In Ganoderma lucidum, Glsnf1 regulates cellulose degradation by inhibiting GlCreA during the utilization of cellulose.

Cellulose is a by-product of agricultural production and an abundant waste. As a carbon source, cellulose can be degraded and utilized by fungi. Carbon sources, which act as nutrients, not only provide energy but also serve as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Nutrient-sensing pathways prioritize the use of preferred carbon sources and regulate the production of cellulose-degrading enzymes when necessary. Understanding the regulation of the fungal cellulolytic response will become increasingly important because we strive to increase the efficiency of the utilization of these renewable energy sources. Here, we show that Glsnf1, a sucrose-nonfermenting serine-threonine-protein kinase 1 (Snf1)/AMP-activated protein kinase homologue in medicinal macro basidiomycete Ganoderma lucidum, actively responds to carbon alterations and positively regulates cellulase activity and cellulase-related gene transcription. The carbon catabolite repressor CreA, a zinc binuclear cluster transcription factor that mediates the sensing of nutrients and suppression of the transcription of a number of genes necessary for the consumption of a less preferred carbon source, participates in the Glsnf1-mediated regulation of cellulases. Glsnf1 not only negatively regulates the transcription level of the CreA gene but also hinders its localization in the nucleus. Overall, our findings reveal a key nutrient-sensing mechanism that is critical for the modulation of carbon source adaptation in G. lucidum.

Black carbon emissions in gasoline vehicle exhaust: a measurement and instrument comparison.

UNLABELLED: A pilot study was conducted to evaluate the performance and agreement of several commercially available black carbon (BC) measurement instruments, when applied to the quantification of BC in light-duty vehicle (LDV) exhaust. Samples from six vehicles, three fuels, and three driving cycles were used. The pilot study included determinations of the method detection limit (MDL) and repeatability. With respect to the MDL, the real-time instruments outperformed the time-integrated instruments, with MDL = 0.12 mg/mi for the AE51 Aethalometer, and 0.15 mg/mi for the Micro Soot Sensor (MSS), versus 0.38 mg/mi for the IMPROVE_A thermal/ optical method, and 0.35 mg/mi for the OT21_T Optical Transmissometer. The real-time instruments had repeatability values ranging from 30% to 35%, which are somewhat better than those of the time-integrated instruments (40-41%). These results suggest that, despite being less resource intensive, real-time methods can be equivalent or superior to time-integrated methods in terms of sensitivity and repeatability. BC mass data, from the photoacoustic and light attenuation instruments, were compared against same-test EC data, determined using the IMPROVE_A method. The MSS BC data was well correlated with EC, with R2 = 0.85 for the composite results and R2 = 0.86 for the phase-by-phase (PBP) results. The correlation of BC, by the AE51, AE22, and OT21_T with EC was moderate to weak. The weaker correlation was driven by the inclusion of US06 test data in the linear regression analysis. We hypothesize that test-cycle-dependent BC:EC ratios are due to the different physicochemical properties of particulate matter (PM) in US06 and Federal Test Procedure (FTP) tests. Correlation amongst the real-time MSS, PASS-1, AE51, and AE22 instruments was excellent (R2 = 0.83-0.95), below 1 mg/mi levels. In the process of investigating these BC instruments, we learned that BC emissions at sub-1 mg/mi levels can be measured and are achievable by current-generation gasoline engines. IMPLICATIONS: Most comparison studies of black carbon (BC) measurement methods were carried out in the ambient air. This study assesses the agreement among various BC measurement instrument in emissions from light-duty gasoline vehicles (LDGVs) on standard test cycles, and evaluates applicability of these methods under various fuel types, driving cycles, and engine combustion technologies. This research helps to fill in the knowledge gap of BC method standardization as stated in the U.S. Environmental Protection Agency (EPA) 2011 Report to Congress on Black Carbon, and these results demonstrate the feasibility of quantification of BC at the 1 mg/mi PM standard in California Low Emission Vehicle III regulations.

Observation of Elevated Air Pollutant Concentrations in a Residential Neighborhood of Los Angeles California Using a Mobile Platform.

