Pure Culture Conditions Research Articles

Inactivation of Escherichia coli, Salmonella enterica, and Listeria monocytogenes using the Contamination Sanitization Inspection and Disinfection (CSI-D) device

The Contamination Sanitization Inspection and Disinfection (CSI-D) device is a handheld fluorescence-based imaging system designed to disinfect food contact surfaces using ultraviolet-C (UVC) illumination. This study aimed to determine the optimal CSI-D parameters (i.e., UVC exposure time and intensity) for the inactivation of the following foodborne bacteria plated on non-selective media: generic Escherichia coli (indicator organism) and the pathogens enterohemorrhagic E. coli, enterotoxigenic E. coli, Salmonella enterica, and Listeria monocytogenes. Each bacterial strain was spread-plated on non-selective agar and exposed to high-intensity (10 mW/cm2) or low-intensity (5 mW/cm2) UVC for 1–5 s. Control plates were not exposed to UVC. The plates were incubated overnight at 37 °C and then enumerated. Three trials for each bacterial strain were conducted. Statistical analysis was carried out to determine if there were significant differences in bacterial growth between UVC intensities and exposure times. Overall, exposure to low or high intensity for 3–5 s resulted in consistent inhibition of bacterial growth, with reductions of 99.9–100 % for E. coli, 96.8–100 % for S. enterica, and 99.2–100 % for L. monocytogenes. The 1 s exposure time showed inconsistent results, with a 66.0–100 % reduction in growth depending on the intensity and bacterial strain. When the results for all strains within each species were combined, the 3–5 s exposure times showed significantly greater (p < 0.05) growth inhibition than the 1 s exposure time. However, there were no significant differences (p > 0.05) in growth inhibition between the high and low UVC intensities. The results of this study show that, in pure culture conditions, exposure to UVC with the CSI-D device for ≥3 s is required to achieve consistent reduction of E. coli, S. enterica, and L. monocytogenes.

Effects of Graphene-Based Nanomaterials on Microorganisms and Soil Microbial Communities.

The past decades have witnessed intensive research on the biological effects of graphene-based nanomaterials (GBNs) and the application of GBNs in different fields. The published literature shows that GBNs exhibit inhibitory effects on almost all microorganisms under pure culture conditions, and that this inhibitory effect is influenced by the microbial species, the GBN's physicochemical properties, the GBN's concentration, treatment time, and experimental surroundings. In addition, microorganisms exist in the soil in the form of microbial communities. Considering the complex interactions between different soil components, different microbial communities, and GBNs in the soil environment, the effects of GBNs on soil microbial communities are undoubtedly intertwined. Since bacteria and fungi are major players in terrestrial biogeochemistry, this review focuses on the antibacterial and antifungal performance of GBNs, their antimicrobial mechanisms and influencing factors, as well as the impact of this effect on soil microbial communities. This review will provide a better understanding of the effects of GBNs on microorganisms at both the individual and population scales, thus providing an ecologically safe reference for the release of GBNs to different soil environments.

Morphogenesis and metabolomics reveal the compatible relationship among Suillus bovinus, Phialocephala fortinii, and their co-host, Pinus massoniana.

