Two-Stage Continuous Conversion of Carbon Monoxide to Ethylene by Whole Cells of Azotobacter vinelandii.

Azotobacter vinelandii is an obligate aerobic diazotroph with a verified transient ability to reduce carbon monoxide to ethylene by its vanadium nitrogenase. In this study, we implemented an industrially relevant continuous two-stage stirred-tank system for in vivo biotransformation of a controlled supply of air enriched with 5% carbon monoxide to 302 µg ethylene g-1 glucose consumed. To attain this value, the process required overcoming critical oxygen limitations during cell proliferation while simultaneously avoiding the A. vinelandii respiratory protection mechanism that negatively impacts in vivo nitrogenase activity. Additionally, process conditions allowed the demonstration of carbon monoxide's solubility as a reaction-limiting factor and a competitor with dinitrogen for the vanadium nitrogenase active site, implying that excess intracellular carbon monoxide could lead to a cessation of cell proliferation and ethylene formation as shown genetically using a new strain of A. vinelandii deficient in carbon monoxide dehydrogenase.IMPORTANCE Ethylene is an essential commodity feedstock used for the generation of a variety of consumer products, but its generation demands energy-intensive processes and is dependent on nonrenewable substrates. This work describes a continuous biological method for investigating the nitrogenase-mediated carbon monoxide reductive coupling involved in ethylene production using whole cells of Azotobacter vinelandii If eventually adopted by industry, this technology has the potential to significantly reduce the total energy input required and the ethylene recovery costs, as well as decreasing greenhouse gas emissions associated with current production strategies.

Azotobacter vinelandii: the source of 100 years of discoveries and many more to come.

Azotobacter vinelandii has been studied for over 100 years since its discovery as an aerobic nitrogen-fixing organism. This species has proved useful for the study of many different biological systems, including enzyme kinetics and the genetic code. It has been especially useful in working out the structures and mechanisms of different nitrogenase enzymes, how they can function in oxic environments and the interactions of nitrogen fixation with other aspects of metabolism. Interest in studying A. vinelandii has waned in recent decades, but this bacterium still possesses great potential for new discoveries in many fields and commercial applications. The species is of interest for research because of its genetic pliability and natural competence. Its features of particular interest to industry are its ability to produce multiple valuable polymers - bioplastic and alginate in particular; its nitrogen-fixing prowess, which could reduce the need for synthetic fertilizer in agriculture and industrial fermentations, via coculture; its production of potentially useful enzymes and metabolic pathways; and even its biofuel production abilities. This review summarizes the history and potential for future research using this versatile microbe.

Azotobacter vinelandii Nitrogenase Activity, Hydrogen Production, and Response to Oxygen Exposure.

Azotobacter vinelandii selectively utilizes three types of nitrogenase (molybdenum, vanadium, and iron only) to fix N2, with their expression regulated by the presence or absence of different metal cofactors in its environment. Each alternative nitrogenase isoenzyme is predicted to have different electron flux requirements based on in vitro measurements, with the molybdenum nitrogenase requiring the lowest flux and the iron-only nitrogenase requiring the highest. Here, prior characterized strains, derepressed in nitrogenase synthesis and also deficient in uptake hydrogenase, were further modified to generate new mutants lacking the ability to produce poly-ß-hydroxybutyrate (PHB). PHB is a storage polymer generated under oxygen-limiting conditions and can represent up to 70% of the cells' dry weight. The absence of such granules facilitated the study of relationships between catalytic biomass and product molar yields across different adaptive respiration conditions. The released hydrogen gas observed during growth, due to the inability of the mutants to recapture hydrogen, allowed for direct monitoring of in vivo nitrogenase activity for each isoenzyme. The data presented here show that increasing oxygen exposure limits equally the in vivo activities of all nitrogenase isoenzymes, while under comparative conditions, the Mo nitrogenase enzyme evolves more hydrogen per unit of biomass than the alternative isoenzymes.IMPORTANCEA. vinelandii has been a focus of intense research for over 100 years. It has been investigated for a variety of functions, including agricultural fertilization and hydrogen production. All of these endeavors are centered around A. vinelandii's ability to fix nitrogen aerobically using three nitrogenase isoenzymes. The majority of research up to this point has targeted in vitro measurements of the molybdenum nitrogenase, and robust data contrasting how oxygen impacts the in vivo activity of each nitrogenase isoenzyme are lacking. This article aims to provide in vivo nitrogenase activity data using a real-time evaluation of hydrogen gas released by derepressed nitrogenase mutants lacking an uptake hydrogenase and PHB accumulation.

