Martina Cappelletti

Microbial degradation of chloroform

Chloroform (CF) is largely produced by both anthropogenic and natural sources. It is detected in ground and surface water sources and it represents the most abundant halocarbon in the atmosphere. Microbial CF degradation occurs under both aerobic and anaerobic conditions. Apart from a few reports describing the utilization of CF as a terminal electron acceptor during growth, CF degradation was mainly reported as a cometabolic process. CF aerobic cometabolism is supported by growth on short-chain alkanes (i.e., methane, propane, butane, and hexane), aromatic hydrocarbons (i.e., toluene and phenol), and ammonia via the activity of monooxygenases (MOs) operatively divided into different families. The main factors affecting CF cometabolism are (1) the inhibition of CF degradation exerted by the growth substrate, (2) the need for reductant supply to maintain MO activity, and (3) the toxicity of CF degradation products. Under anaerobic conditions, CF degradation was mainly associated to the activity of methanogens, although some examples of CF-degrading sulfate-reducing, fermenting, and acetogenic bacteria are reported in the literature. Higher CF toxicity levels and lower degradation rates were shown by anaerobic systems in comparison to the aerobic ones. Applied physiological and genetic aspects of microbial cometabolism of CF will be presented along with bioremediation perspectives.

Analyses of both the alkB gene transcriptional start site and alkB promoter-inducing properties of Rhodococcus sp. strain BCP1 grown on n-alkanes.

ABSTRACT Rhodococcus sp. strain BCP1, known for its capacity to grow on short-chain n-alkanes (C2 to C7) and to cometabolize chlorinated solvents, was found to also utilize medium- and long-chain n-alkanes (C12 to C24) as energy and carbon sources. To examine this feature in detail, a chromosomal region which includes the alkB gene cluster encoding a non-heme di-iron monooxygenase (alkB), two rubredoxins, and one rubredoxin reductase was cloned from the BCP1 genome. Furthermore, the activity of the alkB gene promoter (P alkB ) was examined in the presence of gaseous, liquid, and solid n-alkanes along with intermediates of the putative n-alkane degradation pathway. A recombinant plasmid, pTPalkBLacZ, was constructed by inserting the lacZ gene downstream of P alkB , and it was used to transform Rhodococcus sp. strain BCP1. Measurements of β-galactosidase activity showed that P alkB is induced by C6 to C22 n-alkanes. Conversely, C2 to C5 and >C22 n-alkanes and alkenes, such as hexene, were not inducers of alkB expression. The effects on P alkB expression induced by alternative carbon sources along with putative products of n-hexane metabolism were also evaluated. This report highlights the great versatility of Rhodococcus sp. strain BCP1 and defines for the first time the alkB gene transcriptional start site and the alkB promoter-inducing capacities for substrates different from n-alkanes in a Rhodococcus strain.

A kinetic study of biohydrogen production from glucose, molasses and cheese whey by suspended and attached cells of Thermotoga neapolitana.

Batch tests of H2 production from glucose, molasses and cheese whey by suspended and immobilized cells of Thermotoga neapolitana were conducted to develop a kinetic model of the process. H2 production was inhibited by neither H2 (up to 0.7 mg L(-1)) nor O2 (up to 0.2 mg L(-1)). The H2 specific rates obtained at different substrate concentrations were successfully interpolated with Andrews inhibition model. With glucose and molasses, biofilms performed better than suspended cells. The suspended-cell process was successfully scaled-up to a 19-L bioreactor. Assays co-fed with molasses and cheese whey led to higher H2 productivities and H2/substrate yields than the single-substrate tests. The simulation of the suspended-cell continuous-flow process indicated the potential attainment of H2 productivities higher than those of the batch tests (up to 3.6 mmol H2 h(-1) L(-1) for molasses and 0.67 mmol H2 h(-1) L(-1) for cheese whey) and allowed the identification of the optimal dilution rate.

Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.

OPA1 Isoforms in the Hierarchical Organization of Mitochondrial Functions

OPA1 is a GTPase that controls mitochondrial fusion, cristae integrity, and mtDNA maintenance. In humans, eight isoforms are expressed as combinations of long and short forms, but it is unclear whether OPA1 functions are associated with specific isoforms and/or domains. To address this, we expressed each of the eight isoforms or different constructs of isoform 1 in Opa1-/- MEFs. We observed that any isoform could restore cristae structure, mtDNA abundance, and energetic efficiency independently of mitochondrial network morphology. Long forms supported mitochondrial fusion; short forms were better able to restore energetic efficiency. The complete rescue of mitochondrial network morphology required a balance of long and short forms of at least two isoforms, as shown by combinatorial isoform silencing and co-expression experiments. Thus, multiple OPA1 isoforms are required for mitochondrial dynamics, while any single isoform can support all other functions. These findings will be useful in designing gene therapies for patients with OPA1 haploinsufficiency.

Members of the Order Thermotogales: From Microbiology to Hydrogen Production

Members of the deep-branching order Thermotogales are widespread in various terrestrial, submarine and subterrestrial extreme environments. This bacterial order included both thermophilic and hyperthermophilic anaerobic microorganisms so far pertaining to ten genera. It is only recently (2011) that cultivation of a mesophilic member of this order belonging to a novel genus, Mesotoga, has been successful. All members, with the exception of Mesotoga spp., are recognized as high hydrogen producers having possible applications in biotechnology with a peculiar emphasis for members of the genus Thermotoga (e.g. T. maritima and T. neapolitana). The ecology, phylogeny and metabolism linked to hydrogen production of these bacteria, are reviewed.

