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The biological treatment of complex industrial wastewater has always been a research hotspot. In this experiment, a salt-tolerant strain Stutzerimonas sp. ZW5 with aerobic denitrification and biomineralization ability was screened, and the optimum conditions of ZW5 were explored by kinetics. The removal efficiencies of nitrate (NO3--N), bisphenol A (BPA), phosphorus (PO43--P), and calcium (Ca2+) were 94.47 %, 100 %, 98.87 %, and 83.04 %, respectively. The removal mechanism of BPA was the adsorption of microbial induced calcium precipitation (MICP) and extracellular polymeric substances (EPS). Moreover, BPA could weaken the electron transfer ability and growth metabolism of microorganisms and affect the structure of biominerals. At the same time, the stress response of microorganisms would increase the secretion of EPS to promote the process of biomineralization. Through nitrogen balance experiments, it was found that the addition of BPA would lead to a decrease in the proportion of gaseous nitrogen. This experiment offers novel perspectives on the treatment of industrial effluents and microbial stress response.

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Evolutionary engineering experiments, in combination with omics technologies, revealed genetic markers underpinning the molecular mechanisms behind acetic acid stress tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii. Here, compared to the ancestral Ent strain, evolved yeast strains could quickly adapt to high acetic acid levels (7 g/L) and displayed a shorter lag phase of growth. Bioinformatic-aided whole-genome sequencing identified genetic changes associated with enhanced strain robustness to acetic acid: a duplicated sequence in the essential endocytotic PAN1 gene, mutations in a cell wall mannoprotein (dan4Thr192del), a lipid and fatty acid transcription factor (oaf1Ser57Pro) and a thiamine biosynthetic enzyme (thi13Thr332Ala). Induction of PAN1 and its associated endocytic complex SLA1 and END3 genes was observed following acetic acid treatment in the evolved-resistant strain when compared to the ancestral strain. Genome-wide transcriptomic analysis of the evolved Ent acid-resistant strain (Ent ev16) also revealed a dramatic rewiring of gene expression among genes associated with cellular transport, metabolism, oxidative stress response, biosynthesis/organization of the cell wall, and cell membrane. Some evolved strains also displayed better growth at high acetic acid concentrations and exhibited adaptive metabolic profiles with altered levels of secreted ethanol (4.0-6.4% decrease), glycerol (31.4-78.5% increase), and acetic acid (53.0-60.3% increase) when compared to the ancestral strain. Overall, duplication/mutations and transcriptional alterations are key mechanisms driving improved acetic acid tolerance in probiotic strains. We successfully used adaptive evolutionary engineering to rapidly and effectively elucidate the molecular mechanisms behind important industrial traits to obtain robust probiotic yeast strains for myriad biotechnological applications. KEY POINTS: •Acetic acid adaptation of evolutionary engineered robust probiotic yeast S. boulardii •Enterol ev16 with altered genetic and transcriptomic profiles survives in up to 7 g/L acetic acid •Improved acetic acid tolerance of S. boulardii ev16 with mutated PAN1, DAN4, OAF1, and THI13 genes.

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The physical environments in which microorganisms naturally reside rarely have homogeneous structure, and changes in their porous architecture may have effects on microbial activities that are not typically captured in conventional laboratory studies. In this study, to investigate the influence of environmental structure on microbial responses to stress, we constructed structured environments with different pore properties (determined by X-ray computed tomography). First, using glass beads in different arrangements and inoculated with the soil yeast Saitozyma podzolica, increases in the average equivalent spherical diameters (ESD) of a structure's porous architecture led to decreased survival of the yeast under a toxic metal challenge with lead nitrate. This relationship was reproduced when yeasts were introduced into additively manufactured lattice structures, comprising regular arrays with ESDs comparable to those of the bead structures. The pore ESD dependency of metal resistance was not attributable to differences in cell density in microenvironments delimited by different pore sizes, supporting the inference that pore size specifically was the important parameter in determining survival of stress. These findings highlight the importance of the physical architecture of an organism's immediate environment for its response to environmental perturbation, while offering new tools for investigating these interactions in the laboratory. IMPORTANCE Interactions between cells and their structured environments are poorly understood but have significant implications for organismal success in both natural and nonnatural settings. This work used a multidisciplinary approach to develop laboratory models with which the influence of a key parameter of environmental structure-pore size-on cell activities can be dissected. Using these new methods in tandem with additive manufacturing, we demonstrated that resistance of yeast soil isolates to stress (from a common metal pollutant) is inversely related to pore size of their environment. This has important ramifications for understanding how microorganisms respond to stress in different environments. The findings also establish new pathways for resolving the effects of physical environment on microbial activity, enabling important understanding that is not readily attainable with traditional bulk sampling and analysis approaches.