Characterization of Clostridium ljungdahlii OTA1: a non-autotrophic hyper ethanol-producing strain.

A Clostridium ljungdahlii lab-isolated spontaneous-mutant strain, OTA1, has been shown to produce twice as much ethanol as the C. ljungdahlii ATCC 55383 strain when cultured in a mixotrophic medium containing fructose and syngas. Whole-genome sequencing identified four unique single nucleotide polymorphisms (SNPs) in the C. ljungdahlii OTA1 genome. Among these, two SNPs were found in the gene coding for AcsA and HemL, enzymes involved in acetyl-CoA formation from CO/CO2. Homology models of the respective mutated enzymes revealed alterations in the size and hydrogen bonding of the amino acids in their active sites. Failed attempts to grow OTA1 autotrophically suggested that one or both of these mutated genes prevented acetyl-CoA synthesis from CO/CO2, demonstrating that its activity was required for autotrophic growth by C. ljungdahlii. An inoperable Wood-Ljungdahl pathway resulted in higher CO2 and ethanol yields and lower biomass and acetate yields compared to WT for multiple growth conditions including heterotrophic and mixotrophic conditions. The two other SNPs identified in the C. ljungdahlii OTA1 genome were in genes coding for transcriptional regulators (CLJU_c09320 and CLJU_c18110) and were found to be responsible for deregulated expression of co-localized arginine catabolism and 2-deoxy-D-ribose catabolism genes. Growth medium supplementation experiments suggested that increased arginine metabolism and 2-deoxy-D-ribose were likely to have minor effects on biomass and fermentation product yields. In addition, in silico flux balance analysis simulating mixotrophic and heterotrophic conditions showed no change in flux to ethanol when flux through HemL was changed whereas limited flux through AcsA increased the ethanol flux for both simulations. In characterizing the effects of the SNPs identified in the C. ljungdahlii OTA1 genome, a non-autotrophic hyper ethanol-producing strain of C. ljungdahlii was identified that has utility for further physiology and strain performance studies and as a biocatalyst for industrial applications.

A novel N-terminal region of the membrane ß-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris.

The ß-hexosyltransferase (BHT) from Sporobolomyces singularis is a membrane-bound enzyme that catalyses transgalactosylation reactions to synthesize galacto-oligosaccharides (GOSs). To increase the secretion of the active soluble version of this protein, we examined the uncharacterized novel N-terminal region (amino acids 1-110), which included two predicted endogenous structural domains. The first domain (amino acids 1-22) may act as a classical leader while a non-classical signal was located within the remaining region (amino acids 23-110). A functional analysis of these domains was performed by evaluating the amounts of the rBHT forms secreted by recombinant P. pastoris strains carrying combinations of the predicted structural domains and the α mating factor (MFα) from Saccharomyces cerevisiae as positive control. Upon replacement of the leader domain (amino acids 1-22) by MFα (MFα-rBht(23-594)), protein secretion increased and activity of both soluble and membrane-bound enzymes was improved 53- and 14-fold, respectively. Leader interference was demonstrated when MFα preceded the putative classical rBHT(1-22) leader (amino acids 1-22), explaining the limited secretion of soluble protein by P. pastoris (GS115 : : MFα-rBht(1-594)). To validate the role of the N-terminal domains in promoting protein secretion, we tested the domains using a non-secreted protein, the anti-ß-galactosidase single-chain variable antibody fragment scFv13R4. The recombinants carrying chimeras of the N-terminal 1-110 regions of rBHT preceding scFv13R4 correlated with the secretion strength of soluble protein observed with the rBHT recombinants. Finally, soluble bioactive HIS-tagged and non-tagged rBHT (purified to homogeneity) obtained from the most efficient recombinants (GS115 : : MFα-rBht(23-594)-HIS and GS115 : : MFα-rBht(23-594)) showed comparable activity rates of GOS generation.

