Short-term responses and resistance of soil microbial community structure to elevated CO2 and N addition in grassland mesocosms

Nitrogen (N) addition is known to affect soil microbial communities, but the interactive effects of N addition with otherdrivers of global change remain unclear. The impacts of multiple global changes on the structure of microbial communities may be mediated by specific microbial groups with different life-history strategies. Here, we investigated the combined effects of elevated CO2 and N addition on soil microbial communities using PLFA profiling in a short-term grassland mesocosm experiment. We also examined the linkages between the relative abundance of r- and K-strategist microorganisms and resistance of the microbial community structure to experimental treatments. N addition had a significant effect on microbial community structure, likely driven by concurrent increases in plant biomass and in soil labile C and N. In contrast, microbial community structure did not change under elevated CO2 or show significant CO2 × N interactions. Resistance of soil microbial community structure decreased with increasing fungal/bacterial ratio, but showed a positive relationship with the Gram-positive/Gram-negative bacterial ratio. Our findings suggest that the Grampositive/ Gram-negative bacteria ratio may be a useful indicator of microbial community resistance and that K-strategist abundance may play a role in the short-term stability of microbial communities under global change

Patterns in soil ammonia-oxidizer response to global change

Background/Question/Methods
The Domains Archaea and Bacteria contain the vast majority of Earth’s biodiversity and biomass, and their members play critical, often exclusive, roles in many biogeochemical cycles and ecosystem services. Human-induced global change, particularly with respect to increased nitrogen deposition, has the potential to drastically alter how soil nitrifying communities perform their biogeochemical function. Additionally, multi-factor global change can alter how microbial communities interact with each other and with the associated plant communities. This study, performed in the context of the long-term Jasper Ridge Global Change Experiment (JRGCE) in a California grassland ecosystem, examines how ammonia-oxidizing Archaea and Bacteria (AOA and AOB, respectively) respond to multi-factor global change. Manipulations at the JRGCE include simultaneous increases in CO2, warming, precipitation and nitrogen deposition. Past studies have utilized DNA-fingerprinting methods to assess ammonia-oxidizer response to multi-factor global change. This study compares how seed bank (DNA-based) versus metabolically active (RNA-based) ammonia-oxidizing communities respond to global change manipulations over several seasons. We have employed ultra-deep 454-pyrosequencing techniques to examine these communities using the ammonia monooxygenase (amoA) functional gene marker. Effects of global change have been examined at several phylogenetic levels and linked this community information to gross rates of nitrification using 15N stable isotopic methods. Ammonia-oxidizer and plant communities have been compared using multivariate statistical methods.

Results/Conclusions
Our results show that both AOB and AOA communities are highly influenced by nitrogen deposition in both their abundance and community structure. These changes are linked to increased nitrification rate in the elevated nitrogen deposition plots. Our results further show that the relationship between the AOB and plant communities fundamentally changes under long-term nitrogen deposition manipulation. This positive feedback loop may enhance the rate of change in ammonia-oxidizer communities, which may further elevate nitrification rates. This study provides strong evidence that incorporating microbial community and abundance information into global change predictions is crucial for understanding how ecosystem-level nutrient cycling rates may change.

*The audio track for talks in this symposium may be obtained at the following web address:*

*https://sites.google.com/site/esa2010symposium13audiocontent/esa2010-symposium13-audio-content

Testing interactive effects of global environmental changes on soil nitrogen cycling

Responses of soil nitrogen (N) cycling to simultaneous and potentially interacting global environmental changes are uncertain. Here, we investigated the combined effects of elevated CO2, warming, increased precipitation and enhanced N supply on soil N cycling in an annual grassland ecosystem as part of the Jasper Ridge Global Change Experiment (CA, USA). This field experiment included four treatments-CO2, temperature, precipitation, nitrogen-with two levels per treatment (ambient and elevated), and all their factorial combinations replicated six times. We collected soil samples after 7 and 8 years of treatments, and measured gross rates of N mineralization, N immobilization and nitrification, along with potential rates of ammonia oxidation, nitrite oxidation and denitrification. We also determined the main drivers of these microbial activities (soil ammonium and nitrate concentrations, soil moisture, soil temperature, soil pH, and soil CO2 efflux, as an indicator of soil heterotrophic activity). We found that gross N mineralization responded to the interactive effects of the CO2, precipitation and N treatments: N addition increased gross N mineralization when CO2 and precipitation were either both at ambient or both at elevated levels. However, we found limited evidence for interactions among elevated CO2, warming, increased precipitation, and enhanced N supply on the other N cycling processes examined: statistically significant interactions, when found, tended not to persist across multiple dates. Soil N cycling responded mainly to single-factor effects: long-term N addition increased gross N immobilization, potential ammonia oxidation and potential denitrification, while increased precipitation depressed potential nitrite oxidation and increased potential ammonia oxidation and potential denitrification. In contrast, elevated CO2 and modest warming did not significantly affect any of these microbial N transformations. These findings suggest that global change effects on soil N cycling are primarily additive, and therefore generally predictable from single factor studies

