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At least 847 records · Page 47

Optimization of 15 parameters influencing the long-term survival of bacteria in aquatic systems

NASA is presently engaged in the design and development of a water reclamation system for the future space station. A major concern in processing water is the control of microbial contamination. As a means of developing an optimal microbial control strategy, studies were undertaken to determine the type and amount of contamination which could be expected in these systems under a variety of changing environmental conditions. A laboratory-based Taguchi optimization experiment was conducted to determine the ideal settings for 15 parameters which influence the survival of six bacterial species in aquatic systems. The experiment demonstrated that the bacterial survival period could be decreased significantly by optimizing environmental conditions.

Obenhuber, D. C.↗

Human life support during interplanetary travel and domicile. VI - Generic modular flow schematic for hybrid physical/chemical-biological life support systems

An extension is proposed for the NASA Space Exploration Initiative's Generic Modular Flow Schematics for physical/chemical life support systems which involves the addition of biological processes. The new system architecture includes plant, microbial, and animal habitat, as well as the human habitat subsystem. Major Feedstock Production and Food Preparation and Packaging components have also been incorporated. Inedible plant, aquaculture, microbial, and animal solids are processed for recycling.

Ganapathi, Gani B.↗

Chemical sensors for space applications

The payload of the Space Station Freedom will include sensors for frequent monitoring of the water recycling process and for measuring the many biochemical parameters related to onboard experiments. This paper describes the sensor technologies and the types of transducers and selectors considered for these sensors. Particular attention is given to such aspects of monitoring of the water recycling process as the types of water use, the sources of water and their hazards, the sensor systems for monitoring, microbial monitoring, and monitoring toxic metals and organics. An approach for monitoring water recycling is suggested, which includes microbial testing with a potentiometric device (which should be in first line of tests), the use of an ion-selective electrode for inorganic ion determinations, and the use of optic fiber techniques for the determination of total organic carbon.

Bonting, Sjoerd L.↗

Soil nitrogen cycling and nitrous oxide flux in a Rocky Mountain Douglas-fir forest - Effects of fertilization, irrigation and carbon addition

Nitrous oxide fluxes and soil nitrogen transformations were measured in experimentally-treated high elevation Douglas-fir forests in northwestern New Mexico. On an annual basis, forests that were fertilized with 200 kg N/ha emitted an average of 0.66 kg/ha of N2O-N, with highest fluxes occurring in July and August when soils were both warm and wet. Control, irrigated, and woodchip treated plots were not different from each other, and annual average fluxes ranged from 0.03 to 0.23 kg/ha. Fertilized soil mineralized 277 kg/ha per year in contrast to 18 kg/ha per year in control plots. Relative recovery of (N-15)H4-N applied to soil in laboratory incubations was principally in the form of NO3-N in the fertilized soils, while recovery was mostly in microbial biomass-N in the other treatments. Fertilization apparently added nitrogen that exceeded the heterotrophic microbial demand, resulting in higher rates of nitrate production and higher nitrous oxide fluxes. Global inputs of nitrogen into forests are not currently contributing significantly to the increasing concentrations of nitrous oxide in the atmosphere.

Matson, Pamela A.↗

Soil nitrogen cycling and nitrous oxide flux in a Rocky Mountain Douglas-fir forest - Effects of fertilization, irrigation and carbon addition

Nitrous oxide fluxes and soil nitrogen transformations were measured in experimentally-treated high elevation Douglas-fir forests in northwestern New Mexico, USA. On an annual basis, forests that were fertilized with 200 kg N/ha emitted an average of 0.66 kg/ha of N2O-N, with highest fluxes occurring in July and August when soils were both warm and wet. Control, irrigated, and woodchip treated plots did not differ, and annual average fluxes ranged from 0.03 to 0.23 kg/ha. Annual net nitrogen mineralization and nitrate production were estimated in soil and forest floor using in situ incubations; fertilized soil mineralized 277 kg/ha/y in contrast to 18 kg/ha/y in control plots. Relative recovery of 15NH4-N applied to soil in laboratory incubations was principally in the form of NO3-N in the fertilized soils, while recovery was mostly in microbial biomass-N in the other treatments. Fertilization apparently added nitrogen that exceeded the heterotrophic microbial demand, resulting in higher rates of nitrate production and higher nitrous oxide fluxes. Despite the elevated nitrous oxide emission resulting from fertilization, we estimate that global inputs of nitrogen into forests are not currently contributing significantly to the increasing concentrations of nitrous oxide in the atmosphere.