Ectomycorrhizal (ECM) fungi and dark septate endophytes (DSEs) can both form a symbiotic relationship with the same host plant. However, the interactions that occur among these two types of fungi and their co-hosts are largely unknown. Here, we investigated interactions that occur among the ECM fungus Suillus bovinus, the DSE Phialocephala fortinii, and their co-host Pinus massoniana. We used both scanning electron microscopy and optical microscopy to characterize the morphogenesis of the two symbionts and employed the ultra-high-performance liquid chromatography-tandem mass spectrometry technique to assess the effects of fungal inoculation on the root metabolome. Under pure culture conditions, no synergistic or antagonistic effects were observed between Phi. fortinii and S. bovinus. Generally, S. bovinus and Phi. fortinii can simultaneously colonize P. massoniana roots without affecting each other's symbiotic processes. S. bovinus can colonize the root locus where Phi. fortinii has already invaded but not vice versa, which may be due to the physical barrier effect of the mantle. Both fungi can significantly promote the growth of P. massoniana, and they have a synergistic effect on host N and K uptake. Metabolite accumulation patterns in roots inoculated with Phi. fortinii and/or S. bovinus were greatly altered, especially with respect to organic acids, flavonoids, lipids, and phenolic acids. S. bovinus inoculation significantly enhanced root flavonoid biosynthesis, whereas Phi. fortinii and dual-inoculation treatments mainly induced phenylpropanoid biosynthesis. These findings reveal compatible relationships among P. massoniana, S. bovinus, and Phi. fortinii, and suggest a theoretical basis for ECM fungi and DSE co-application when cultivating seedlings. IMPORTANCE The prevalence of both ectomycorrhizal fungi and dark septate endophytes in the roots of a wide spectrum of tree species is well recognized. In this study, we investigated the interactions that occur among the ECM fungus S. bovinus, the DSE Phi. fortinii, and their co-host, P. massoniana. The two fungi can simultaneously colonize P. massoniana roots without affecting each other's symbiotic processes. S. bovinus appears to be superior to Phi. fortinii in microniche competition, which may be due to the physical barrier effect of the mantle. The two fungi have different effects on root metabolite accumulation patterns. S. bovinus inoculation significantly enhanced root flavonoid biosynthesis, whereas Phi. fortinii and dual-inoculation treatments mainly induced phenylpropanoid biosynthesis. This is the first study revealing the morphological and metabolic mechanisms that contribute to the compatible relationship among ECM fungi, DSEs, and their co-host.

Preliminary exploration on the ectomycorrhizal status of a wild edible Gomphus species from Southwest China.

A wild edible Gomphus species was discovered at local wild mushroom markets from May to November in Southwest China, where it was eaten for hundreds of years. However, litter information on the taxonomy is available. Whether Gomphus is a saprotrophic, parasitic, or ectomycorrhizal (ECM) fungus is unclear. In the present study, field investigation, fungi isolation, optimum medium, morphological description, molecular analyses, and preliminary exploration on mycorrhizal synthesis were carried out. The morphological and molecular analyses showed that the same species between Gomphus matijun and Gomphus sp. (zituoluo) might be the related species of Gomphus purpuraceus. Moreover, the root dry weight and first-lateral root number of inoculated seedlings were significantly enhanced by evaluating Pinus massoniana seedlings inoculated with G. matijun. Meanwhile, the levels of nine phytohormones, including five new phytohormones, in the roots of inoculated seedlings were upregulated. This study explored the mycorrhizal synthesis of the wild edible Gomphus species from Southwest China with P. massoniana Lamb. We concluded that G. matijun might be an ECM fungus. The mycorrhizal synthesis of G. matijun under pure culture conditions provided the basis for the next inoculation under controlled soil conditions, making the conservation and cultivation of G. matijun feasible in the future.

Non-Conventional Yeast: Behavior under Pure Culture, Sequential and Aeration Conditions in Beer Fermentation.

The use of wild yeasts, isolated from different environments, is becoming the most interesting option for the production of new beers. The objective of this study is to evaluate the potential of seven non-conventional yeast strains from five different species (Saccharomyces cerevisiae, Hanseniaspora guilliermondii, Metschnikowia pulcherrima, Torulaspora delbrueckii, and Zygosaccharomyces bailii) isolated from Madrid agriculture to produce type ale beer. Wild yeast strains were evaluated at laboratory and pilot plant scales under different fermentation conditions (pure, aerated, and sequential culture). Strain S. cerevisiae SafAle S-04 was used as a reference. Throughout the fermentation of beer, volatile compounds were determined by GC and residual sugars by HPLC, among other parameters. The yeast strains used for the fermentation in pure culture conditions were unable to ferment maltose and maltotriose (0.73-1.18% v/v of ethanol). The results of the study under aerated conditions showed varying levels of higher alcohol and ester concentrations. It should be noted that the strain CLI 1057 (S. cerevisiae) fermented maltose in the presence of oxygen (Kluyver effect). This strain also showed a high production of 4-vinyl guaiacol, making it suitable for producing beers with a phenolic profile. Finally, three strains (H. guilliermondii, Z. bailii, and T. delbrueckii) were evaluated in sequential culture together with commercial strain and found to improve the organoleptic characteristics of the brewed beer. These approaches offer the opportunity to add new product characteristics to the beers.

