Growth kinetics of Lactococcus lactis and Lactobacillus casei in liquid culture medium containing as prebiotics inulin or fructose.

BACKGROUND: Predictive microbiology is a tool that allows us to evaluate the behavior of the concentration of biomass and estimated cells under extrinsic conditions, providing scientific and industrial benefits. In the present study, the growth of L. lactis and L. casei combined with inulin and fructose was modeled using the Gompertz sigmoidal growth functions and plotted using data obtained from batch culture in relation to biomass and cell concentration expressed as estimates in ln N (OD600nm and cells mL-1 ) as a function of time. RESULTS: The results of the kinetic modeling indicated that (T1) A1B1 = L. lactis + fructose and (T4) A2B2 = L. casei + inulin presented the best function coefficients and best fits in most cases compared to the rest. The specific growth rate of the maximum acceleration was from 0.364 to 0.473 h-1 and 0.100 to 0.129 h-1 , the concentration of bacterial cells (A) was from 0.556 to 0.713 and 0.425 to 0.548 respectively and the time where (µ) occurred with a greater magnitude (L) fluctuated between 0.854 and 0.802 and when this time in (L) is very fast, it presents values of ≤0.072 to ≤0.092. Its coefficient of determination and/or multiple regression (R2 ) obtained in the two adjustments was 0.97. CONCLUSION: It was possible to predict the influence of the carbon source on the behavior of maximum growth rates, higher consumption due to nutrient affinity and shorter growth time. © 2023 Society of Chemical Industry.

Progress in bioleaching: fundamentals and mechanisms of microbial metal sulfide oxidation - part A.

Bioleaching of metal sulfides is performed by diverse microorganisms. The dissolution of metal sulfides occurs via two chemical pathways, either the thiosulfate or the polysulfide pathway. These are determined by the metal sulfides' mineralogy and their acid solubility. The microbial cell enables metal sulfide dissolution via oxidation of iron(II) ions and inorganic sulfur compounds. Thereby, the metal sulfide attacking agents iron(III) ions and protons are generated. Cells are active either in a planktonic state or attached to the mineral surface, forming biofilms. This review, as an update of the previous one (Vera et al., 2013a), summarizes some recent discoveries relevant to bioleaching microorganisms, contributing to a better understanding of their lifestyle. These comprise phylogeny, chemical pathways, surface science, biochemistry of iron and sulfur metabolism, anaerobic metabolism, cell-cell communication, molecular biology, and biofilm lifestyle. Recent advances from genetic engineering applied to bioleaching microorganisms will allow in the future to better understand important aspects of their physiology, as well as to open new possibilities for synthetic biology applications of leaching microbial consortia. KEY POINTS: • Leaching of metal sulfides is strongly enhanced by microorganisms • Biofilm formation and extracellular polymer production influences bioleaching • Cell interactions in mixed bioleaching cultures are key for process optimization.

Reverse engineering directed gene regulatory networks from transcriptomics and proteomics data of biomining bacterial communities with approximate Bayesian computation and steady-state signalling simulations.

BACKGROUND: Network inference is an important aim of systems biology. It enables the transformation of OMICs datasets into biological knowledge. It consists of reverse engineering gene regulatory networks from OMICs data, such as RNAseq or mass spectrometry-based proteomics data, through computational methods. This approach allows to identify signalling pathways involved in specific biological functions. The ability to infer causality in gene regulatory networks, in addition to correlation, is crucial for several modelling approaches and allows targeted control in biotechnology applications. METHODS: We performed simulations according to the approximate Bayesian computation method, where the core model consisted of a steady-state simulation algorithm used to study gene regulatory networks in systems for which a limited level of details is available. The simulations outcome was compared to experimentally measured transcriptomics and proteomics data through approximate Bayesian computation. RESULTS: The structure of small gene regulatory networks responsible for the regulation of biological functions involved in biomining were inferred from multi OMICs data of mixed bacterial cultures. Several causal inter- and intraspecies interactions were inferred between genes coding for proteins involved in the biomining process, such as heavy metal transport, DNA damage, replication and repair, and membrane biogenesis. The method also provided indications for the role of several uncharacterized proteins by the inferred connection in their network context. CONCLUSIONS: The combination of fast algorithms with high-performance computing allowed the simulation of a multitude of gene regulatory networks and their comparison to experimentally measured OMICs data through approximate Bayesian computation, enabling the probabilistic inference of causality in gene regulatory networks of a multispecies bacterial system involved in biomining without need of single-cell or multiple perturbation experiments. This information can be used to influence biological functions and control specific processes in biotechnology applications.