Chloroform aerobic cometabolism by butane-growing Rhodococcus aetherovorans BCP1 in continuous-flow biofilm reactors.

This work focuses on chloroform (CF) cometabolism by a butane-grown aerobic pure culture (Rhodococcus aetherovorans BCP1) in continuous-flow biofilm reactors. The goals were to obtain preliminary information on the feasibility of CF biodegradation by BCP1 in biofilm reactors and to evaluate the applicability of the pulsed injection of growth substrate and oxygen to biofilm reactors. The attached-cell tests were initially conducted in a 0.165-L bioreactor and, then, scaled-up to a 1.772-L bioreactor. Glass cylinders were utilized as biofilm carriers. The continuous supply of growth substrate (butane), which led to the attainment of the highest CF degradation rate (8.4 mgCFxa0day−1xa0mbiofilm surface−2), was compared with four schedules of butane and oxygen pulsed feeding. The pulsed injection technique allowed the attainment of a ratio of CF mass degraded per unit mass of butane supplied equal to 0.16xa0mgCFxa0mgbutane−1, a value 4.4 times higher than that obtained with the continuous substrate supply. A procedure based on the utilization of integral mass balances and of average concentrations along the bioreactors resulted in a satisfactory match between the predicted and the experimental CF degradation performances, and can therefore be utilized to provide a guideline for optimizing the substrate pulsed injection schedule.

Rhodococcus aetherivorans BCP1 as cell factory for the production of intracellular tellurium nanorods under aerobic conditions

BackgroundTellurite (TeO32−) is recognized as a toxic oxyanion to living organisms. However, mainly anaerobic or facultative-anaerobic microorganisms are able to tolerate and convert TeO32− into the less toxic and available form of elemental Tellurium (Te0), producing Te-deposits or Te-nanostructures. The use of TeO32−-reducing bacteria can lead to the decontamination of polluted environments and the development of “green-synthesis” methods for the production of nanomaterials. In this study, the tolerance and the consumption of TeO32− have been investigated, along with the production and characterization of Te-nanorods by Rhodococcus aetherivorans BCP1 grown under aerobic conditions.ResultsAerobically grown BCP1 cells showed high tolerance towards TeO32− with a minimal inhibitory concentration (MIC) of 2800xa0μg/mL (11.2xa0mM). TeO32− consumption has been evaluated exposing the BCP1 strain to either 100 or 500xa0μg/mL of K2TeO3 (unconditioned growth) or after re-inoculation in fresh medium with new addition of K2TeO3 (conditioned growth). A complete consumption of TeO32− at 100xa0μg/mL was observed under both growth conditions, although conditioned cells showed higher consumption rate. Unconditioned and conditioned BCP1 cells partially consumed TeO32− at 500xa0μg/mL. However, a greater TeO32− consumption was observed with conditioned cells. The production of intracellular, not aggregated and rod-shaped Te-nanostructures (TeNRs) was observed as a consequence of TeO32− reduction. Extracted TeNRs appear to be embedded in an organic surrounding material, as suggested by the chemical–physical characterization. Moreover, we observed longer TeNRs depending on either the concentration of precursor (100 or 500xa0μg/mL of K2TeO3) or the growth conditions (unconditioned or conditioned grown cells).ConclusionsRhodococcus aetherivorans BCP1 is able to tolerate high concentrations of TeO32− during its growth under aerobic conditions. Moreover, compared to unconditioned BCP1 cells, TeO32−conditioned cells showed a higher oxyanion consumption rate (for 100xa0μg/mL of K2TeO3) or to consume greater amount of TeO32− (for 500xa0μg/mL of K2TeO3). TeO32− consumption by BCP1 cells led to the production of intracellular and not aggregated TeNRs embedded in an organic surrounding material. The high resistance of BCP1 to TeO32− along with its ability to produce Te-nanostructures supports the application of this microorganism as a possible eco-friendly nanofactory.

Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes

Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD—coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation—were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria–Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step.

Application of Pyrolysis-Gas Chromatography-Mass Spectrometry and Multivariate Analysis to Study Bacteria and Fungi in Biofilms Used for Bioremediation

Biofilms are communities of microorganisms adhering to a surface and embedded in an extracellular polymeric matrix, frequently associated with disease and contamination, and also used for engineering applications such as bioremediation. A mixed biofilm formed by bacteria and fungi may provide an optimal habitat for addressing contaminated areas. To exploit the potential of natural microbial communities consisting of bacteria and fungi, it is essential to understand and control their formation. In this work, a method to discriminate among bacteria of genera Bacillus, Pseudomonas, Rhodococcus with respect to the fungus Pleorotus in a biofilm by means of pyrolysis-gaschromatography-mass spectrometry and multivariate analysis is reported. Methylated fatty acids were chosen as biomarkers of microorganisms in the pyrolysates. In situ thermal hydrolysis and methylation was applied. Pyrograms were used as fingerprints, thus allowing for the characterization of whole cells analyzed without any sample pretreatment. Normalized pyrographic peak areas were chosen as variables for chemometric data processing. Principal components analysis was applied as a data exploration tool. Satisfactory results were obtained in analyzing a real biofilm. The influence of growth medium on whole bacteria fatty acid cell composition was also explored.