The Widespread Use of Nanomaterials: The Effects on the Function and Diversity of Environmental Microbial Communities.

In recent years, as an emerging material, nanomaterials have rapidly expanded from laboratories to large-scale industrial productions. Along with people’s productive activities, these nanomaterials can enter the natural environment of soil, water and atmosphere through various ways. At present, a large number of reports have proved that nanomaterials have certain toxic effects on bacteria, algae, plants, invertebrates, mammalian cell lines and mammals in these environments, but people still know little about the ecotoxicology of nanomaterials. Most relevant studies focus on the responses of model strains to nanomaterials in pure culture conditions, but these results do not fully represent the response of microbial communities to nanomaterials in natural environments. Over the years, the effect of nanomaterials infiltrated into the natural environment on the microbial communities has become a popular topic in the field of nano-ecological environment research. It was found that under different environmental conditions, nanomaterials have various effects on the microbial communities. The medium; the coexisting pollutants in the environment and the structure, particle size and surface modification of nanomaterials may cause changes in the structure and function of microbial communities. This paper systematically summarizes the impacts of different nanomaterials on microbial communities in various environments, which can provide a reference for us to evaluate the impacts of nanomaterials released into the environment on the microecology and has certain guiding significance for strengthening the emission control of nanomaterials pollutants.

Exploring AI-2-mediated interspecies communications within rumen microbial communities

BackgroundThe rumen is an ecosystem with a complex microbial microflora in which microbes initiate biofilm formation by attaching to plant surfaces for plant degradation and are capable of converting feed to nutrients and energy via microbial processes. Quorum sensing (QS) is a cell-to-cell communication mechanism that allows microbes to synchronize the expression of multiple genes in the group to perform social behaviors such as chemotaxis and biofilm formation using self-synthesized QS signaling molecules. Whereas QS has been extensively studied in model microorganisms under pure culture conditions, QS mechanisms are poorly understood in complex bacterial communities, such as the rumen microflora, in which cell-to-cell communication may be common.ResultsHere, we analyzed 981 rumens bacterial and archaeal genomes from the Joint Genome Institute (JGI) and GenBank databases and identified 15 types of known QS signaling molecule-related genes. The analysis of the prevalence and abundance of genes involved in QS showed that 767 microbial genomes appeared to possess QS-related genes, including 680 bacterial genomes containing autoinducer-2 (AI-2) synthase- or receptor-encoding genes. Prevotella, Butyivibrio, Ruminococcus, Oribacterium, Selenomonas, and Treponema, known abundant bacterial genera in the rumen, possessed the greatest numbers of AI-2-related genes; these genes were highly expressed within the metatranscriptome dataset, suggesting that intra- and interspecies communication mediated by AI-2 among rumen microbes was universal in the rumen. The QS processes mediated by the dCache_1-containing AI-2 receptors (CahRs) with various functional modules may be essential for degrading plants, digesting food, and providing energy and nutrients to the host. Additionally, a universal natural network based on QS revealed how rumen microbes coordinate social behaviors via the AI-2-mediated QS system, most of which may potentially function via AI-2 binding to the extracellular sensor dCache_1 domain to activate corresponding receptors involved in different signal transduction pathways, such as methyl-accepting chemotaxis proteins, histidine kinases, serine phosphatases, c-di-GMP synthases and phosphodiesterases, and serine/threonine kinases in the rumen.ConclusionsThe exploration of AI-2-related genes, especially CahR-type AI-2 receptors, greatly increased our insight into AI-2 as a potentially “universal” signal mediating social behaviors and will help us better understand microbial communication networks and the function of QS in plant-microbe interactions in complex microecosystems.DGdc6xosz2q-6EKgKy6aZ-Video

Occurrence, hyphal growth rate, and carbon source utilization of fungi from continental Antarctica