Resolution-of-the-identity second-order Møller-Plesset perturbation theory with complex basis functions: Benchmark calculations and applications to strong-field ionization of polyacenes.

We study the performance of the resolution-of-the-identity (RI) approximation for complex basis functions that we recently introduced [M. Hernández Vera and T.-C. Jagau, J. Chem. Phys. 151, 111101 (2019)] for second-order Møller-Plesset (MP2) perturbation theory as well as for the Coulomb and exchange contributions in Hartree-Fock theory. The sensitivity of this new RI-MP2 method toward the basis set and the auxiliary basis set is investigated, and computation times are analyzed. We show that the auxiliary basis set can be chosen purely real, that is, no complex-scaled functions need to be included. This approximation enables a further speedup of the method without compromising accuracy. We illustrate the application range of our implementation by computing static-field ionization rates of several polyacenes up to pentacene (C22H18) at the RI-MP2 level of theory. Pronounced anisotropies are observed for the ionization rates of these molecules.

Resolution-of-the-identity approximation for complex-scaled basis functions.

A resolution-of-the-identity (RI) approximation for two-electron integrals over Gaussian basis functions with a complex-scaled exponent is presented. Such functions are used in non-Hermitian quantum mechanics to represent electronic resonances by L2 integrable wave functions with complex energies. We have implemented this new RI approximation for second-order Møller-Plesset perturbation (MP2) theory as well as for the Coulomb and exchange contributions in Hartree-Fock (HF) theory. We discuss the differences to the standard RI approximation of Hermitian quantum mechanics and demonstrate the utility of the non-Hermitian RI-MP2 and RI-HF methods by computations of the orientation-dependent ionization rates of CO, C6H6, and C10H8 in static electric fields. Our results illustrate that RI-MP2 correctly describes correlation effects in molecular electronic resonances while the computational cost is low enough to allow for investigations of medium-sized molecules.

Automated Microscopic Analysis of Metal Sulfide Colonization by Acidophilic Microorganisms.

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.

Collisional relaxation kinetics for ortho and para NH2- under photodetachment in cold ion traps.

The collisional cooling of the internal rotational states of the nonlinear anion NH2- (1A1), occurring at the low temperature of a cold ion trap under helium buffer gas cooling, is examined via quantum dynamics calculations and ion decay rate measurements. The calculations employ a novel ab initio potential energy surface that describes the interaction anisotropy and range of action between the molecular anions and the neutral He atoms. The state changing integral cross sections are employed to obtain the state-to-state rate coefficients, separately for the ortho- and the para-NH2- ions. These rates are in turn used to compute the state population evolution in the trap for both species, once photodetachment by a laser is initiated in the trap. The present work shows results for the combined losses of both species after the photodetachment laser is switched on and analyzes the differences of loss kinetics between the two hyperfine isomers.

NH2- in a cold ion trap with He buffer gas: Ab initio quantum modeling of the interaction potential and of state-changing multichannel dynamics.

We present an extensive range of accurate ab initio calculations, which map in detail the spatial electronic potential energy surface that describes the interaction between the molecular anion NH2- (1A1) in its ground electronic state and the He atom. The time-independent close-coupling method is employed to generate the corresponding rotationally inelastic cross sections, and then the state-changing rates over a range of temperatures from 10 to 30 K, which is expected to realistically represent the experimental trapping conditions for this ion in a radio frequency ion trap filled with helium buffer gas. The overall evolutionary kinetics of the rotational level population involving the molecular anion in the cold trap is also modelled during a photodetachment experiment and analyzed using the computed rates. The present results clearly indicate the possibility of selectively detecting differences in behavior between the ortho- and para-anions undergoing photodetachment in the trap.

Rotationally inelastic collisions of H2+ ions with He buffer gas: Computing cross sections and rates.