We observed elevated air pollutant concentrations, especially of ultrafine particles (UFP), black carbon (BC) and NO, across the residential neighborhood of the Boyle Heights Community (BH) of Los Angeles, California. Using an electric vehicle mobile platform equipped with fast response instruments, real-time air pollutant concentrations were measured in BH in spring and summer of 2008. Pollutant concentrations varied significantly in the two seasons, on different days, and by time of day, with an overall average UFP concentration in the residential areas of ~33 000 cm-3. The averaged UFP, BC, and NO concentrations measured on Soto St, a major surface street in BH, were 57 000 cm-3, 5.1 µg m-3, and 67 ppb, respectively. Concentrations of UFP across the residential areas in BH were nearly uniform spatially, in contrast to other areas in the greater metropolitan area of Los Angeles where UFP concentrations exhibit strong gradients downwind of roadways. We attribute this "UFP cloud" to high traffic volumes, including heavy duty diesel trucks on the freeways which surround and traverse BH, and substantial numbers of high-emitting vehicles (HEVs) on the surface streets traversing BH. Additionally, the high density of stop signs and lights and short block lengths, requiring frequent accelerations of vehicles, may contribute. The data also support a role for photochemical production of UFP in the afternoon. UFP concentration peaks (5 s average) of up to 9 million particles cm-3 were also observed immediately behind HEVs when they accelerated from stop lights in the BH neighborhood and areas immediately adjacent. Although encounters with HEV during mornings accounted for only about 6% and 17% of time spent monitoring residential areas and major surface streets, HEV contributed to about 28% and 53% of total ultrafine particles measured on the route, respectively. The observation of elevated pollutant number concentrations across the Boyle Heights community highlights how multiple factors combine to create high pollutant levels, and has important human exposure assessment implications, including the potential utility of our data as inputs to epidemiological studies.

Roles of water in improved production of mycelial biomass and lignocellulose-degrading enzymes by water-supply solid-state fermentation of Ganoderma lucidum.

The liquid phase was proved to be a significant influencing factor among the three phases in solid-state fermentation (SSF), which determined water control was crucial. However, obvious water loss was caused by microbial utilization and moisture evaporation. Super absorbent polymer (SAP) was utilized to supply water in SSF owing to its high water-holding capacity. Adding 0.15% SAP could significantly increase the biomass of Ganoderma lucidum by 33.59% and promote filter paper activity (FPA), endocellulase activity and laccase activity by 27.11%, 29.14% and 47.39%, respectively. Water states of fermentation substrates were detected by the low-field nuclear magnetic resonance (LF-NMR). Results revealed that water present and lost was dominated by the capillary water. At the end of fermentation, the capillary water content (Ccw) in water-supply SSF was 20.48% and 17.20% higher than that in static SSF and cold-model SSF. The relaxation time of the capillary water was reduced by 56.53% in water-supply SSF and by 53.40% in static SSF, but it just reduced by 6.82% in cold-model SSF. In addition, the Ccw in SSF had a high correlation with the biomass and lignocellulose-degrading enzyme activities of G. lucidum. These results clearly demonstrated that capillary water played a very important role in improved production of G. lucidum.

Real-world particle and NOx emissions from hybrid electric vehicles under cold weather conditions.

Hybrid electric vehicle (HEV) technology is critical to reduce the impact of the internal combustion engines on air pollution and greenhouse gases. HEVs have an advantage in market penetration due to their lower cost and higher driving range compared to battery electric vehicles (BEVs). On the other hand, HEVs use an internal combustion engine and still emit air pollutants. It is hypothesized that HEV performance is impacted by the weather conditions as a result of many factors. It was beyond the scope of this work to systematically evaluate all factors so instead we measured emissions from two vehicles driving city and highway routes in Minneapolis, Minnesota in the winter (-5 °C) and looked for major differences in emissions relative to each vehicle and relative to results that would be obtained from a chassis dynamometer in a controlled laboratory setting at a higher temperature approximately 20 °C). The study then looked to associate differences in emissions with the prevailing conditions to gain new insights. Emissions of interest included the total particle number (TPN), solid particle number (SPN), particulate matter mass (PM), and NOx. One key difference in vehicle engine technology was PFI (port fuel injection) versus GDI (gasoline direct injection). We found the frequency at which the Prius hybrid engine reignited was much higher than the Sonata for city and highway driving, although for both vehicles the catalyst temperature remained high and appeared to be unaffected by the reignitions, despite the cold weather. For most conditions, the Prius emitted more NOx but fewer particles than the Sonata. In some cases, NOx and particle emissions exceeded the most comparable laboratory-based emissions standards.

Effect of pesticide residues on simulated beer brewing and its inhibition elimination by pesticide-degrading enzyme.

Four different pesticide residues used in barley planting were selected to investigate their effects on simulated beer brewing. The influences were found to be different by varied agricultural chemicals. Among the four types of pesticides, at 25 µg/mL, triadimefon or carbendazim barely affected the brewing progress. However, ethametsulfuron-methyl and carbaryl (15 µg/mL and 2.5 µg/mL, respectively) exhibited slightly inhibition on saccharification and significantly negative impacts on yeast growth and alcohol fermentation. After pretreated by 50 µL carbaryl-degrading enzyme with the Kcat value of 2.12 s-1 at 30°C for 90 min, the negative influence on simulated beer brewing brought by carbaryl can be eliminated in the fermentation system containing 2.5 µg/mL carbaryl. The efficiency of ethanol fermentation was improved, and the removal rate of carbaryl in the brewing system was greatly accelerated. Taken together, this study suggested a potential method for solving the fermentation inhibition by pesticide residues.