Reported data on the diversity and physiological capabilities of fungi inhabiting ice-free outcrops of continental Antarctica are limited. The present study aimed at investigating the occurrence of fungi on fragments of microbial mats on the lakeshore and lake water and compared it with fungal occurrence on terrestrial moss, using a culture-dependent method. Another purpose was to evaluate the responses of hyphal growth to temperature and the profiles of substrate utilization of fungal isolates under pure culture conditions. A total of 46 fungal isolates were obtained and grouped into eight taxa, with Phoma herbarum being the most frequent taxon, the occurrence of which was closely related to the nitrogen level of the substrata. The colony diameter growth rates of most fungal isolates under pure culture conditions increased linearly with temperature to reach the maximum at 20–25 °C, indicating that these fungi were mesophilic. Moreover, fungal isolates exhibited diversity and redundancy in their utilization of 71 carbon sources, having potentials to utilize an array of substrates including sugars, sugar alcohols, hexose-phosphates, amino acids, hexose acids, and carboxylic acids.

Antibiotic-accelerated cyanobacterial growth and aquatic community succession towards the formation of cyanobacterial bloom in eutrophic lake water

Antibiotics can stimulate the growth of model cyanobacterial species under pure culture conditions, but their influence on cyanobacterial blooms in natural aquatic ecosystems remains unclear. In this study, three commonly detected antibiotics (sulfamethoxazole, tetracycline, and ciprofloxacin) and their ternary mixture were proved to selectively stimulate (p < 0.05) the growth and photosynthetic activity of cyanobacteria in an aquatic microcosm at an environmentally relevant exposure dose of 300 ng/L under both oligotrophic and eutrophic conditions. Under the eutrophic condition, cyanobacteria reached a bloom density of 1.61 × 106 cells/mL in 15 days without antibiotics, while the cyanobacteria exposed to tetracycline, sulfamethoxazole, ciprofloxacin, and their ternary mixture exceeded this bloom density within only 10, 8, 7, and 6 days, respectively. Principal coordinate analysis indicated that the antibiotic contaminants accelerated the prokaryotic community succession towards the formation of a cyanobacterial bloom by promoting the dominance of Microcystis, Synechococcus, and Oscillatoria under the eutrophic condition. After 15 days of culture, the antibiotic exposure increased the density of cyanobacteria by 1.38–2.31-fold and 2.28–3.94-fold under eutrophic and oligotrophic conditions, respectively. Antibiotic exposure generated higher stimulatory effects on cyanobacterial growth under the oligotrophic condition, but the antibiotic(s)-treated cyanobacteria did not form a bloom due to nutrient limitation. Redundancy analysis indicated that the three target antibiotics and their ternary mixture affected the prokaryotic community structure in a similar manner, while tetracycline showed some differences compared to sulfamethoxazole, ciprofloxacin, and the ternary antibiotic mixture with regard to the regulation of the eukaryotic community structure. This study demonstrates that antibiotic contaminants accelerate the formation of cyanobacterial blooms in eutrophic lake water and provides insights into the ecological effects of antibiotics on aquatic microbial communities.

Cartap hydrochloride induced stress response in Anabaena variabilis ARM 441

Cartap hydrochloride is a moderately hazardous nereistoxin analogue insecticide that is predominantly applied in paddy fields of India, at a recommended dose of 10 μg ml−1 to kill chewing and sucking insect pests of rice crop. Toxicity of cartap hydrochloride was studied on non-target free-living nitrogen fixing cyanobacterium Anabaena variabilis ARM 441 commonly used as algal biofertilizer in rice cultivation. Anabaena sp. could tolerate commercial grade insecticide up to 30 μg ml−1. However, at the recommended dose of 10 μg ml−1, it caused reduction in algal growth, total nitrogen and heterocyst frequency by 47.28, 24.29 and 17.72% respectively, as well as photosynthetic pigments under pure culture conditions. Scanning electron micrographs revealed cell rupture and breakage in filaments due to cartap exposure with the formation of akinetes. Cartap hydrochloride induced stress, since level of superoxide dismutase, peroxidase and catalase were increased by 108.57, 187.5 and 117% respectively. Generation of superoxide radicals and hydrogen peroxide were also increased by 152.48 and 34% respectively. Lipid peroxidation was increased by 31.03%, whereas there was decline in ascorbate content by 48.45%, however the glutathione content was increased by 128.57%. Increase in osmolytes such as proline from 8.6 to 32.8% and sucrose from 61.22 to 90.13% indicates their possible role in overcoming cartap induced oxidative stress and can be helpful in assessing its detrimental effect on Anabaena variabilis ARM 441, since cyanobacterial biofertilizers are purposely used in paddy fields as nitrogen contributors.