We present quantum calculations for the inelastic collisions between H2+ molecules, in rotationally excited internal states, and He atoms. This work is motivated by the possibility of experiments in which the molecular ions are stored and translationally cooled in an ion trap and a He buffer gas is added for deactivation of the internal rotational population, in particular at low (cryogenic) translational temperatures. We carry out an accurate representation of the forces at play from an ab initio description of the relevant potential energy surface, with the molecular ion in its ground vibrational state, and obtain the cross sections for state-changing rotationally inelastic collisions by solving the coupled channel quantum scattering equations. The presence of hyperfine and fine structure effects in both ortho- and para-H2+ molecules is investigated and compared to the results where such a contribution is disregarded. An analysis of possible propensity rules that may predict the relative probabilities of inelastic events involving rotational state-changing is also carried out, together with the corresponding elastic cross sections from several initial rotational states. Temperature-dependent rotationally inelastic rates are then computed and discussed in terms of relative state-changing collisional efficiency under trap conditions. The results provide the essential input data for modeling different aspects of the experimental setups which can finally produce internally cold molecular ions interacting with a buffer gas.

Omics on bioleaching: current and future impacts.

Bioleaching corresponds to the microbial-catalyzed process of conversion of insoluble metals into soluble forms. As an applied biotechnology globally used, it represents an extremely interesting field of research where omics techniques can be applied in terms of knowledge development, but moreover in terms of process design, control, and optimization. In this mini-review, the current state of genomics, proteomics, and metabolomics of bioleaching and the major impacts of these analytical methods at industrial scale are highlighted. In summary, genomics has been essential in the determination of the biodiversity of leaching processes and for development of conceptual and functional metabolic models. Proteomic impacts are mostly related to microbe-mineral interaction analysis, including copper resistance and biofilm formation. Early steps of metabolomics in the field of bioleaching have shown a significant potential for the use of metabolites as industrial biomarkers. Development directions are given in order to enhance the future impacts of the omics in biohydrometallurgy.

Manipulation of pyrite colonization and leaching by iron-oxidizing Acidithiobacillus species.

In this study, the process of pyrite colonization and leaching by three iron-oxidizing Acidithiobacillus species was investigated by fluorescence microscopy, bacterial attachment, and leaching assays. Within the first 4-5 days, only the biofilm subpopulation was responsible for pyrite dissolution. Pyrite-grown cells, in contrast to iron-grown cells, were able to oxidize iron(II) ions or pyrite after 24 h iron starvation and incubation with 1 mM H2O2, indicating that these cells were adapted to the presence of enhanced levels of reactive oxygen species (ROS), which are generated on metal sulfide surfaces. Acidithiobacillus ferrivorans SS3 and Acidithiobacillus ferrooxidans R1 showed enhanced pyrite colonization and biofilm formation compared to A. ferrooxidans (T). A broad range of factors influencing the biofilm formation on pyrite were also identified, some of them were strain-specific. Cultivation at non-optimum growth temperatures or increased ionic strength led to a decreased colonization of pyrite. The presence of iron(III) ions increased pyrite colonization, especially when pyrite-grown cells were used, while the addition of 20 mM copper(II) ions resulted in reduced biofilm formation on pyrite. This observation correlated with a different extracellular polymeric substance (EPS) composition of copper-exposed cells. Interestingly, the addition of 1 mM sodium glucuronate in combination with iron(III) ions led to a 5-fold and 7-fold increased cell attachment after 1 and 8 days of incubation, respectively, in A. ferrooxidans (T). In addition, sodium glucuronate addition enhanced pyrite dissolution by 25%.

Visualization and analysis of EPS glycoconjugates of the thermoacidophilic archaeon Sulfolobus metallicus.