Protons and pleomorphs: aerobic hydrogen production in Azotobacters.

As obligate aerobic soil organisms, the ability of Azotobacter species to fix nitrogen is unusual given that the nitrogenase complex requires a reduced cellular environment. Molecular hydrogen is an unavoidable byproduct of the reduction of dinitrogen; at least one molecule of H2 is produced for each molecule of N2 fixed. This could be considered a fault in nitrogenase efficiency, essentially a waste of energy and reducing equivalents. Wild-type Azotobacter captures this hydrogen and oxidizes it with its membrane-bound uptake hydrogenase complex. Strains lacking an active hydrogenase complex have been investigated for their hydrogen production capacities. What is the role of H2 in the energy metabolism of nitrogen-fixing Azotobacter? Is hydrogen production involved in Azotobacter species' protection from or tolerance to oxygen, or vice versa? What yields of hydrogen can be expected from hydrogen-evolving strains? Can the yield of hydrogen be controlled or increased by changing genetic, environmental, or physiological conditions? We will address these questions in the following mini-review.

Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6.

The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6's genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6's yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound.

Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052.

Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis d-glucose and d-fructose and only d-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l(-1) of butanol and 26.3 g l(-1) of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g(-1), respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in d-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the d-fructose phosphotransferase transport system and delayed sporulation.

Heterologous expression of a bioactive ß-hexosyltransferase, an enzyme producer of prebiotics, from Sporobolomyces singularis.

Galacto-oligosaccharides (GOS) are indigestible dietary fibers that are able to reach the lower gastrointestinal tract to be selectively fermented by health-promoting bacteria. In this report, we describe the heterologous expression of an optimized synthetically produced version of the ß-hexosyltransferase gene (Bht) from Sporobolomyces singularis. The Bht gene encodes a glycosyl hydrolase (EC 3.2.1.21) that acts as galactosyltransferase, able to catalyze a one-step conversion of lactose to GOS. Expression of the enzyme in Escherichia coli yielded an inactive insoluble protein, while the methylotrophic yeast Pichia pastoris GS115 produced a bioactive ß-hexosyltransferase (rBHT). The enzyme exhibited faster kinetics at pHs between 3.5 and 6 and at temperatures between 40 and 50°C. Enzyme stability improved at temperatures lower than 40°C, and glucose was found to be a competitive inhibitor of enzymatic activity. P. pastoris secreted a fraction of the bioactive rBHT into the fermentation broth, while the majority of the enzyme remained associated with the outer membrane. Both the secreted and the membrane-associated forms were able to efficiently convert lactose to GOS. Additionally, resting cells with membrane-bound enzyme converted 90% of the initial lactose into GOS at 68% yield (g/g) (the maximum theoretical is 75%) with no secondary residual (glucose or galactose) products. This is the first report of a bioactive BHT from S. singularis that has been heterologously expressed.

Structural analysis and functional evaluation of the disordered ß-hexosyltransferase region from Hamamotoa (Sporobolomyces) singularis.

Hamamotoa (Sporobolomyces) singularis codes for an industrially important membrane bound ß-hexosyltransferase (BHT), (BglA, UniprotKB: Q564N5) that has applications in the production of natural fibers such as galacto-oligosaccharides (GOS) and natural sugars found in human milk. When heterologously expressed by Komagataella phaffii GS115, BHT is found both membrane bound and soluble secreted into the culture medium. In silico structural predictions and crystal structures support a glycosylated homodimeric enzyme and the presence of an intrinsically disordered region (IDR) with membrane binding potential within its novel N-terminal region (1-110 amino acids). Additional in silico analysis showed that the IDR may not be essential for stable homodimerization. Thus, we performed progressive deletion analyses targeting segments within the suspected disordered region, to determine the N-terminal disorder region's impact on the ratio of membrane-bound to secreted soluble enzyme and its contribution to enzyme activity. The ratio of the soluble secreted to membrane-bound enzyme shifted from 40% to 53% after the disordered N-terminal region was completely removed, while the specific activity was unaffected. Furthermore, functional analysis of each glycosylation site found within the C-terminal domain revealed reduced total secreted protein activity by 58%-97% in both the presence and absence of the IDR, indicating that glycosylation at all four locations is required by the host for the secretion of active enzyme and independent of the removed disordered N-terminal region. Overall, the data provides evidence that the disordered region only partially influences the secretion and membrane localization of BHT.