Matson, Pamela A.↗

Design and testing of a unique randomized gravity, continuous flow bioreactor

A rotating, null gravity simulator, or Couette bioreactor was successfully used for the culture of mammalian cells in a simulated microgravity environment. Two limited studies using Lipomyces starkeyi and Streptomyces clavuligerus were also conducted under conditions of simulated weightlessness. Although these studies with microorganisms showed promising preliminary results, oxygen limitations presented significant limitations in studying the biochemical and cultural characteristics of these cell types. Microbial cell systems such as bacteria and yeast promise significant potential as investigative models to study the effects of microgravity on membrane transport, as well as substrate induction of inactive enzyme systems. Additionally, the smaller size of the microorganisms should further reduce the gravity induced oscillatory particle motion and thereby improve the microgravity simulation on earth. Focus is on the unique conceptual design, and subsequent development of a rotating bioreactor that is compatible with the culture and investigation of microgravity effects on microbial systems. The new reactor design will allow testing of highly aerobic cell types under simulated microgravity conditions. The described reactor affords a mechanism for investigating the long term effects of reduced gravity on cellular respiration, membrane transfer, ion exchange, and substrate conversions. It offers the capability of dynamically altering nutrients, oxygenation, pH, carbon dioxide, and substrate concentration without disturbing the microgravity simulation, or Couette flow, of the reactor. All progeny of the original cell inoculum may be acclimated to the simulated microgravity in the absence of a substrate or nutrient. The reactor has the promise of allowing scientists to probe the long term effects of weightlessness on cell interactions in plants, bacteria, yeast, and fungi. The reactor is designed to have a flow field growth chamber with uniform shear stress, yet transfer high concentrations of oxygen into the culture medium. The system described allows for continuous, on line sampling for production of product without disturbing fluid and particle dynamics in the reaction chamber. It provides for the introduction of substrate, or control substances after cell adaptation to simulated microgravity has been accomplished. The reactor system provides for the nondisruptive, continuous flow replacement of nutrient and removal of product. On line monitoring and control of growth conditions such as pH and nutrient status are provided. A rotating distribution valve allows cessation of growth chamber rotation, thereby preserving the simulated microgravity conditions over longer periods of time.

Lassiter, Carroll B.↗

Continuous monitoring of bacterial attachment

A major concern with the Space Station Freedom (SSF) water supply system is the control of longterm microbial contamination and biofilm development in the water storage and distribution systems. These biofilms have the potential for harboring pathogens as well as microbial strains containing resistance factors that could negatively influence crew health. The proposed means for disinfecting the water system on SSF (iodine) may encourage the selection of resistant strains. In fact, biofilm bacteria were observed in water lines from the Space Shuttle Columbia (OV-102); therefore, an alternative remediation method is required to disinfect spacecraft water lines. A thorough understanding of colonization events and the physiological parameters that will influence bacteria adhesion is required. The limiting factor for development of this technology is the ability to continuously monitor adhesion events and the effects of biocides on sessile bacteria. Methods were developed to allow bacterial adhesion and subsequent biocidal treatment to be monitored continuously. This technique couples automated image analysis with a continuous flow of a bacterial suspension through an optical flow cell. A strain of Pseudomonas cepacia isolated from the water supply of the Space Shuttle Discovery (OV-103) during STS-39 was grown in a nitrogen-limited continuous culture. This culture was challenged continuously with iodine during growth, and the adhesion characteristics of this strain was measure with regard to flow rate. Various biocides (ozone, hypochlorite, and iodine) were added to the flow stream to evaluate how well each chemical removed the bacteria. After biocide treatment, a fresh bacterial suspension was introduced into the flow cell, and the attachment rate was evaluated on the previously treated surface. This secondary fouling was again treated with biocide to determine the efficacy of multiple batch chemical treatments in removing biofilm.