Glsnf1-mediated metabolic rearrangement participates in coping with heat stress and influencing secondary metabolism in Ganoderma lucidum.

The AMP-activated protein kinase (AMPK)/Sucrose-nonfermenting serine-threonine protein kinase 1 (Snf1) plays an important role in metabolic remodelling in response to energy stress. However, the role of AMPK/Snf1 in responding to other environmental stresses and metabolic remodelling in microorganisms was unclear. Heat stress (HS), which is one important environmental factor, could induce the production of reactive oxygen species and the accumulation of ganoderic acids (GAs) in Ganoderma lucidum. Here, the functions of AMPK/Snf1 were analysed under HS condition in G. lucidum. We observed that Glsnf1 was rapidly and strongly activated when G. lucidum was exposed to HS. HS significantly increased intracellular H2O2 levels (by approximately 1.6-fold) and decreased the dry weight of G. lucidum (by approximately 45.6%). The exogenous addition of N-acetyl-l-cysteine (NAC) and ascorbic acid (VC), which function as ROS scavengers, partially inhibited the HS-mediated reduction in biomass. Adding the AMPK/Snf1 inhibitor compound C (20 µM) under HS conditions increased the H2O2 content (by approximately 2.3-fold of that found in the strain without HS treatment and 1.5-fold of that found in the strain under HS treatment without compound C) and decreased the dry weight of G. lucidum (an approximately 28.5% decrease compared with that of the strain under HS conditions without compound C). Similar results were obtained by silencing the Glsnf1 gene. Further study found that Glsnf1 meditated metabolite distribution from respiration to glycolysis, which is considered a protective mechanism against oxidative stress. In addition, Glsnf1 negatively regulated the biosynthesis of GA by removing ROS. In conclusion, our results suggest that Glsnf1-mediated metabolic remodelling is involved in heat stress adaptability and the biosynthesis of secondary metabolites in G. lucidum.

Influence of PacC on the environmental stress adaptability and cell wall components of Ganoderma lucidum.

The transcription factor PacC/Rim101 participates in environmental pH adaptation, development and secondary metabolism in many fungi, but whether PacC/Rim101 contributes to fungal adaptation to environmental stress remains unclear. In our previous study, a homologous gene of PacC/Rim101 was identified, and PacC-silenced strains of the agaricomycete Ganoderma lucidum were constructed. In this study, we further investigated the functions of PacC in G. lucidum and found that PacC-silenced strains were hypersensitive to environmental stresses, such as osmotic stress, oxidative stress and cell wall stress, compared with wild-type (WT) and empty-vector control (CK) strains. In addition, transmission electron microscopy images of the cell wall structure showed that the cell walls of the PacC-silenced strains were thinner (by approximately 25-30%) than those of the WT and CK strains. Further analysis of cell wall composition showed that the ß-1,3-glucan content in the PacC-silenced strains was only approximately 78-80% of that in the WT strain, and the changes in ß-1,3-glucan content were consistent with downregulation of glucan synthase gene expression. The ability of PacC to bind to the promoters of glucan synthase-encoding genes confirms that PacC transcriptionally regulates these genes.

A Wide Area of Air Pollutant Impact Downwind of a Freeway during Pre-Sunrise Hours.

We have observed a wide area of air pollutant impact downwind of a freeway during pre-sunrise hours in both winter and summer seasons. In contrast, previous studies have shown much sharper air pollutant gradients downwind of freeways, with levels above background concentrations extending only 300 m downwind of roadways during the day and up to 500 m at night. In this study, real-time air pollutant concentrations were measured along a 3 600 m transect normal to an elevated freeway 1-2 hours before sunrise using an electric vehicle mobile platform equipped with fast-response instruments. In winter pre-sunrise hours, the peak ultrafine particle (UFP) concentration (~95 000 cm-3) occurred immediately downwind of the freeway. However, downwind UFP concentrations as high as ~ 40 000 cm-3 extended at least 1 200 m from the freeway, and did not reach background levels (~15 000 cm-3) until a distance of about 2 600 m. UFP concentrations were also elevated over background levels up to 600 m upwind of the freeway. Other pollutants, such as NO and particle-bound polycyclic aromatic hydrocarbons, exhibited similar long-distance downwind concentration gradients. In contrast, air pollutant concentrations measured on the same route after sunrise, in the morning and afternoon, exhibited the typical daytime downwind decrease to background levels within ~300 m as found in earlier studies. Although pre-sunrise traffic volumes on the freeway were much lower than daytime congestion peaks, downwind UFP concentrations were significantly higher during pre-sunrise hours than during the daytime; UFP and NO concentrations were also strongly correlated with traffic counts on the freeway. We associate these elevated pre-sunrise concentrations over a wide area with a nocturnal surface temperature inversion, low wind speeds, and high relative humidity. Observation of a wide air pollutant impact area downwind of a major roadway prior to sunrise has important exposure assessment implications since it demonstrates extensive roadway impacts on residential areas during pre-sunrise hours, when most people are at home.