Design and testing of low intensity laser biostimulator

BACKGROUND: The non-invasive nature of laser biostimulation has made lasers an attractive alternative in Medical Acupuncture at the last 25 years. However, there is still an uncertainty as to whether they work or their effect is just placebo. Although a plethora of scientific papers published about the topic showing positive clinical results, there is still a lack of objective scientific proofs about the biostimulation effect of lasers in Medical Acupuncture. The objective of this work was to design and build a low cost portable laser device for stimulation of acupuncture points, considered here as small localized biosources (SLB), without stimulating any sensory nerves via shock or heat and to find out a suitable method for objectively evaluating its stimulating effect. The design is aimed for studying SLB potentials provoked by laser stimulus, in search for objective proofs of the biostimulation effect of lasers used in Medical Acupuncture. METHODS: The proposed biostimulator features two operational modes: program mode and stimulation mode and two output polarization modes: linearly and circularly polarized laser emission. In program mode, different user-defined stimulation protocols can be created and memorized. The laser output can be either continuous or pulse modulated. Each stimulation session consists of a pre-defined number of successive continuous or square pulse modulated sequences of laser emission. The variable parameters of the laser output are: average output power, pulse width, pulse period, and continuous or pulsed sequence duration and repetition period. In stimulation mode the stimulus is automatically applied according to the pre-programmed protocol. The laser source is 30 mW AlGaInP laser diode with an emission wavelength of 685 nm, driven by a highly integrated driver. The optical system designed for beam collimation and polarization change uses single collimating lens with large numerical aperture, linear polarizer and a quarter-wave retardation plate. The proposed method for testing the device efficiency employs a biofeedback from the subject by recording the biopotentials evoked by the laser stimulus at related distant SLB sites. Therefore measuring of SLB biopotentials caused by the stimulus would indicate that a biopotential has been evoked at the irradiated site and has propagated to the measurement sites, rather than being caused by local changes of the electrical skin conductivity. RESULTS: A prototype device was built according to the proposed design using relatively inexpensive and commercially available components. The laser output can be pulse modulated from 0.1 to 1000 Hz with a duty factor from 10 to 90 %. The average output power density can be adjusted in the range 24 – 480 mW/cm2, where the total irradiation is limited to 2 Joule per stimulation session. The device is controlled by an 8-bit RISC Flash microcontroller with internal RAM and EEPROM memory, which allows for a wide range of different stimulation protocols to be implemented and memorized. The integrated laser diode driver with its onboard light power control loop provides safe and consistent laser modulation. The prototype was tested on the right Tri-Heater (TH) acupuncture meridian according to the proposed method. Laser evoked potentials were recorded from most of the easily accessible SLB along the meridian under study. They appear like periodical spikes with a repetition rate from 0.05 to 10 Hz and amplitude range 0.1 – 1 mV. CONCLUSION: The prototype's specifications were found to be better or comparable to those of other existing devices. It features low component count, small size and low power consumption. Because of the low power levels used the possibility of sensory nerve stimulation via the phenomenon of shock or heat is excluded. Thus senseless optical stimulation is achieved. The optical system presented offers simple and cost effective way for beam collimation and polarization change. The novel method proposed for testing the device efficiency allows for objectively recording of SLB potentials evoked by laser stimulus. Based on the biopotential records obtained with this method, a scientifically based conclusion can be drawn about the effectiveness of the commercially available devices for low-level laser therapy used in Medical Acupuncture. The prototype tests showed that with the biostimulator presented, SLB could be effectively stimulated at low power levels. However more studies are needed to derive a general conclusion about the SLB biostimulation mechanism of lasers and their most effective power and optical settings