Selenium-oxidizing Agrobacterium sp. T3F4 steadily colonizes in soil promoting selenium uptake by pak choi (Brassica campestris)

Selenium (Se) deficiency in soil is linked to its low content in edible crops, resulting in adverse impacts on the health of 15% of the global population. The crop mainly absorbs oxidized selenate and selenite from soil, then converts them into organic Se. However, the role of Se-oxidizing bacteria in soil Se oxidation, Se bioavailability and Se absorption into plants remains unclear. The strain Agrobacterium sp. T3F4, isolated from seleniferous soil, was able to oxidize elemental Se into selenite under pure culture conditions. The green fluorescent protein (gfp)-gene-marked strain (T3F4-GFP) and elemental Se or selenite (5 mg·kg−1) were added to pak choi (Brassica campestris ssp. chinensis) pot cultures. Observation of the fluorescence and viable counting indicated that GFP-expressing bacterial cells steadily colonized the soil in the pots and the leaves of the pak choi, reaching up to 6.6 × 106 and 2.0 × 105 CFU g−1 at 21 days post cultivation, respectively. Moreover, the total Se content (mostly organic Se) was significantly increased in the pak choi under T3F4 inoculated pot culture, with elemental Se(0) being oxidized into Se(IV), and soil Se(IV) being dissolved before being absorbed by the crop. After strain T3F4 was inoculated, no significant differences in microbial diversity were observed in the soils and roots, whereas the abundance of Rhizobium spp. was significantly increased. To our knowledge, this is the first time that Se-oxidizing Agrobacterium sp. T3F4 has been found to steadily colonize soil and plant tissues, and that its addition to soil increases the absorption of Se in plants. This study provides a potential strategy for Se biofortification.

Effects of Co-culture on Improved Productivity and Bioresource for Microalgal Biomass Using the Floc-Forming Bacteria Melaminivora Jejuensis

Bacterial and algal floc formation was induced by inoculating three species of wastewater-derived bacteria (Melaminivora jejuensis, Comamonas flocculans, and Escherichia coli) into algal cultures (Chlorella sorokiniana). Bacterial and algal flocs formed in algal cultures inoculated with M. jejuensis and C. flocculans, and these flocs showed higher sedimentation rates than pure algal culture. The floc formed by M. jejuensis (4988.46 ± 2589.81 μm) was 10-fold larger than the floc formed by C. flocculans (488.60 ± 226.22 μm), with a three-fold higher sedimentation rate (M. jejuensis, 91.08 ± 2.32% and C. flocculans, 32.55 ± 6.33%). Biomass and lipid productivity were improved with M. jejuensis inoculation [biomass, 102.25 ± 0.35 mg/(L·day) and 57.80 ± 0.20 mg/(L·day)] compared with the productivity obtained under pure algal culture conditions [biomass, 78.00 ± 3.89 mg/(L·day) and lipids, 42.26 ± 2.11 mg/(L·day)]. Furthermore, the fatty acid composition of the biomass produced under pure algal culture conditions was mainly composed of C16:0 (43.67%) and C18:2 (45.99%), whereas the fatty acid composition of the biomass produced by M. jejuensis was mainly C16:0 (31.80%), C16:1 (24.45%), C18:1 (20.23%), and C18:2 (16.11%). These results suggest the possibility of developing an efficient method for harvesting microalgae using M. jejuensis and provide information on how to improve biomass productivity using floc-forming bacteria.