Directed Evolution of Sulfonylurea Esterase and Characterization of a Variant with Improved Activity.

Esterase SulE detoxicates a variety of sulfonylurea herbicides through de-esterification. SulE exhibits high activity against thifensulfuron-methyl but low activity against other sulfonylureas. In this study, two variants, m2311 (P80R) and m0569 (P80R and G176A), with improved activity were screened from a mutation library constructed by error-prone PCR. Variant m2311 showed a higher activity against sulfonylureas in comparison variant m0569 and was further investigated. The kcat/ Km value of variant m2311 for metsulfuron-methyl, sulfometuron-methyl, chlorimuron-ethyl, tribenuron-methyl, and ethametsulfuron-methyl increased by 3.20-, 1.72-, 2.94-, 2.26- and 2.96-fold, respectively, in comparison with the wild type. Molecular modeling suggested that the activity improvement of variant m2311 is due to the substitution of Pro80 by arginine, leading to the formation of new hydrogen bonds between the enzyme and substrate. This study facilitates further elucidation of the structure and function of SulE and provides an improved gene resource for the detoxification of sulfonylurea residues and the genetic engineering of sulfonylurea-resistant crops.

Humidity Control Strategies for Solid-State Fermentation: Capillary Water Supply by Water-Retention Materials and Negative-Pressure Auto-controlled Irrigation.

Solid-state fermentation (SSF) has regained interest owing to its advantages in solid waste treatment and fermentation industries. However, heterogeneous heat and mass transfer are often caused by the absence of free water and noticeable water loss from microbial utilization and moisture evaporation in SSF. It is necessary to explore more effective ways to solve issues of water loss and water supplement in SSF based on online capillary water monitoring, because capillary water is the dominant form of water that is present and lost in substrate. Two novel capillary-water supply strategies were proposed, established and evaluated using three selected reference strains, including water-retention materials and negative-pressure auto-controlled irrigation (NPACI). This study employed superabsorbent polymer, a kind of water-retention material to enhance enzyme productivity with the most significant increase of 2.47 times. Moreover, the combination of NPACI and 0.1% superabsorbent polymers increased productivity by 2.80-fold, together with lowered gradients of temperature, moisture and products. Furthermore, a modified liquid-supply SSF was constructed through successful capillary water control by proposed humidity control strategies. This modified SSF system could address the shortcomings of inhomogeneous culture of traditional SSF.

Aircraft emission impacts in a neighborhood adjacent to a general aviation airport in southern California.

Real time air pollutant concentrations were measured downwind of Santa Monica Airport (SMA), using an electric vehicle mobile platform equipped with fast response instruments in spring and summer of 2008. SMA is a general aviation airport operated for private aircraft and corporate jets in Los Angeles County, California. An impact area of elevated ultrafine particle (UFP) concentrations was observed extending beyond 660 m downwind and 250 m perpendicular to the wind on the downwind side of SMA. Aircraft operations resulted in average UFP concentrations elevated by factors of 10 and 2.5 at 100 and 660 m downwind, respectively, over background levels. The long downwind impact distance (i.e., compared to nearby freeways at the same time of day) is likely primarily due to the large volumes of aircraft emissions containing higher initial concentrations of UFP than on-road vehicles. Aircraft did not appreciably elevate average levels of black carbon (BC), particle-bound polycyclic aromatic hydrocarbons (PB-PAH), although spikes in concentration of these pollutants were observed associated with jet takeoffs. Jet departures resulted in peak 60-s average concentrations of up to 2.2 x 10(6) cm(-3), 440 ng m(-3), and 30 microg m(-3) for UFP, PB-PAH, and BC, respectively, 100 m downwind of the takeoff area. These peak levels were elevated by factors of 440, 90, and 100 compared to background concentrations. Peak UFP concentrations were reasonably correlated (r(2) = 0.62) with fuel consumption rates associated with aircraft departures, estimated from aircraft weights and acceleration rates. UFP concentrations remained elevated for extended periods associated particularly with jet departures, but also with jet taxi and idle, and operations of propeller aircraft. UFP measured downwind of SMA had a median mode of about 11 nm (electric mobility diameter), which was about half of the 22 nm median mode associated with UFP from heavy duty diesel trucks. The observation of highly elevated ultrafine particle concentrations in a large residential area downwind of this local airport has potential health implications for persons living near general aviation airports.