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Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.

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Antibiotics elicit drastic changes in microbial gene expression, including the induction of stress response genes. While certain stress responses are known to "cross-protect" bacteria from other stressors, it is unclear whether cellular responses to antibiotics have a similar protective role. By measuring the genome-wide transcriptional response dynamics of Escherichia coli to four antibiotics, we found that trimethoprim induces a rapid acid stress response that protects bacteria from subsequent exposure to acid. Combining microfluidics with time-lapse imaging to monitor survival and acid stress response in single cells revealed that the noisy expression of the acid resistance operon gadBC correlates with single-cell survival. Cells with higher gadBC expression following trimethoprim maintain higher intracellular pH and survive the acid stress longer. The seemingly random single-cell survival under acid stress can therefore be predicted from gadBC expression and rationalized in terms of GadB/C molecular function. Overall, we provide a roadmap for identifying the molecular mechanisms of single-cell cross-protection between antibiotics and other stressors.

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When anode-respiring bacteria (ARB) respire electrons to an anode in microbial electrochemical cells (MXCs), they harvest only a small amount of free energy. This means that ARB must have a high substrate-oxidation rate coupled with a high ratio of electrons used for respiration compared to total electrons removed by substrate utilization. It also means that they are especially susceptible to inhibition that slows anode respiration or lowers their biomass yield. Using several electrochemical techniques, we show that a relatively high total ammonium-nitrogen (TAN) concentration (2.2 g TAN/L) induced significant stress on the ARB biofilms, lowering their true yield and forcing the ARB to boost the ratio of electrons respired per electrons consumed from the substrate. In particular, a higher respiration rate, measured as current density (j), was associated with slower growth and a lower net yield, compared to an ARB biofilm grown with a lower ammonium concentration (0.2 g TAN/L). Further increases in influent TAN (to 3 and then to 4.4 g TAN/L) caused nearly complete inhibition of anode respiration. However, the ARB could recover from high-TAN inhibition after a shift of the MXC's feed to 0.2 g TAN/L. In summary, ARB biofilms were inhibited by a high TAN concentration, but could divert more electron flow toward anode respiration with modest inhibition and recover when severe inhibition was relieved. Biotechnol. Bioeng. 2017;114: 1151-1159. © 2017 Wiley Periodicals, Inc.

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The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility.

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Infectious salmon anemia virus (ISAV) is a salmon pathogen causing serious outbreaks in fish farms world-wide. There is currently no effective commercially available vaccine and there is a need for better understanding of host pathogen interactions with this virus. Various strains can cause both acute and persistent infections and therefore establish a balance with the host immune responses. We have studied host responses to this infection by analyzing the main branches of the unfolded protein response (UPR) in salmon cells in vitro and in tissues from infected fish to gain a better understanding of virus-host interactions. ISAV induce the main symptoms and signaling pathways of UPR (ATF6, PERK and IRE1) without inducing translational attenuation. This may be due to concomitant induction of an important negative feedback loop via the phosphatase regulator GADD34. The host cells can therefore respond with translation of cytokine and antiviral proteins to curb or control infection.

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