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.

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.

Development of an implantable three-dimensional model of a functional pathogenic multispecies biofilm to study infected wounds.

Chronic wounds cannot heal due to impairment of regeneration, mainly caused by the persistent infection of multispecies biofilms. Still, the effects of biofilm wound infection and its interaction with the host are not fully described. We aimed to study functional biofilms in physiological conditions in vitro, and their potential effects in health and regeneration in vivo. Therefore, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis were seeded in collagen-based scaffolds for dermal regeneration. After 24 h, scaffolds had bacterial loads depending on the initial inoculum, containing viable biofilms with antibiotic tolerance. Afterwards, scaffolds were implanted onto full skin wounds in mice, together with daily supervision and antibiotic treatment. Although all mice survived their health was affected, displaying fever and weight loss. After ten days, histomorphology of scaffolds showed high heterogeneity in samples and within groups. Wounds were strongly, mildly, or not infected according to colony forming units, and P. aeruginosa had higher identification frequency. Biofilm infection induced leucocyte infiltration and elevated interferon-γ and interleukin-10 in scaffolds, increase of size and weight of spleen and high systemic pro-calcitonin concentrations. This functional and implantable 3D biofilm model allows to study host response during infection, providing a useful tool for infected wounds therapy development.

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.

Biofilm formation of Ancylobacter sp. TS-1 on different granular materials and its ability for chemolithoautotrophic As(III)-oxidation at high concentrations.

The oxidation of arsenic (As) is a key step in its removal from water, and biological oxidation may provide a cost-effective and sustainable method. The biofilm-formation ability of Ancylobacter sp. TS-1, a novel chemolithoautotrophic As oxidizer, was studied for four materials: polypropylene, graphite, sand, and zeolite. After seven days under batch mixotrophic conditions, with high concentrations of As(III) (225 mg·L-1), biofilm formation was detected on all materials except for polypropylene. The results demonstrate As(III)-oxidation of TS-1 biofilms and suggest that the number of active cells was similar for graphite, sand, and zeolite. However, the biofilm biomass follows the specific surface area of each material: 7.0, 2.4, and 0.4 mg VSS·cm-3 for zeolite, sand, and graphite, respectively. Therefore, the observed biofilm-biomass differences were probably associated with different amounts of EPS and inert biomass. Lastly, As(III)-oxidation kinetics were assessed for the biofilms formed on graphite and zeolite under chemolithoautotrophic conditions. The normalized oxidation rate for biofilms formed on these materials was 3.6 and 1.0 mg·L-1·h-1·cm-3, resulting among the highest reported values for As(III)-oxidizing biofilms operated at high-As(III) concentrations. Our findings suggest that biofilm reactors based on Ancylobacter sp. TS-1 are highly promising for their utilization in As(III)-oxidation pre-treatment of high-As(III) polluted waters.

Diffusible signal factor signaling controls bioleaching activity and niche protection in the acidophilic, mineral-oxidizing leptospirilli.

Bioleaching of metal sulfide ores involves acidophilic microbes that catalyze the chemical dissolution of the metal sulfide bond that is enhanced by attached and planktonic cell mediated oxidation of iron(II)-ions and inorganic sulfur compounds. Leptospirillum spp. often predominate in sulfide mineral-containing environments, including bioheaps for copper recovery from chalcopyrite, as they are effective primary mineral colonizers and oxidize iron(II)-ions efficiently. In this study, we demonstrated a functional diffusible signal factor interspecies quorum sensing signaling mechanism in Leptospirillum ferriphilum and Leptospirillum ferrooxidans that produces (Z)-11-methyl-2-dodecenoic acid when grown with pyrite as energy source. In addition, pure diffusible signal factor and extracts from supernatants of pyrite grown Leptospirillum spp. inhibited biological iron oxidation in various species, and that pyrite grown Leptospirillum cells were less affected than iron grown cells to self inhibition. Finally, transcriptional analyses for the inhibition of iron-grown L. ferriphilum cells due to diffusible signal factor was compared with the response to exposure of cells to N- acyl-homoserine-lactone type quorum sensing signal compounds. The data suggested that Leptospirillum spp. diffusible signal factor production is a strategy for niche protection and defense against other microbes and it is proposed that this may be exploited to inhibit unwanted acidophile species.