Randomized placebo-controlled trial of feline-origin Enterococcus hirae probiotic effects on preventative health and fecal microbiota composition of fostered shelter kittens.

Introduction: Diarrhea is the second most common cause of mortality in shelter kittens. Studies examining prevention strategies in this population are lacking. Probiotics are of particular interest but studies in cats are largely limited to healthy adults or those with induced disease. Only one study in domestic cats describes the use of host-derived bacteria as a probiotic. We previously identified Enterococcus hirae as a dominant species colonizing the small intestinal mucosa in healthy shelter kittens. Oral administration of a probiotic formulation of kitten-origin E. hirae (strain 1002-2) mitigated the increase in intestinal permeability and fecal water loss resulting from experimental enteropathogenic E. coli infection in purpose-bred kittens. Based on these findings, we hypothesized that administration of kitten-origin E. hirae to weaned fostered shelter kittens could provide a measurable preventative health benefit. Methods: We conducted a randomized, placebo-controlled, blinded clinical trial to determine the impact of a freeze-dried E. hirae probiotic on body weight gain, incidence of diarrhea, carriage of potential diarrheal pathogens, and composition of the intestinal microbiota in weaned fostered shelter kittens. Results: One-hundred thirty kittens completed the study. Fifty-eight kittens received the probiotic and 72 received the placebo. There were no significant differences in age, weight upon initiation of the study, number of days in the study, average daily gain in body weight, or weight at completion of the study. Kittens treated with E. hirae were 3.4 times less likely to develop diarrhea compared to kittens treated with placebo (odds ratio = 0.294, 95% CI 0.109-0.792, p = 0.022). A significant impact of E. hirae was not observed on the presence or abundance of 30 different bacterial, viral, protozoal, fungal, algal, and parasitic agents in feces examined by qPCR. With exception to a decrease in Megamonas, administration of the E. hirae probiotic did not alter the predominant bacterial phyla present in feces based on 16S rRNA gene amplicon sequencing. Discussion: Decreased incidence of diarrhea associated with preventative administration of E. hirae to foster kittens supports a rationale for use of E. hirae for disease prevention in this young population at high risk for intestinal disease though additional studies are warranted.

The intestinal microbiota, gastrointestinal environment and colorectal cancer: a putative role for probiotics in prevention of colorectal cancer?

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States, and, even though 5-15% of the total CRC cases can be attributed to individual genetic predisposition, environmental factors could be considered major factors in susceptibility to CRC. Lifestyle factors increasing the risks of CRC include elevated body mass index, obesity, and reduced physical activity. Additionally, a number of dietary elements have been associated with higher or lower incidence of CRC. In this context, it has been suggested that diets high in fruit and low in meat might have a protective effect, reducing the incidence of colorectal adenomas by modulating the composition of the normal nonpathogenic commensal microbiota. In addition, it has been demonstrated that changes in abundance of taxonomic groups have a profound impact on the gastrointestinal physiology, and an increasing number of studies are proposing that the microbiota mediates the generation of dietary factors triggering colon cancer. High-throughput sequencing and molecular taxonomic technologies are rapidly filling the knowledge gaps left by conventional microbiology techniques to obtain a comprehensive catalog of the human intestinal microbiota and their associated metabolic repertoire. The information provided by these studies will be essential to identify agents capable of modulating the massive amount of gut bacteria in safe noninvasive manners to prevent CRC. Probiotics, defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (219), are capable of transient modulation of the microbiota, and their beneficial effects include reinforcement of the natural defense mechanisms and protection against gastrointestinal disorders. Probiotics have been successfully used to manage infant diarrhea, food allergies, and inflammatory bowel disease; hence, the purpose of this review was to examine probiotic metabolic activities that may have an effect on the prevention of CRC by scavenging toxic compounds or preventing their generation in situ. Additionally, a brief consideration is given to safety evaluation and production methods in the context of probiotics efficacy.

Meta-analysis and functional validation of nutritional requirements of solventogenic Clostridia growing under butanol stress conditions and coutilization of D-glucose and D-xylose.

