double knock-out mutants of phenol hydroxylases were able to attack substituted phenols via the classical ortho-route. In contrast, the triple knock-out mutant expressed meta-pathway genes to act on p-cresol, indicating that this pathway serves as a reserve in strain 1CP. Growth of 1CP on phenol, p-cresol, and styrene induces several gene clusters that are associated with lignin metabolization. Catechol, protocatechuate, and phenylacetic acid are major key intermediates that are funneled into central metabolic pathways, which enable strain 1CP to degrade acetophenone, benzoate, phenol, 2-phenylethanol, and styrene. Strain 1CP possesses an alternative, modified ortho-cleavage pathway that allows it to degrade 2-chlorophenol. Interestingly, in almost all cases, redundant genes were identified; however, only in a few cases, such as phenol hydroxylases, were they found to be active and simultaneously involved in metabolic activities. The transcriptomic and kinetic data showed that the redundant styrene oxide isomerase is upregulated and involved in styrene degradation.IMPORTANCERhodococcus opacus 1CP is a model organism for various biotechnological applications due to its capabilities to metabolize a vast range of aromatic and xenobiotic compounds. Although strain 1CP has been used for decades in bioremediation, the complete metabolic pathways underlying degradation have never been elucidated. In this study, the ability of the strain to bypass phenol hydroxylase deletions and degrade substituted phenols is described using genomics, transcriptomics, and gene knock-out analyses. Despite its metabolic versatility, strain 1CP has been reported only for the ortho-cleavage pathway. No enzymatic or metabolic evidence has supported the presence of a meta-cleavage pathway to degrade aromatic compounds. Genes associated with such meta-pathways have been identified, but are not clustered with known degradation operons. In this study, we demonstrate that a triple knock-out mutant can utilize a meta-cleavage pathway for the degradation of substituted phenols.

Bioleaching of metals from sulphide minerals is an interfacial process that is promoted by biofilm formation. Since sulphide minerals spontaneously produce H2O2, this study evaluated the role of this compound in the early adherence of the acidophilic bioleaching bacterium Leptospirillum sp. CF-1 to different substrates. During 8 h of incubation under acidic conditions, the minerals produced micromolar amounts of H2O2 (pyrite > chalcopyrite > sphalerite). As expected, suspensions containing quartz did not produce detectable amounts of H2O2. In agreement, adherence to pyrite, chalcopyrite, and sphalerite steadily increased during the 8 h of the assays, while no adherence to quartz was detected. After 8 h of incubation, the highest and lowest adherence were observed for pyrite (94 % +/- 1.8) and sphalerite (73 % +/- 4), respectively. Furthermore, incubation of the strain CF-1 in the presence of 10 mu M H2O2 for 2 and 6 h led to an increase in early-stage biofilm formation compared to non-exposed biofilms. In agreement, 10 mu M H2O2 also significantly stimulated the adherence of the strain to various sulphide minerals and quartz, suggesting that H2O2 itself has a key effect on attachment to different substrates. Exposure of strain CF-1 to 10 mu M H2O2 also resulted in an increase in the expression of genes for flagellin (flaA), type IV pili (pilV), diffusible signal factor (dfs), and genes related to the synthesis of EPS (epsDHI). Altogether, these results suggest that H2O2 could represent an important environmental signal that favours the adherence of the microorganism to the mineral by regulating the expression of genes related to adherence and biofilm formation.

Two phenylacetaldehyde dehydrogenases, originating from Escherichia coli K-12 (FeaB-K-12) and Sphingopyxis fribergensis Kp5.2 (FeaB-Kp5.2), were immobilized on powdery silica carrier with various functionalization. First, the suitability of these carriers for application in combination with phenylacetaldehydes and phenylacetic acids was studied. Out of two carriers functionalized differently, mesoporous cellular foam, whose surface was modified with 3-glycidyloxypropyl groups (MCF-G), showed promising results. Hence, this carrier was further tested at 17 different immobilization conditions. Despite both enzymes showing high immobilization efficiency, the initial activities were relatively low compared to the free enzymes. Interestingly, the immobilized FeaB-Kp5.2 on MCF-G-Kw showed about 80% of retained activity after two months of incubation at 0 degrees C, indicating that the immobilization enhances the stability of this enzyme. In contrast, no changes in the temperature stability of FeaB-Kp5.2 due to immobilization could be noted. However, relative enzyme activities towards all three substituted phenylacetaldehydes could be increased by the immobilization to approximately 130%. The most active and stable powdery immobilizate was MCF-G-Kw-FeaB-Kp5.2 at pH 8. In addition, FeaB-Kp5.2 was also immobilized and tested on monolith silica carrier for continuous catalysis to produce phenylacetic acids.

Arsenic is a toxic metalloid. It is released to the environment normally as arsenite [As(III)], largely due to mining activities. As(III) is more toxic and in many acidic mining environments more mobile than arsenate [As(V)] and therefore the oxidation of As(III) to As(V) is fundamental to immobilize and treat arsenic waste. Here, we report on the isolation and the phenotypic as well as genomic characterization of a novel extremely acidophilic As(III)-oxidizing strain from Reiche Zeche, a former silver mining site (Freiberg, Germany). The isolated strain CJR1 was identified as Acidiphilium acidophilum by 99.7

Dismantling of printed circuit boards is a promising pretreatment step for the processing of electronic waste. It allows the separation and subsequent sorting of the different electronic components (ECs) and eases the downstream processes by enriching ECs and the elements contained in them. Biodismantling is a novel process using iron-oxidizing microorganisms to generate an oxidative ferric iron-rich solution to dissolve solder joints, thus releasing ECs. This study evaluates the influence of pH and iron concentration that proved to be rate- limiting factors of the dismantling reaction. Since microbial iron oxidation and dismantling may be performed in separate reactors, it also investigates the effect of sonication on dismantling and thus allows to optimize parameters for industrial applications. At pH 1.2 and 500 mM ferric iron, dismantling times were down to 10 h, but the addition of ultrasound treatment improved dismantling rates even further, reaching complete dismantling within 1.9 h.

Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophilic bacterium belonging to microbial communities involved in sulfide ore bioleaching. This microorganism possesses redundancy of genes encoding ATP-independent chaperone holdases like Hsp20 (hps20.1, hsp20.2, and hsp20.3), Hsp31, Hsp33, RidA (ridA.1 and ridA.2), and Lon (lon.1, lon.2, and lon.3), and single copy genes encoding SlyD and CnoX. We evaluated the response of these holdases to short and long-term stresses induced by changes in temperature (30° to 37 °C), pH (1.6 to 1.2 or 2.0), and oxidative status (1 mM H2O2) as well as to different energy sources (iron, sulfur, pyrite, sphalerite or chalcopyrite). Cells adapted under thermal and oxidative stress conditions showed a generalized upregulation of holdase genes, while short-term stress led to more discrete increases in transcript levels, with only hsp20.2 and hsp31 showing higher mRNA levels. hsp31 was also upregulated under acidic stresses, sulfur and sulfides. hsp20 variants showed different mRNA levels under different conditions, and cnoX was induced under oxidative conditions. Cells cultured on chalcopyrite had similar responses to those grown with peroxide. With some exceptions, stresses led to significant increases in intracellular ROS content, and decreases in ATP. These results pave the way to understanding proteostasis systems in extreme acidophilic bacteria.

While the presence of chloride ions strongly inhibits most bioleaching bacteria, the newly isolated acidophilic, iron-oxidizing bacterium Alicyclobacillus sp. SO9 even requires chloride. The strain has so far been maintained with different electron donors, but always in the presence of 0.02

Bioleaching as a method for extracting metals from low-grade ores is gaining attention due to its potential as an alternative to energy-intensive and less-efficient metallurgical processes. Furthermore, increasing the chloride concentration in bioleaching environments may enhance the efficiency, thus raising the interest also in halotolerant acidophilic iron oxidizers like Sulfobacillus thermosulfidooxidans. This study aimed to compare the chloride tolerance of various Sulfobacillus species. The results showed that Sb. thermosulfidooxidans exhibited the highest tolerance compared to other tested strains. Based on the knowledge, that yeast extract (YE) may have a positive impact on the chloride tolerance of microorganisms, the importance of single YE components on bacterial respiratory activity was also investigated. The experiments showed that in Sb. thermosulfidooxidans casamino acids with potassium and glucose can be applied to replace the yeast extract. Moreover, ultrafiltration of casamino acids indicated the importance of small peptides or amino acids (<1 kDa). This study contributes to the understanding of the requirements for creating a defined medium for an efficient bioleaching process in the presence of chloride ions involving Sb. thermosulfidooxidans.

In previous investigations to treat acidic As(III)-containing solutions, an autotrophic acidophilic arsenite oxidizer, Acidiphilium acidophilum CJR1, has been isolated. Nonetheless, its physiological response toward As(III) remained unknown. An RNA-Seq approach was employed to obtain more insight. Though capable of autotrophic growth with arsenite, due to considerably better growth in the presence of yeast extract, A. acidophilum CJR1 was cultured under two heterotrophic conditions, one with additional arsenite and as a control one with yeast extract and 20 μM Fe2+. The results indicated the expression of arsenite oxidase genes under both conditions, suggesting that the enzyme may be constitutively expressed. In the As(III) condition, the COG categorization indicated that most strongly upregulated genes were energy-related or chaperones. These results provide the first transcriptomic insight into the arsenite response of an Acidiphilium acidophilum strain.

Many acidophilic iron-oxidizing bacteria used in the mining industry for the bioleaching of sulfidic minerals are intolerant to high chloride concentrations, resulting in problems where chloride occurs in the deposit at high concentrations or only seawater is available. In search for strains tolerating such conditions a tetrathionate- and iron-oxidizing bacterium was isolated from a tailings-contaminated beach sample at Portman Bay, Cartagena-La Union mining district, Spain, in the presence of 20 g l-1 (0.34 M) sodium chloride. The isolate was able to form spores, did not grow in the absence of NaCl, and oxidized ferrous iron in the presence of up to 1.5 M (∼87 g l-1) NaCl. Genome sequencing based on a combination of Illumina and PacBio reads revealed two contigs, a circular bacterial chromosome of 5.2 Mbp and a plasmid of 90 kbp, respectively. The chromosome comprised seven different 16S rRNA genes. Submission of the chromosome to the Type (Strain) Genome Server (TYGS) without preselection of similar sequences revealed exclusively type strains of the genus Alicyclobacillus. In the TYGS analyses the respective most similar species were dependent on whether the final tree was derived from just 16S rRNA, from the genomes, or from the proteomes. Thus, TYGS analysis clearly showed that isolate SO9 represents a novel species of the genus Alicyclobacillus. In the presence of artificial seawater with almost 0.6 M chloride, the addition of Alicyclobacillus sp. SO9 improved copper dissolution from chalcopyrite (CuFeS2) compared to abiotic leaching without bacteria. The new isolate SO9, therefore, has potential for bioleaching at elevated chloride concentrations.

The distribution and concentration of chemicals in the environment depends on the chemical behavior and transport as well as transformation processes. General principles and concepts of assessment of chemicals includes degradability, toxicity and mutagenicity tests.