Mapping the intracellular metabolome of yeast biocapsules - Spherical structures of yeast attached to fungal pellets

Co-culture conditions are beneficial for study due to the advances which arise from symbiotic interactions and which cannot be replicated under pure culture conditions. Here, the focus is on the connection between two fungi – a yeast, Saccharomyces cerevisiae, and a filamentous fungus, Penicillium chrysogenum – in a yeast immobilization system termed’ yeast biocapsules’, where the yeast and filamentous fungus are strongly attached to one another, forming spherical structures. This co-culture condition hinders filamentous fungal biomass growth, while immobilization of yeast cells continues to increase. The effect of the co-culture condition on endometabolites or intracellular metabolites were tracked during the beginning and end of the yeast biocapsule formation period, and metabolites analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD). Distinct metabolite profiles were found between single culture conditions, involving each organism separately, and with the co-culture condition, where there were differences in 54 endometabolites. Specifically, co-culture condition compounds such as fructose, glycolic acid and glyceric acid were present in higher concentrations at the end of biocapsule formation. These results shed light on the mechanisms and biochemical impact of the interaction between the yeast and filamentous fungus and serve as a basis to apply and further develop yeast biocapsules as a new biotechnological tool with benefits for industry.

Effects of copper on nitrous oxide (N2O) reduction in denitrifiers and N2O emissions from agricultural soils

Biochemical reduction of nitrous oxide (N2O) to dinitrogen (N2) by N2O reductase (N2OR) is the only known sink to consume N2O. Copper (Cu) and pH are two key factors determining the activity of the enzyme N2OR. We hypothesized that changes in the Cu level could enhance the reduction of N2O to N2 in denitrifier strains and decrease the N2O emissions from agricultural soils. To test this hypothesis, Cu-modified culture medium was applied to denitrifier strains, and Cu-modified organic fertilizer was applied to both soil microcosms and fields. Of 46 denitrifier strains, 25 showed higher denitrifying activities and 30N2/(46N2O + 30N2) after the addition of Cu under pure culture conditions. Among 10 genera, Azospirillum and Herbaspirillum were the most responsive to the Cu level changes. The N2O flux was significantly reduced 4 or 8 days onwards after the application of 130 mM CuSO4-modified organic fertilizer (vol:wt = 1:1) into Andosol or Fluvisol, respectively, under soil microcosm conditions. In addition, the cumulative N2O emissions were significantly reduced after the application of 130 mM CuSO4-modified organic fertilizer. They were moderately reduced after the application of 130 mM CuSO4-modified organic fertilizer (vol:wt = 1:1) into a Fluvisol field. They were significantly lower in Azospirillum sp. UNPF1-inoculated soils after the application of 130 mM CuSO4-modified organic fertilizer when compared with that in dual non-inoculated and unmodified soils. Soils inoculated with Herbaspirillum sp. UKPF54 showed results similar to non-inoculated Fluvisol fields. These results suggest that Cu may enhance N2O conversion to N2 in denitrifiers and that Cu-modified organic fertilizer may enhance N2O consumption or decrease N2O emissions in agricultural soils.

Fungal endophyte Phomopsis liquidambari biodegrades soil resveratrol: a potential allelochemical in peanut monocropping systems

Most allelochemicals are secondary products released from root excretions or plant residues that accumulate in continuous cropping systems and cause severe decline in peanut yield. Resveratrol is a plant-derived stilbene that is released from peanut residues and accumulates in the soil; however, its allelopathic effects on peanut production are overlooked. Effective management solutions need to be developed to relieve allelopathy caused by soil resveratrol. Here, the biodegradation of resveratrol by the fungal endophyte Phomopsis liquidambari was investigated in a mineral salt medium and a soil trial. Resveratrol and its metabolites (produced by degradation by P. liquidambari) were detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Resveratrol released from peanut residues reached a maximum concentration of 0.18 μg g-1 soil in litterbag experiments. Exogenous resveratrol inhibited peanut growth, nodule formation, and soil dehydrogenase activity, and reduced the soil microbial biomass carbon content and bacterial abundance, indicating an allelopathic role in peanut growth. More than 97% of the resveratrol was degraded within 72 and 168 h by P. liquidambari in pure culture and soil conditions, respectively. Resveratrol was first cleaved to 3,5-dihydroxybenzaldehyde and 4-hydroxybenzaldehyde, which were subsequently oxidized into 3,5-dihydroxybenzoic acid and 4-hydroxybenzoic acid, respectively. Fungal resveratrol cleavage oxygenase and the related gene expression were enhanced when P. liquidambari was induced by the resveratrol during the incubation. Our results indicate that the practical application of the fungal endophyte P. liquidambari has strong potential for biodegrading soil resveratrol, which can cause allelopathy in peanut continuous cropping systems. © 2019 Society of Chemical Industry.