Recent advances in systems biology, omics, and computational studies allow us to carry out data mining for improving biofuel production bioprocesses. Of particular interest are bioprocesses that center on microbial capabilities to biotransform both the hexose and pentose fractions present in crop residues. This called for a systematic exploration of the components of the media to obtain higher-density cultures and more-productive fermentation operations than are currently found. By using a meta-analysis approach of the transcriptional responses to butanol stress, we identified the nutritional requirements of solvent-tolerant strain Clostridium beijerinckii SA-1 (ATCC 35702). The nutritional requirements identified were later validated using the chemostat pulse-and-shift technique. C. beijerinckii SA-1 was cultivated in a two-stage single-feed-stream continuous production system to test the proposed validated medium formulation, and the coutilization of D-glucose and D-xylose was evaluated by taking advantage of the well-known ability of solventogenic clostridia to utilize a large variety of carbon sources such as mono-, oligo-, and polysaccharides containing pentose and hexose sugars. Our results indicated that C. beijerinckii SA-1 was able to coferment hexose/pentose sugar mixtures in the absence of a glucose repression effect. In addition, our analysis suggests that the solvent and acid resistance mechanisms found in this strain are differentially regulated compared to strain NRRL B-527 and are outlined as the basis of the analysis toward optimizing butanol production.

Safety and Modulatory Effects of Humanized Galacto-Oligosaccharides on the Gut Microbiome.

Complex dietary carbohydrate structures including ß(1-4) galacto-oligosaccharides (GOS) are resistant to digestion in the upper gastrointestinal (GI) tract and arrive intact to the colon where they benefit the host by selectively stimulating microbial growth. Studies have reported the beneficial impact of GOS (alone or in combination with other prebiotics) by serving as metabolic substrates for modulating the assembly of the infant gut microbiome while reducing GI infections. N-Acetyl-D-lactosamine (LacNAc, Galß1,4GlcNAc) is found in breast milk as a free disaccharide. This compound is also found as a component of human milk oligosaccharides (HMOs), which have repeating and variably branched lactose and/or LacNAc units, often attached to sialic acid and fucose monosaccharides. Human glycosyl-hydrolases do not degrade most HMOs, indicating that these structures have evolved as natural prebiotics to drive the proper assembly of the infant healthy gut microbiota. Here, we sought to develop a novel enzymatic method for generating LacNAc-enriched GOS, which we refer to as humanized GOS (hGOS). We showed that the membrane-bound ß-hexosyl transferase (rBHT) from Hamamotoa (Sporobolomyces) singularis was able to generate GOS and hGOS from lactose and N-Acetyl-glucosamine (GlcNAc). The enzyme catalyzed the regio-selective, repeated addition of galactose from lactose to GlcNAc forming the ß-galactosyl linkage at the 4-position of the GlcNAc and at the 1-position of D-galactose generating, in addition to GOS, LacNAc, and Galactosyl-LacNAc trisaccharides which were produced by two sequential transgalactosylations. Humanized GOS is chemically distinct from HMOs, and its effects in vivo have yet to be determined. Thus, we evaluated its safety and demonstrated the prebiotic's ability to modulate the gut microbiome in 6-week-old C57BL/6J mice. Longitudinal analysis of gut microbiome composition of stool samples collected from mice fed a diet containing hGOS for 5 weeks showed a transient reduction in alpha diversity. Differences in microbiome community composition mostly within the Firmicutes phylum were observed between hGOS and GOS, compared to control-fed animals. In sum, our study demonstrated the biological synthesis of hGOS, and signaled its safety and ability to modulate the gut microbiome in vivo, promoting the growth of beneficial microorganisms, including Bifidobacterium and Akkermansia.

The pleiotropic effects of prebiotic galacto-oligosaccharides on the aging gut.