Koeing, D. W.↗

Methane production and consumption in grassland and boreal ecosystems

The objectives of the this project were to develop a mechanistic understanding of methane production and oxidation suitable for incorporation into spatially explicit models for spatial extrapolation. Field studies were undertaken in Minnesota, Canada, and Colorado to explore the process controls over the two microbial mediated methane transformations in a range of environments. Field measurements were done in conjunction with ongoing studies in Canada (the Canadian Northern Wetlands Projects: NOWES) and in Colorado (The Shortgrass Steppe Long Term Ecological Research Project: LTER). One of the central hypotheses of the proposal was that methane production should be substrate limited, as well as being controlled by physical variables influencing microbial activity (temperature, oxidation status, and pH). Laboratory studies of peats from Canada and Minnesota (Northern and Southern Boreal) were conducted with amendments of a methanogenic substrate at multiple temperatures and at multiple pHs (the latter by titrating samples). The studies showed control by substrate, pH, and temperature in order in anaerobic samples. Field and laboratory manipulations of natural plant litter, rather than an acetogenic substrate, showed similarly large effects. The studies concluded that substrate is an important control over methanogenesis, that substrate availability in the field is closely coupled to the chemistry of the dominant vegetation influencing its decomposition rate, that most methane is produced from recent plant litter, and that landscape changes in pH are an important control, highly correlated with vegetation.

Schimel, David S.↗

Use of lunar regolith as a substrate for plant growth

Regenerative Life Support Systems (RLSS) will be required to regenerate air, water, and wastes, and to produce food for human consumption during long-duration missions to the Moon and Mars. It may be possible to supplement some of the materials needed for a lunar RLSS from resources on the Moon. Natural materials at the lunar surface may be used for a variety of lunar RLSS needs, including (1) soils or solid-support substrates for plant growth, (2) sources for extraction of essential, plant-growth nutrients, (3) substrates for microbial populations in the degradation of wastes, (4) sources of O2 and H2, which may be used to manufacture water, (5) feed stock materials for the synthesis of useful minerals (e.g., molecular sieves), and (6) shielding materials surrounding the outpost structure to protect humans, plants, and microorganisms from harmful radiation. Use of indigenous lunar regolith as a terrestrial-like soil for plant growth could offer a solid support substrate, buffering capacity, nutrient source/storage/retention capabilities, and should be relatively easy to maintain. The lunar regolith could, with a suitable microbial population, play a role in waste renovation; much like terrestrial waste application directly on soils. Issues associated with potentially toxic elements, pH, nutrient availability, air and fluid movement parameters, and cation exchange capacity of lunar regolith need to be addressed before lunar materials can be used effectively as soils for plant growth.

Ming, D. W.↗

Portable Immune-Assessment System

Portable immune-assessment system developed for use in rapidly identifying infections or contaminated environment. System combines few specific fluorescent reagents for identifying immune-cell dysfunction, toxic substances, buildup of microbial antigens or microbial growth, and potential identification of pathogenic microorganisms using fluorescent microplate reader linked to laptop computer. By using few specific dyes for cell metabolism, DNA/RNA conjugation, specific enzyme activity, or cell constituents, one makes immediate, onsite determination of person's health or of contamination of environment.

Pierson, Duane L.↗

Gene-Based Detection of Microorganisms in Environmental Samples Using PCR

Contaminating microorganisms pose a serious potential risk to the crew's well being and water system integrity aboard the International Space Station (ISS). We are developing a gene-based microbial monitor that functions by replicating specific segments of DNA as much as 10(exp 12) x. Thus a single molecule of DNA can be replicated to detectable levels, and the kinetics of that molecule's accumulation can be used to determine the original concentration of specific microorganisms in a sample. Referred to as the polymerase chain reaction (PCR), this enzymatic amplification of specific segments of the DNA or RNA from contaminating microbes offers the promise of rapid, sensitive, quantitative detection and identification of bacteria, fungi, viruses, and parasites. We envision a small instrument capable of assaying an ISS water sample for 48 different microbes in a 24 hour period. We will report on both the developments in the chemistry necessary for the PCR assays to detect microbial contaminants in ISS water, and on progress towards the miniaturization and automation of the instrumentation.