CO2 conversion to methane and biomass in obligate methylotrophic methanogens in marine sediments

Methyl substrates are important compounds for methanogenesis in marine sediments but diversity and carbon utilization by methylotrophic methanogenic archaea have not been clarified. Here, we demonstrate that RNA-stable isotope probing (SIP) requires 13C-labeled bicarbonate as co-substrate for identification of methylotrophic methanogens in sediment samples of the Helgoland mud area, North Sea. Using lipid-SIP, we found that methylotrophic methanogens incorporate 60–86% of dissolved inorganic carbon (DIC) into lipids, and thus considerably more than what can be predicted from known metabolic pathways (~40% contribution). In slurry experiments amended with the marine methylotroph Methanococcoides methylutens, up to 12% of methane was produced from CO2, indicating that CO2-dependent methanogenesis is an alternative methanogenic pathway and suggesting that obligate methylotrophic methanogens grow in fact mixotrophically on methyl compounds and DIC. Although methane formation from methanol is the primary pathway of methanogenesis, the observed high DIC incorporation into lipids is likely linked to CO2-dependent methanogenesis, which was triggered when methane production rates were low. Since methylotrophic methanogenesis rates are much lower in marine sediments than under optimal conditions in pure culture, CO2 conversion to methane is an important but previously overlooked methanogenic process in sediments for methylotrophic methanogens.

Construction of a Geobacter Strain With Exceptional Growth on Cathodes.

Insoluble extracellular electron donors are important sources of energy for anaerobic respiration in biogeochemical cycling and in diverse practical applications. The previous lack of a genetically tractable model microorganism that could be grown to high densities under anaerobic conditions in pure culture with an insoluble extracellular electron donor has stymied efforts to better understand this form of respiration. We report here on the design of a strain of Geobacter sulfurreducens, designated strain ACL, which grows as thick (ca. 35 μm) confluent biofilms on graphite cathodes poised at -500 mV (versus Ag/AgCl) with fumarate as the electron acceptor. Sustained maximum current consumption rates were >0.8 A/m2, which is >10-fold higher than the current consumption of the wild-type strain. The improved function on the cathode was achieved by introducing genes for an ATP-dependent citrate lyase, completing the complement of enzymes needed for a reverse TCA cycle for the synthesis of biosynthetic precursors from carbon dioxide. Strain ACL provides an important model organism for elucidating the mechanisms for effective anaerobic growth with an insoluble extracellular electron donor and may offer unique possibilities as a chassis for the introduction of synthetic metabolic pathways for the production of commodities with electrons derived from electrodes.

Metabolic switching of sake yeast by kimoto lactic acid bacteria through the [GAR+] non-genetic element