BACKGROUND: Prebiotic galacto-oligosaccharides (GOS) have an extensively demonstrated beneficial impact on intestinal health. In this study, we determined the impact of GOS diets on hallmarks of gut aging: microbiome dysbiosis, inflammation, and intestinal barrier defects ("leaky gut"). We also evaluated if short-term GOS feeding influenced how the aging gut responded to antibiotic challenges in a mouse model of Clostridioides difficile infection. Finally, we assessed if colonic organoids could reproduce the GOS responder-non-responder phenotypes observed in vivo. RESULTS: Old animals had a distinct microbiome characterized by increased ratios of non-saccharolytic versus saccharolytic bacteria and, correspondingly, a lower abundance of ß-galactosidases compared to young animals. GOS reduced the overall diversity, increased the abundance of specific saccharolytic bacteria (species of Bacteroides and Lactobacillus), increased the abundance of ß-galactosidases in young and old animals, and increased the non-saccharolytic organisms; however, a robust, homogeneous bifidogenic effect was not observed. GOS reduced age-associated increased intestinal permeability and increased MUC2 expression and mucus thickness in old mice. Clyndamicin reduced the abundance Bifidobacterium while increasing Akkermansia, Clostridium, Coprococcus, Bacillus, Bacteroides, and Ruminococcus in old mice. The antibiotics were more impactful than GOS on modulating serum markers of inflammation. Higher serum levels of IL-17 and IL-6 were observed in control and GOS diets in the antibiotic groups, and within those groups, levels of IL-6 were higher in the GOS groups, regardless of age, and higher in the old compared to young animals in the control diet groups. RTqPCR revealed significantly increased gene expression of TNFα in distal colon tissue of old mice, which was decreased by the GOS diet. Colon transcriptomics analysis of mice fed GOS showed increased expression of genes involved in small-molecule metabolic processes and specifically the respirasome in old animals, which could indicate an increased oxidative metabolism and energetic efficiency. In young mice, GOS induced the expression of binding-related genes. The galectin gene Lgals1, a ß-galactosyl-binding lectin that bridges molecules by their sugar moieties and is an important modulator of the immune response, and the PI3K-Akt and ECM-receptor interaction pathways were also induced in young mice. Stools from mice exhibiting variable bifidogenic response to GOS injected into colon organoids in the presence of prebiotics reproduced the response and non-response phenotypes observed in vivo suggesting that the composition and functionality of the microbiota are the main contributors to the phenotype. CONCLUSIONS: Dietary GOS modulated homeostasis of the aging gut by promoting changes in microbiome composition and host gene expression, which was translated into decreased intestinal permeability and increased mucus production. Age was a determining factor on how prebiotics impacted the microbiome and expression of intestinal epithelial cells, especially apparent from the induction of galectin-1 in young but not old mice. Video abstract.

Role of antioxidant enzymes in bacterial resistance to organic acids.

Growth in aerobic environments has been shown to generate reactive oxygen species (ROS) and to cause oxidative stress in most organisms. Antioxidant enzymes (i.e., superoxide dismutases and hydroperoxidases) and DNA repair mechanisms provide protection against ROS. Acid stress has been shown to be associated with the induction of Mn superoxide dismutase (MnSOD) in Lactococcus lactis and Staphylococcus aureus. However, the relationship between acid stress and oxidative stress is not well understood. In the present study, we showed that mutations in the gene coding for MnSOD (sodA) increased the toxicity of lactic acid at pH 3.5 in Streptococcus thermophilus. The inclusion of the iron chelators 2,2'-dipyridyl (DIP), diethienetriamine-pentaacetic acid (DTPA), and O-phenanthroline (O-Phe) provided partial protection against 330 mM lactic acid at pH 3.5. The results suggested that acid stress triggers an iron-mediated oxidative stress that can be ameliorated by MnSOD and iron chelators. These findings were further validated in Escherichia coli strains lacking both MnSOD and iron SOD (FeSOD) but expressing a heterologous MnSOD from S. thermophilus. We also found that, in E. coli, FeSOD did not provide the same protection afforded by MnSOD and that hydroperoxidases are equally important in protecting the cells against acid stress. These findings may explain the ability of some microorganisms to survive better in acidified environments, as in acid foods, during fermentation and accumulation of lactic acid or during passage through the low pH of the stomach.

Cell immobilization for production of lactic acid biofilms do it naturally.