Glass, John I.↗

Meteorites, Microfossils and Exobiology

The discovery of evidence for biogenic activity and possible microfossils in a Martian meteorite may have initiated a paradigm shift regarding the existence of extraterrestrial microbial life. Terrestrial extremophiles that live in deep granite and hydrothermal vents and nanofossils in volcanic tuffs have altered the premise that microbial life and microfossils are inconsistent with volcanic activity and igneous rocks. Evidence for biogenic activity and microfossils in meteorites can no longer be dismissed solely because the meteoritic rock matrix is not sedimentary. Meteorite impact-ejection and comets provide mechanisms for planetary cross-contamination of biogenic chemicals, microfossils, and living microorganisms. Hence, previously dismissed evidence for complex indigenous biochemicals and possible microfossils in carbonaceous chondrites must be re-examined. Many similar, unidentifiable, biological-like microstructures have been found in different carbonaceous chondrites and the prevailing terrestrial contaminant model is considered suspect. This paper reports the discovery of microfossils indigenous to the Murchison meteorite. These forms were found in-situ in freshly broken, interior surfaces of the meteorite. Environmental Scanning Electron Microscope (ESEM) and optical microscopy images indicate that a population of different biological-like forms are represented. Energy Dispersive Spectroscopy reveals these forms have high carbon content overlaying an elemental distribution similar to the matrix. Efforts at identification with terrestrial microfossils and microorganisms were negative. Some forms strongly resemble bodies previously isolated in the Orgueil meteorite and considered microfossils by prior researchers. The Murchison forms are interpreted to represent an indigenous population of the preserved and altered carbonized remains (microfossils) of microorganisms that lived in the parent body of this meteorite at diverse times during the past 4.5 billion years (Gy).

Hoover, Richard B.↗

Asteroid Impacts, Microbes, and the Cooling of the Atmosphere

Earth's surface temperature constrained microbial evolution, according to Schwartzman et al. (1993). Their hypothesis states that the maximal temperature that extant organisms of a given type tolerate is the surface temperature occurring when that type of organism arose. Schwartzman and his colleagues concluded that the temperature changed from 100 C to 50 C between 3.75 billion years ago (BYA) and 1 BYA. These temperatures are consistent with those derived from oxygen isotope ratios in ancient sediments (Karhu and Epstein 1986, Knauth and Lowe 1978). The 100 C surface temperature they derive for 3.75 BYA is also the same as Earth's surface temperature 4.4 BYA (Kosting and Ackerman 1986). In this article, we address the cause of the delay in surface cooling until 3.75 BYA, and we explore the implications for microbial evolution of a high temperature on early Earth. We propose that three effects of the early heavy bombardment of Earth by asteroids and comets, until 3.8 BYA, could have delayed onset of surface cooling.

Oberbeck, Verne R.↗

Biodegradation of Fresh vs. Oven-Dried Inedible Crop Residue in a Continuously Stirred Tank Reactor

The degradation of soluble organics and mineral recovery from fresh and oven-dried biomass were compared in an Intermediate-Scale Aerobic Bioreactor (8 L working volume) to determine if drying crop residue improves performance in a continuously stirred tank reactor (CSTR). The study was conducted in an Intermediate-Scale Aerobic Bioreactor (ISAB) CSTR with dimensions of 390 mm height x 204 mm diameter. The pH in the bioreactor was controlled at 6.0, temperature at 30 C, and aeration at 7.0 L/min. Gases monitored were CO2 evolution and dissolved oxygen. Homogeneously mixed wheat cultures, used either fresh or oven-dried biomass and were leached, then placed in the ISAB for a 4-day degradation period. Studies found that mineral recovery was greater for leached oven-dried crop residue. However, after activity by the mixed microbial communities in the ISAB CSTR, there were little notable differences in the measured mineral recovery and degradation of soluble organic compounds. Degradation of soluble organic compounds was also shown to improve for leached oven-dried crop residue, but after mixing in the CSTR the degradation of the fresh biomass seemed to be slightly greater. Time for the biomass to turn in the CSTR appeared to be one factor for the experimental differences between the fresh and oven-dried biomass. Other factors, although not as defined, were the differing physical structures in the cell walls and varying microbial components of the fresh and oven-dried treatments due to changes in chemical composition after drying of the biomass.