In traditional kimoto-type sake production, cells of Saccharomyces cerevisiae sake yeast are grown in a starter mash generated by lactate fermentation by lactic acid bacteria (LAB) such as Leuconostoc mesenteroides and Lactobacillus sakei. However, the microbial interactions between sake yeast and kimoto LAB have not been well analyzed. Since the formation of a prion-like element (designated [GAR+]) in yeast cells is promoted by bacteria, we here examined the associated phenotype (i.e., increased glucosamine resistance) in sake yeast strains K701 (a representative sake strain) and Km67 (a strain isolated from kimoto-type sake mash). Approximately 0.5% of K701 and Km67cells, as well as 0.2% of laboratory strain X2180cells, exhibited increased glucosamine resistance under pure culture conditions, and the frequency of this metabolic switching was further enhanced by coculture with kimoto LAB. The LAB-promoted emergence of the glucosamine-resistant cells was the most prominent in Km67, suggesting that this strain possesses an advanced mechanism for response to LAB. While the glucosamine-resistant clones of X2180 and K701 exhibited lower rates of alcoholic fermentation under high-glucose conditions than did the respective naive strains, glucosamine resistance did not severely affect alcoholic fermentation in Km67. The population of dead cells after alcoholic fermentation was decreased in the glucosamine-resistant clones of X2180, K701, and Km67. These results suggested that the formation of [GAR+] in Km67 may be beneficial in kimoto-type sake making, since [GAR+] may increase cell viability in the sake starter mash without impairing alcoholic fermentation performance.

Effect of maize root exudates on indole-3-acetic acid production by rice endophytic bacteria under influence of L-tryptophan

Background: It is assumed that plant growth regulators produced by beneficial bacterial species could also influence plant growth. IAA is a major plant growth regulator responsible for stimulation of plant growth. There are several microorganisms which are naturally responsible for L- tryptophan metabolism. Methods: In total, 56 indigenous morphologically distinct isolates from rice roots were selected and subsequently characterized with biochemical tests, 16S rRNA sequencing and plant growth promoting activities. Pseudomonas fluorescens RE1 (GenBank: MF102882.1) and RE17 (GenBank: MF103672.1) endophytes resulted in better PGP activity against the other 54 isolates. Both endophytes were tested to screen indole-3-acetic acid production ability in pure culture conditions with L-tryptophan at 0, 50, 100, 200 and 500µg/ml concentrations. Results: P. fluorescens RE1 was recorded efficient for indole production in comparison to P. fluorescens RE17 at various L-tryptophan concentrations. P. fluorescens RE1 was shown to produce between 0.8 µg/ml and 11.5µg/ml of indole at various tryptophan concentrations, while RE17 produced between 1.2µg/ml and 10.2µg/ml. At 200 and 500µg/ml tryptophan concentration, P. fluorescens RE17 produced 7.4pmol/ml and 9.3pmol/ml IAA, respectively. Conclusions: Inoculation of maize seed with P. fluorescens RE1 and RE17 showed a significantly higher level of IAA production in comparison to non-inoculated seeds. Current study outcomes proved that plant growth regulators produced by Pseudomonas species could also play a critical role in plant growth promotion.

Effect of maize root exudates on indole-3-acetic acid production by rice endophytic bacteria under influence of L-tryptophan.

Background: It is assumed that plant growth regulators produced by beneficial bacterial species could also influence plant growth. IAA is a major plant growth regulator responsible for stimulation of plant growth. There are several microorganisms which are naturally responsible for L- tryptophan metabolism. Methods: In total, 56 indigenous morphologically distinct isolates from rice roots were selected and subsequently characterized with biochemical tests, 16S rRNA sequencing and plant growth promoting activities. Pseudomonasfluorescens RE1 (GenBank: MF102882.1) and RE17 (GenBank: MF103672.1) endophytes resulted in better PGP activity against the other 54 isolates. Both endophytes were tested to screen indole-3-acetic acid production ability in pure culture conditions with L-tryptophan at 0, 50, 100, 200 and 500µg/ml concentrations. Results: P.fluorescens RE1 was recorded efficient for indole production in comparison to P. fluorescens RE17 at various L-tryptophan concentrations. P. fluorescens RE1 was shown to produce between 0.8 µg/ml and 11.5µg/ml of indole at various tryptophan concentrations, while RE17 produced between 1.2µg/ml and 10.2µg/ml. At 200 and 500µg/ml tryptophan concentration, P. fluorescens RE17 produced 7.4pmol/ml and 9.3pmol/ml IAA, respectively. Conclusions: Inoculation of maize seed with P. fluorescens RE1 and RE17 showed a significantly higher level of IAA production in comparison to non-inoculated seeds. Current study outcomes proved that plant growth regulators produced by Pseudomonas species could also play a critical role in plant growth promotion.