Interest in natural cell immobilization or biofilms for lactic acid fermentation has developed considerably over the last few decades. Many studies report the benefits associated with biofilms as industrial methods for food production and for wastewater treatment, since the formation represents a protective means of microbial growth offering survival advantages to cells in toxic environments. The formation of biofilms is a natural process in which microbial cells adsorb to a support without chemicals or polymers that entrap the cells and is dependent on the reactor environment, microorganism, and characteristics of the support. These unique characteristics enable biofilms to cause chronic infections, disease, food spoilage, and devastating effects as in microbial corrosion. Their distinct resistance to toxicity, high biomass potential, and improved stability over cells in suspension make biofilms a good tool for improving the industrial economics of biological lactic acid production. Lactic acid bacteria and specific filamentous fungi are the main sources of biological lactic acid. Over the past two decades, studies have focused on improving the lactic acid volumetric productivity through reactor design development, new support materials, and improvements in microbial production strains. To illustrate the operational designs applied to the natural immobilization of lactic acid producing microorganisms, this chapter presents the results of a search for optimum parameters and how they are affected by the physical, chemical, and biological variables of the process. We will place particular emphasis upon the relationship between lactic acid productivity attained by various types of reactors, supports, media formulations, and lactic acid producing microorganisms.

Draft Genome Sequence of Lactobacillus rhamnosus NCB 441, Isolated from Egyptian White Domiati Cheese.

Here, we report the draft genome sequence of Lactobacillus rhamnosus NCB 441, which was isolated from pickled white cheese samples gathered at Farafra Oasis in New Valley Governorate, Egypt. The genome size is 2,969,245 bp with a G+C content of 46.7%.

Accelerated Biodegradation of the Agrochemical Ametoctradin by Soil-Derived Microbial Consortia.

Pesticide-resistant plant pathogens are an increasing threat to the global food supply and have generated a need for novel, efficacious agrochemicals. The current regulatory process for approving new agrochemicals is a tedious but necessary process. One way to accelerate the safety evaluation process is to utilize in vitro systems to demonstrate pesticide degradation by soil microbes prior to ex vivo soil evaluations. This approach may have the capability to generate metabolic profiles free of inhibitory substances, such as humic acids, commonly present in ex vivo soil systems. In this study, we used a packed-bed microbial bioreactor to assess the role of the natural soil microbial community during biodegradation of the triazolopyrimidine fungicide, ametoctradin. Metabolite profiles produced during in vitro ametoctradin degradation were similar to the metabolite profiles obtained during environmental fate studies and demonstrated the degradation of 81% of the parent compound in 72 h compared to a half-life of 2 weeks when ametoctradin was left in the soil. The microbial communities of four different soil locations and the bioreactor microbiome were compared using high throughput sequencing. It was found that biodegradation of ametoctradin in both ex vivo soils and in vitro in the bioreactor correlated with an increase in the relative abundance of Burkholderiales, well characterized microbial degraders of xenobiotic compounds.

Influence of the intestinal microbiota on disease susceptibility in kittens with experimentally-induced carriage of atypical enteropathogenic Escherichia coli.

Typical enteropathogenic E. coli (tEPEC) carries the highest hazard of death in children with diarrhea and atypical EPEC (aEPEC) was recently identified as significantly associated with diarrheal mortality in kittens. In both children and kittens there is a significant association between aEPEC burden and diarrheal disease, however the infection can be found in individuals with and without diarrhea. It remains unclear to what extent, under what conditions, or by what mechanisms aEPEC serves as a primary pathogen in individuals with diarrhea. It seems likely that a combination of host and bacterial factors enable aEPEC to cause disease in some individuals and not in others. The purpose of this study was to determine the impact of aEPEC on intestinal function and diarrhea in kittens following experimentally-induced carriage and the influence of a disrupted intestinal microbiota on disease susceptibility. Results of this study identify aEPEC as a potential pathogen in kittens. In the absence of disruption to the intestinal microbiota, kittens are resistant to clinical signs of aEPEC carriage but demonstrate significant occult changes in intestinal absorption and permeability. Antibiotic-induced disruption of the intestinal microbiota prior to infection increases subsequent intestinal water loss as determined by % fecal wet weight. Enrichment of the intestinal microbiota with a commensal member of the feline mucosa-associated microbiota, Enterococcus hirae, ameliorated the effects of aEPEC experimental infection on intestinal function and water loss. These observations begin to unravel the mechanisms by which aEPEC infection may be able to exploit susceptible hosts.