Biofilms are surface-associated colonies of microorganisms embedded in a matrix of extracellular polymeric substances (EPS). As EPS mediate the contact between cells and surfaces, an understanding of their composition and production is of particular interest. In this study, the EPS components of Sulfolobus metallicus DSM 6482(T) forming biofilms on elemental sulfur (S(0)) were investigated by confocal laser scanning microscopy (CLSM). In order to visualize cell and EPS distributions, biofilm cells were stained with various dyes specific for glycoconjugates, proteins, nucleic acids and lipids. Biofilm cells on S(0) were heterogeneously distributed and characterized as individual cells, microcolonies, and large clusters up to a hundred micrometers in diameter. The glycoconjugates in biofilms were detected by fluorescence lectin-binding analysis (FLBA). Screening of 72 commercially available lectins resulted in the selection of 21 lectins useful for staining biofilms of S. metallicus (T). Capsular EPS from planktonic cells were mainly composed of carbohydrates and proteins. In contrast, colloidal EPS from planktonic cells were dominated by carbohydrates. Proteins were found to be major components in EPS from biofilms on S(0). Using specific probes combined with CLSM, we showed that extracellular proteins and nucleic acids were present in the EPS matrix. Finally, we showed that S. metallicus (T) cells were embedded in a flexible EPS matrix. This study provides new insights into archaeal biofilms and EPS composition and properties with respect to their interactions with S(0).

Rotational excitation of HCN by para- and ortho-H2.

Rotational excitation of the hydrogen cyanide (HCN) molecule by collisions with para-H2(j = 0, 2) and ortho-H2(j = 1) is investigated at low temperatures using a quantum time independent approach. Both molecules are treated as rigid rotors. The scattering calculations are based on a highly correlated ab initio 4-dimensional (4D) potential energy surface recently published. Rotationally inelastic cross sections among the 13 first rotational levels of HCN were obtained using a pure quantum close coupling approach for total energies up to 1200 cm(-1). The corresponding thermal rate coefficients were computed for temperatures ranging from 5 to 100 K. The HCN rate coefficients are strongly dependent on the rotational level of the H2 molecule. In particular, the rate coefficients for collisions with para-H2(j = 0) are significantly lower than those for collisions with ortho-H2(j = 1) and para-H2(j = 2). Propensity rules in favor of even Δj transitions were found for HCN in collisions with para-H2(j = 0) whereas propensity rules in favor of odd Δj transitions were found for HCN in collisions with H2(j ⩾ 1). The new rate coefficients were compared with previously published HCN-para-H2(j = 0) rate coefficients. Significant differences were found due the inclusion of the H2 rotational structure in the scattering calculations. These new rate coefficients will be crucial to improve the estimation of the HCN abundance in the interstellar medium.

Shotgun proteomics study of early biofilm formation process of Acidithiobacillus ferrooxidans ATCC 23270 on pyrite.

Acidithiobacillus ferrooxidans is a chemolithoautotrophic, mesophilic Gram-negative bacterium able to oxidize ferrous iron, sulfur, and metal sulfides. It forms monolayer biofilms where extracellular polymeric substances are essential for cell attachment and metal sulfide leaching. High-throughput proteomics has been applied to study the early process of biofilm formation on pyrite by At. ferrooxidans ATCC 23270. After 24 h contact with the mineral, planktonic and sessile (biofilm) cell subpopulations were separated and proteins extracted. In total, 1319 proteins were detected in both samples. Sixty-two of these were found to be increased in biofilms. Additionally, 25 proteins were found to be decreased in the biofilm cell subpopulation. Three transcriptional factors were found to be increased or decreased among both cell subpopulations, suggesting their potential involvement in the regulation of these processes. Although no significant differences were observed for the known proteins related to ferrous iron and sulfur oxidation pathways among both cell subpopulations, the results presented here show that the early steps of At. ferrooxidans biofilm formation consist of a set of metabolic adaptations following cell attachment to the mineral surface. Functions such as extracellular polymeric substances biosynthesis seem to be pivotal. This first high-throughput proteomic study may also contribute to the annotation of several unknown At. ferrooxidans proteins found.

Progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation--part A.