Quorum Sensing Signaling Molecules Positively Regulate c-di-GMP Effector PelD Encoding Gene and PEL Exopolysaccharide Biosynthesis in Extremophile Bacterium Acidithiobacillus thiooxidans.

Acidithiobacillus species are fundamental players in biofilm formation by acidophile bioleaching communities. It has been previously reported that Acidithiobacillus ferrooxidans possesses a functional quorum sensing mediated by acyl-homoserine lactones (AHL), involved in biofilm formation, and AHLs naturally produced by Acidithiobacillus species also induce biofilm formation in Acidithiobacillus thiooxidans. A c-di-GMP pathway has been characterized in Acidithiobacillus species but it has been pointed out that the c-di-GMP effector PelD and pel-like operon are only present in the sulfur oxidizers such as A. thiooxidans. PEL exopolysaccharide has been recently involved in biofilm formation in this Acidithiobacillus species. Here, by comparing wild type and ΔpelD strains through mechanical analysis of biofilm-cells detachment, fluorescence microscopy and qPCR experiments, the structural role of PEL exopolysaccharide and the molecular network involved for its biosynthesis by A. thiooxidans were tackled. Besides, the effect of AHLs on PEL exopolysaccharide production was assessed. Mechanical resistance experiments indicated that the loss of PEL exopolysaccharide produces fragile A. thiooxidans biofilms. qRT-PCR analysis established that AHLs induce the transcription of pelA and pelD genes while epifluorescence microscopy studies revealed that PEL exopolysaccharide was required for the development of AHL-induced biofilms. Altogether these results reveal for the first time that AHLs positively regulate pel genes and participate in the molecular network for PEL exopolysaccharide biosynthesis by A. thiooxidans.

Systems biology of acidophile biofilms for efficient metal extraction.

Society's demand for metals is ever increasing while stocks of high-grade minerals are being depleted. Biomining, for example of chalcopyrite for copper recovery, is a more sustainable biotechnological process that exploits the capacity of acidophilic microbes to catalyze solid metal sulfide dissolution to soluble metal sulfates. A key early stage in biomining is cell attachment and biofilm formation on the mineral surface that results in elevated mineral oxidation rates. Industrial biomining of chalcopyrite is typically carried out in large scale heaps that suffer from the downsides of slow and poor metal recoveries. In an effort to mitigate these drawbacks, this study investigated planktonic and biofilm cells of acidophilic (optimal growth pH < 3) biomining bacteria. RNA and proteins were extracted, and high throughput "omics" performed from a total of 80 biomining experiments. In addition, micrographs of biofilm formation on the chalcopyrite mineral surface over time were generated from eight separate experiments. The dataset generated in this project will be of great use to microbiologists, biotechnologists, and industrial researchers.

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.

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 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.

Deep neural networks outperform human expert's capacity in characterizing bioleaching bacterial biofilm composition.

BACKGROUND: Deep neural networks have been successfully applied to diverse fields of computer vision. However, they only outperform human capacities in a few cases. METHODS: The ability of deep neural networks versus human experts to classify microscopy images was tested on biofilm colonization patterns formed on sulfide minerals composed of up to three different bioleaching bacterial species attached to chalcopyrite sample particles. RESULTS: A low number of microscopy images per category (<600) was sufficient for highly efficient computational analysis of the biofilm's bacterial composition. The use of deep neural networks reached an accuracy of classification of ∼90% compared to ∼50% for human experts. CONCLUSIONS: Deep neural networks outperform human experts' capacity in characterizing bacterial biofilm composition involved in the degradation of chalcopyrite. This approach provides an alternative to standard, time-consuming biochemical methods.

Proteomics Reveal Enhanced Oxidative Stress Responses and Metabolic Adaptation in Acidithiobacillus ferrooxidans Biofilm Cells on Pyrite.