Responses of soil nitrite-oxidizers to global environmental changes

Background/Question/Methods
There is increasing evidence that global change can alter the structure of plant
communities with large impacts on the functioning of terrestrial ecosystems. However, little is known about the impact of global change on soil microbial communities. In particular, the response of soil nitrite-oxidizers microorganisms that mediate the second step of nitrification, a key process of the nitrogen cycle, has never been investigated.
Here, we examined the effects of four main global environmental changes on the activity, the abundance and the diversity of soil nitrite-oxidizers in an annual grassland ecosystem as part of the Jasper Ridge Global Change Experiment (CA, USA). This experiment includes four treatments - CO2, temperature, precipitation and nitrogen - with two levels per treatment (ambient and elevated, with elevated treatment based on prediction for the end of the century), and all of their factorial combinations. We measured potential nitrite oxidation, the abundance of soil Nitrobacter- and Nitrospiralike nitrite-oxidizers (using quantitative PCR targeting nxrA and 16S rRNA gene, respectively) and the diversity of soil Nitrobacter-like nitrite-oxidizers (using cloning sequencing targeting the nxrA gene) in each treatment combination at the end of the 7th and 8th growing seasons under treatments. Furthermore, we analyzed to what extent changes in the activity of the soil nitrite-oxidizers result from changes in their abundance or diversity.

Results/Conclusions
Simulated global environmental changes significantly altered the activity, as well as the abundance and the diversity of soil nitrite-oxidizers. Potential nitrite oxidation decreased with increased precipitation and increased with elevated CO2 when combined with added nitrogen or precipitation. The abundance of soil Nitrobacter-like nitrite-oxidizers also decreased with increased precipitation and increased with elevated levels of CO2 and nitrogen. In contrast, the abundance of soil Nitrospira-like nitrite-oxidizers increased with enhanced precipitation and decreased with elevated levels of CO2 and temperature. Finally, the structure of the soil Nitrobacter-like nitrite-oxidizers was significantly altered by the treatments. Consistent with results reported by Attard et al. (2010) for agroecosystems, we found that changes in potential rates of nitrite oxidation in response to treatments were partly explained by changes in the abundance of soil Nitrobacter-like nitrite-oxidizers, but not by changes in the abundance of soil Nitrospiralike nitrite-oxidizers, suggesting that Nitrobacter-like nitrite-oxidizers were the main functional players of the soil nitrite-oxidizing microbial community.
Our study provides evidence that global change could alter the abundance and diversity of soil nitrite-oxidizers, with potential impacts for soil nitrogen cycling.

*The audio track for talks in this symposium may be obtained at the following web address:*

*https://sites.google.com/site/esa2010symposium13audiocontent/esa2010-symposium13-audio-content

Global Change Could Amplify Fire Effects on Soil Greenhouse Gas Emissions

Background: Little is known about the combined impacts of global environmental changes and ecological disturbances on ecosystem functioning, even though such combined impacts might play critical roles in shaping ecosystem processes that can in turn feed back to climate change, such as soil emissions of greenhouse gases.[br/] Methodology/Principal Findings: We took advantage of an accidental, low-severity wildfire that burned part of a long-term global change experiment to investigate the interactive effects of a fire disturbance and increases in CO(2) concentration, precipitation and nitrogen supply on soil nitrous oxide (N(2)O) emissions in a grassland ecosystem. We examined the responses of soil N(2)O emissions, as well as the responses of the two main microbial processes contributing to soil N(2)O production - nitrification and denitrification - and of their main drivers. We show that the fire disturbance greatly increased soil N(2)O emissions over a three-year period, and that elevated CO(2) and enhanced nitrogen supply amplified fire effects on soil N(2)O emissions: emissions increased by a factor of two with fire alone and by a factor of six under the combined influence of fire, elevated CO(2) and nitrogen. We also provide evidence that this response was caused by increased microbial denitrification, resulting from increased soil moisture and soil carbon and nitrogen availability in the burned and fertilized plots. [br/] Conclusions/Significance: Our results indicate that the combined effects of fire and global environmental changes can exceed their effects in isolation, thereby creating unexpected feedbacks to soil greenhouse gas emissions. These findings highlight the need to further explore the impacts of ecological disturbances on ecosystem functioning in the context of global change if we wish to be able to model future soil greenhouse gas emissions with greater confidence

Electrical impedance along connective tissue planes associated with acupuncture meridians