Bioleaching of metal sulfides is performed by a diverse group of microorganisms. The dissolution chemistry of metal sulfides follows two pathways, which are determined by the mineralogy and the acid solubility of the metal sulfides: the thiosulfate and the polysulfide pathways. Bacterial cells can effect this metal sulfide dissolution via iron(II) ion and sulfur compound oxidation. Thereby, iron(III) ions and protons, the metal sulfide-attacking agents, are available. Cells can be active either in planktonic state or in forming biofilms on the mineral surface; however, the latter is much more efficient in terms of bioleaching kinetics. In the case of Acidithiobacillus ferrooxidans, bacterial exopolymers contain iron(III) ions, each complexed by two uronic acid residues. The resulting positive charge allows an electrostatic attachment to the negatively charged pyrite. Thus, the first function of complexed iron(III) ions is the mediation of cell attachment, while their second function is oxidative dissolution of the metal sulfide, similar to the role of free iron(III) ions in non-contact leaching. In both cases, the electrons extracted from the metal sulfide reduce molecular oxygen via a redox chain forming a supercomplex spanning the periplasmic space and connecting both outer and inner membranes. In this review, we summarize some recent discoveries relevant to leaching bacteria which contribute to a better understanding of these fascinating microorganisms. These include surface science, biochemistry of iron and sulfur metabolism, anaerobic metabolism, and biofilm formation. The study of microbial interactions among multispecies leaching consortia, including cell-to-cell communication mechanisms, must be considered in order to reveal more insights into the biology of bioleaching microorganisms and their potential biotechnological use.

AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans.

Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.

A new ab initio potential energy surface for the collisional excitation of HCN by para- and ortho-H2.

We present a new four-dimensional potential energy surface for the collisional excitation of HCN by H2. Ab initio calculations of the HCN-H2 van der Waals complex, considering both molecules as rigid rotors, were carried out at the explicitly correlated coupled cluster with single, double, and perturbative triple excitations [CCSD(T)-F12a] level of theory using an augmented correlation-consistent triple zeta (aVTZ) basis set. The equilibrium structure is linear HCN-H2 with the nitrogen pointing towards H2 at an intermolecular separation of 7.20 a0. The corresponding well depth is -195.20 cm(-1). A secondary minimum of -183.59 cm(-1) was found for a T-shape configuration with the H of HCN pointing to the center of mass of H2. We also determine the rovibrational energy levels of the HCN-para-H2 and HCN-ortho-H2 complexes. The calculated dissociation energies for the para and ortho complexes are 37.79 cm(-1) and 60.26 cm(-1), respectively. The calculated ro-vibrational transitions in the HCN-H2 complex are found to agree by more than 0.5% with the available experimental data, confirming the accuracy of the potential energy surface.

Membrane vesicles in Acidithiobacillia class extreme acidophiles: influence on collective behaviors of 'Fervidacidithiobacillus caldus'.

Membrane vesicles (MVs) are envelope-derived extracellular sacs that perform a broad diversity of physiological functions in bacteria. While considerably studied in pathogenic microorganisms, the roles, relevance, and biotechnological potential of MVs from environmental bacteria are less well established. Acidithiobacillaceae family bacteria are active players in the sulfur and iron biogeochemical cycles in extremely acidic environments and drivers of the leaching of mineral ores contributing to acid rock/mine drainage (ARD/AMD) and industrial bioleaching. One key aspect of such a role is the ability of these bacteria to tightly interact with the mineral surfaces and extract electrons and nutrients to support their chemolithotrophic metabolism. Despite recent advances in the characterization of acidithiobacilli biofilms and extracellular matrix (ECM) components, our understanding of its architectural and mechanistic aspects remains scant. Using different microscopy techniques and nano-tracking analysis we show that vesiculation is a common phenomenon in distant members of the Acidithiobacillaceae family, and further explore the role of MVs in multicellular colonization behaviors using 'Fervidacidithiobacillus caldus' as a bacterial model. Production of MVs in 'F. caldus' occurred in both planktonic cultures and biofilms formed on sulfur surfaces, where MVs appeared individually or in chains resembling tube-shaped membranous structures (TSMSs) important for microbial communication. Liquid chromatography-mass spectrometry data and bioinformatic analysis of the MV-associated proteome revealed that 'F. caldus' MVs were enriched in proteins involved in cell-cell and cell-surface processes and largely typified the MVs as outer MVs (OMVs). Finally, microbiological assays showed that amendment of 'F. caldus' MVs to cells and/or biofilms affects collective colonizing behaviors relevant to the ecophysiology and applications of these acidophiles, providing grounds for their exploitation in biomining.