Reactive oxygen species (ROS) cause oxidative stress and growth inhibition by inactivation of essential enzymes, DNA and lipid damage in microbial cells. Acid mine drainage (AMD) ecosystems are characterized by low pH values, enhanced levels of metal ions and low species abundance. Furthermore, metal sulfides, such as pyrite and chalcopyrite, generate extracellular ROS upon exposure to acidic water. Consequently, oxidative stress management is especially important in acidophilic leaching microorganisms present in industrial biomining operations, especially when forming biofilms on metal sulfides. Several adaptive mechanisms have been described, but the molecular repertoire of responses upon exposure to pyrite and the presence of ROS are not thoroughly understood in acidophiles. In this study the impact of the addition of H2O2 on iron oxidation activity in Acidithiobacillus ferrooxidans DSM 14882T was investigated. Iron(II)- or sulfur-grown cells showed a higher sensitivity toward H2O2 than pyrite-grown ones. In order to elucidate which molecular responses may be involved, we used shot-gun proteomics and compared proteomes of cells grown with iron(II)-ions against biofilm cells, grown for 5 days in presence of pyrite as sole energy source. In total 1157 proteins were identified. 213 and 207 ones were found to have increased levels in iron(II) ion-grown or pyrite-biofilm cells, respectively. Proteins associated with inorganic sulfur compound (ISC) oxidation were among the latter. In total, 80 proteins involved in ROS degradation, thiol redox regulation, macromolecule repair mechanisms, biosynthesis of antioxidants, as well as metal and oxygen homeostasis were found. 42 of these proteins had no significant changes in abundance, while 30 proteins had increased levels in pyrite-biofilm cells. New insights in ROS mitigation strategies, such as importance of globins for oxygen homeostasis and prevention of unspecific reactions of free oxygen that generate ROS are presented for A. ferrooxidans biofilm cells. Furthermore, proteomic analyses provide insights in adaptations of carbon fixation and oxidative phosphorylation pathways under these two growth conditions.

Biofilm dynamics and EPS production of a thermoacidophilic bioleaching archaeon.

Bioleaching of metal sulfides represents an interfacial process where biofilm formation is important in the initial steps of this process. In technical applications of bioleaching, such as reactor leaching in the temperature range of 50 up to 90 °C and also in (self-heating) heaps, thermophilic archaea play an important role and often are the leaching organisms of choice. Nevertheless, to date there is little information available on the interactions between thermoacidophilic archaea and their natural mineral substrates such as pyrite. Especially for extracellular polymeric substances (EPS) of archaea and their biofilms in bioleaching environments information is rather limited. The present work focused on investigations of biofilm dynamics and EPS production of the thermoacidophilic archaeon Acidianus sp. DSM 29099 under bioleaching conditions. The results show that biofilms are dispersed non-homogeneously on pyrite. Large parts of the pyrite surfaces remain free of cells. Cell detachment from pyrite results in microbial "footprints" which, based on lectin binding assays, consist of mannose, glucose and fucose containing compounds. A monolayer biofilm develops on pyrite after 2-4 days of incubation. In addition, the pyrite surface is covered with a layer of organic compounds. EPS analysis indicates the presence of proteins, polysaccharides and uronic acids, the composition of which varies according to substrate and lifestyle (i.e. planktonic, biofilm cells). This report provides insight into EPS and biofilm characteristics of thermophilic archaea and improves understanding of the mineral-microbial-biofilm interfacial interactions in extreme environments. Moreover, the results on interaction dynamics of archaeal microbial consortia will facilitate the understanding of thermophilic bioleaching.

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.

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.

Insights into the biology of acidophilic members of the Acidiferrobacteraceae family derived from comparative genomic analyses.

The family Acidiferrobacteraceae (order Acidiferrobacterales) currently contains Gram negative, neutrophilic sulfur oxidizers such as Sulfuricaulis and Sulfurifustis, as well as acidophilic iron and sulfur oxidizers belonging to the Acidiferrobacter genus. The diversity and taxonomy of the genus Acidiferrobacter has remained poorly explored. Although several metagenome and bioleaching studies have identified its presence worldwide, only two strains, namely Acidiferrobacter thiooxydans DSM 2932T, and Acidiferrobacter spp. SP3/III have been isolated and made publically available. Using 16S rRNA sequence data publically available for the Acidiferrobacteraceae, we herein shed light into the molecular taxonomy of this family. Results obtained support the presence of three clades Acidiferrobacter, Sulfuricaulis and Sulfurifustis. Genomic analyses of the genome sequences of A. thiooxydansT and Acidiferrobacter spp. SP3/III indicate that ANI relatedness between the SPIII/3 strain and A. thiooxydansT is below 95-96%, supporting the classification of strain SP3/III as a new species within this genus. In addition, approximately 70% of Acidiferrobacter sp. SPIII/3 predicted genes have a conserved ortholog in A. thiooxydans strains. A comparative analysis of iron, sulfur oxidation pathways, genome plasticity and cell-cell communication mechanisms of Acidiferrobacter spp. are also discussed.

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.