BACKGROUND: Acupuncture points and meridians are commonly believed to possess unique electrical properties. The experimental support for this claim is limited given the technical and methodological shortcomings of prior studies. Recent studies indicate a correspondence between acupuncture meridians and connective tissue planes. We hypothesized that segments of acupuncture meridians that are associated with loose connective tissue planes (between muscles or between muscle and bone) visible by ultrasound have greater electrical conductance (less electrical impedance) than non-meridian, parallel control segments. METHODS: We used a four-electrode method to measure the electrical impedance along segments of the Pericardium and Spleen meridians and corresponding parallel control segments in 23 human subjects. Meridian segments were determined by palpation and proportional measurements. Connective tissue planes underlying those segments were imaged with an ultrasound scanner. Along each meridian segment, four gold-plated needles were inserted along a straight line and used as electrodes. A parallel series of four control needles were placed 0.8 cm medial to the meridian needles. For each set of four needles, a 3.3 kHz alternating (AC) constant amplitude current was introduced at three different amplitudes (20, 40, and 80 μAmps) to the outer two needles, while the voltage was measured between the inner two needles. Tissue impedance between the two inner needles was calculated based on Ohm's law (ratio of voltage to current intensity). RESULTS: At the Pericardium location, mean tissue impedance was significantly lower at meridian segments (70.4 ± 5.7 Ω) compared with control segments (75.0 ± 5.9 Ω) (p = 0.0003). At the Spleen location, mean impedance for meridian (67.8 ± 6.8 Ω) and control segments (68.5 ± 7.5 Ω) were not significantly different (p = 0.70). CONCLUSION: Tissue impedance was on average lower along the Pericardium meridian, but not along the Spleen meridian, compared with their respective controls. Ultrasound imaging of meridian and control segments suggested that contact of the needle with connective tissue may explain the decrease in electrical impedance noted at the Pericardium meridian. Further studies are needed to determine whether tissue impedance is lower in (1) connective tissue in general compared with muscle and (2) meridian-associated vs. non meridian-associated connective tissue

An active electrode for biopotential recording from small localized bio-sources

BACKGROUND: Laser bio-stimulation is a well-established procedure in Medical Acupuncture. Nevertheless there is still a confusion as to whether it works or the effect is just placebo. Although a plethora of scientific papers published, showing positive clinical results, there is still a lack of objective scientific proofs about the bio-stimulation effect of lasers used in Acupuncture. The objective of this work was to design and build a body surface electrode and an amplifier for biopotential recording from acupuncture points, considered here as small localized bio-sources (SLB). The design is aimed for studying SLB potentials provoked by laser stimulus, in search for objective proofs of the bio-stimulation effect of lasers used in Medical Acupuncture. METHODS: The active electrode presented features a new adjustable anchoring system and fractionation of the biopotential amplifier between the electrode and the cabinet's location. The new adjustable electrode anchoring system is designed to reduce the electrode-skin contact impedance, its variation and motion artifacts. That is achieved by increasing the electrode-skin tension and decreasing its relative movement. Additionally the sensing element provides local constant skin stretching thus eliminating the contribution of the skin potential artifact. The electrode is attached to the skin by a double-sided adhesive pad, where the sensing element is a stainless steel, 4 mm in diameter. The fractionation of the biopotential amplifier is done by incorporating the amplifier's front-end op-amps at the electrodes, thus avoiding the use of extra buffers. The biopotential amplifier features two selectable modes of operation: semi-AC-mode with a -3 dB bandwidth of 0.32–1000 Hz and AC-mode with a bandwidth of 0.16–1000 Hz. RESULTS: The average measured DC electrode-skin contact impedance of the proposed electrode was 450 kΩ, with electrode tension of 0.3 kg/cm(2 )on an unprepared skin of the inner forearm. The peak-to-peak noise voltage measured at the amplifier output, with input terminals connected to common, was 10 mV(p-p), or 2 μV(p-p )referred to the input. The common-mode rejection ratio of the amplifier was 96 dB at 50 Hz, measured with imbalanced electrodes' impedances. The prototype was also tested practically and sample records were obtained after a low intensity SLB laser stimulation. All measurements showed almost a complete absence of 50 Hz interference, although no electrolyte gel or skin preparation was applied. CONCLUSION: The results showed that the new active electrode presented significantly reduced the electrode-skin impedance, its variation and motion artifact influences. This allowed SLB signals with relatively high quality to be recorded without skin preparation. The design offers low noise and major reduction in parts, size and power consumption. The active electrode specifications were found to be better or at least comparable to those of other existing designs