Approaches for Eliminating Bacteria Introduced during In Situ Bioleaching of Fractured Sulfidic Ores in Deep Subsurface

The major objective of the EU Horizon 2020 project “BioMOre” is the technical realization of indirect in situ leaching of Kupferschiefer sandstone and black shale ore by a ferric iron lixiviant generated by a mixed culture of autotrophic, acidophilic, iron-oxidizing bacteria and archaea in a ferric iron-generating bioreactor (FIGB). These organisms could colonize the deeply buried geological formations even under anaerobic conditions as most are able to grow by coupling the reduction of ferric iron to the oxidation of reduced sulfur compounds in the absence of oxygen. Development of an inhibition protocol to eliminate these allochthonous microbial bioreactor populations subsequent to the completion of in situ bioleaching was therefore investigated. Column bioleaching experiments using a laboratory-scale FIGB confirmed not only that metals were solubilised from both the sandstone and shale ores, but also that significant numbers of bacteria were released from the FIGB. The efficacy of 13 different chemical compounds in inhibiting microbial iron oxidation has been tested at different concentrations in shake flask and FIGB-coupled columns. Iron-oxidation activity, microcalorimetrically-determined activity and ATP measurements, in combination with microscopic cell counts and biomolecular analysis (T-RFLP, qPCR), plate counts and most-probable-number (MPN), were used to monitor the inhibiting effects on the acidophiles. Complete inhibition of metabolic activity of iron-oxidizing acidophiles was achieved in the presence of 0.4 mM formate, 300 mM chloride, 100 mM nitrate, 10 mM of primary C6 to C8 alcohols, 100 mM 1-butanol, 100 mM 1-pentanol, 0.1 mM SDS or 0.35 mM benzoic acid. No inhibition was found for 0.6 mM acetic acid and 200 mM methanol. Based on these results a recipe for the chemical composition of the “decommissioning solution” is proposed.</jats:p

Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies

Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291T), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291T. Pseudomonas monteilii (DSM14164) clustered well with P. putida strains

Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement

A transcriptomics and proteomics approach was employed to study the expression changes associated with p-hydroxybenzoate production by the engineered Pseudomonas putida strain S12palB1. To establish p-hydroxybenzoate production, phenylalanine-tyrosine ammonia lyase (pal/tal) was introduced to connect the tyrosine biosynthetic and p-coumarate degradation pathways. In agreement with the efficient p-hydroxybenzoate production, the tyrosine biosynthetic and p-coumarate catabolic pathways were upregulated. Also many transporters were differentially expressed, one of which—a previously uncharacterized multidrug efflux transporter with locus tags PP1271-PP1273—was found to be associated with p-hydroxybenzoate export. In addition to tyrosine biosynthesis, also tyrosine degradative pathways were upregulated. Eliminating the most prominent of these resulted in a 22% p-hydroxybenzoate yield improvement. Remarkably, the upregulation of genes contributing to p-hydroxybenzoate formation was much higher in glucose than in glycerol-cultured cells

Engineering of Solvent-Tolerant Pseudomonas putida S12 for Bioproduction of Phenol from Glucose

Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-dl-phenylalanine and m-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch

Transcriptome Analysis of a Phenol-Producing Pseudomonas putida S12 Construct: Genetic and Physiological Basis for Improved Production▿ †

The unknown genetic basis for improved phenol production by a recombinant Pseudomonas putida S12 derivative bearing the tpl (tyrosine-phenol lyase) gene was investigated via comparative transcriptomics, nucleotide sequence analysis, and targeted gene disruption. We show upregulation of tyrosine biosynthetic genes and possibly decreased biosynthesis of tryptophan caused by a mutation in the trpE gene as the genetic basis for the enhanced phenol production. In addition, several genes in degradation routes connected to the tyrosine biosynthetic pathway were upregulated. This either may be a side effect that negatively affects phenol production or may point to intracellular accumulation of tyrosine or its intermediates. A number of genes identified by the transcriptome analysis were selected for targeted disruption in P. putida S12TPL3. Physiological and biochemical examination of P. putida S12TPL3 and these mutants led to the conclusion that the metabolic flux toward tyrosine in P. putida S12TPL3 was improved to such an extent that the heterologous tyrosine-phenol lyase enzyme had become the rate-limiting step in phenol biosynthesis