Crawford, Kamau↗

Deep Basalt Aquifers in Orcus Patera, Elysium Basin Mars: Perspectives for Exobiology Exploration

Direct indicators of shorelines, spillways, and terraces allowed to determine the extent of the Elysium Paleolake between the contour-lines 1000 and 500 m below the Martian datum. The Elysium Paleolake is bordered north by Orcus Patera (14N/181W), which lies west of the Tartarus Montes and Tartarus Colles. The Orcus Patera displays an ellipse-shaped collapsed caldera of 360-km long and 100-km wide. Viking topographic data show that the bottom of the caldera is located at 2500 below the Martian datum, and surrounded by a steep-walled ram art which crest is located at about 0 m elevation. Considering the localization of Orcus Patera in the Elysium paleolake, its altimetry, and the magmatic origin of this caldera, we propose the existence of a paleolake in Orcus Patera generated (a) by juvenile water from magma during the Noachian period, and (b) by intermittent influx of the Elysium Basin from Hesperian to Amazonian. Results are encouraging to consider this site as a potential high-energy source environment for microbial communities. are circumscribed by a 50-km wide lava field mapped as Noachian material. The structure of Orcus Patera represents the record of material erupted from a magmatic reservoir. The caldera is enclosed by steep inner walls (25% measured from topographic data), values which could be in agreement with the presence of a deep magmatic reservoir, as suggested by the typology of Crumpler et.al. The depth of the caldera might be due to the collapse of the magma reservoir, and the release of gases accompanying the magma thermal evolution. Origins of water for the paleolake(s): The water that generated a paleolake in Orcus Patera may have come from two origins: (1) Juvenile water: Plescia and Crips estimated a magma H20 content by weight between 0.5% and 1.5% using for the first value a comparison with terrestrial basalt, and for the second values from a Martian meteorite. The amount of H20 can be estimated by the volume of erupted lava, and the lava content of the caldera. In this study, we adopt a water content of 1%. The total volume of magma that has been contained in the caldera, and the volume of lava contained in the observed lava field is about 110 x 10(exp 6) cubic km, that gives a total volume of 1.10 x 10(exp 6) cubic km of water. The juvenile water expelled by the overpressure within the magma chamber charged with desolved water-vapor may have moved into the crust. The decrease in overburden pressure led to bubble formation. The ascent of these bubbles generated a pressurization of the magma, which was sufficient to fracture the overlaying magma layer, (2) Water from Elysium paleolake. During the Amazonian, the rise of the Elysium paleolake level generated an overspilling that supplied the caldera with water. The southern portion of the crest shows a deep gap 12-km wide at -1500 m elevation, locating the gap between 500 to 1000 in below the assumed water of Elysium paleolake, thus facilitating the influx of Elysium paleolake water into Orcus Patera. Bathymetric calculations give a floor area of 25,500 sq km at -2000 m elevation, and a water volume of 42,000 cubic km, with a lake-level at -1500 m. A substantial amount of water may have percolated through the fractured lava, and part of the volume may have overspilled the northern crest of Orcus Patera to debouch in the Tartarus Montes region. We envision the formation of a subsurface aqueous environment in basaltic rocks at the contact of the two water-source origins, possibly the percolating surface lake water, and more likely the juvenile water. Similarly to terrestrial calderas, Orcus Patera might be surrounded by ring-fractures caused by the collapse of the magma chamber that followed the release of gases. These ring-fractures may have been covered later by sedimentation in the caldera (lacustrine, aeolian, and volcanic), and by mass wasting. The detumescence of the magma in the caldera, and the vesiculation of the juvenile water may have operated simultaneously. Comparatively to terrestrial melts, Martian iron-rich melts are denser. This greater density implies greater effusion rates (eight-times terrestrial values), and larger fissuration widths (two-times terrestrial ones). With increasing vesiculation of magma, the bubbles interact with one-another because there are of similar pressure. They make a magma froth at the contact with the caldera surface, and on the walls of the fractures. In the saturated magma, froth, where the volume ratio of gases-to-liquid is about 4:1, the bubbles form a huge surface area of interconnected spaces. Bubbles near the caldera surface disrupt the magma, and fragmentation takes place, which moves downward through the magma column. On Earth, the bubbles are likely to grow between 1 and 50 mm in diameter due to the difference between the magma surface tension, and the bubble supersaturation pressure. The Martian low-pressure at surface level is likely to accelerate the expansion of the bubbles, and increase their final diameter and number, creating more voids in the magma. The strong magma froth with enclosed juvenile water bubbles interconnected with exsolved gas bubbles constitute a potential geothermal environment for geochemical energy production from basalt and water that does not require excessive temperatures. This process can start at +20C. Similar types of environments have been shown on Earth as potential energy sources for microbial metabolism, and could have provided deep aqueous basaltic niches for possible Martian microorganisms, even geologically recently. During the Amazonian, combination of volcanism and water activity still existed on Mars. Moreover, this type of potential niches open ways for investigation of possible oases of extinct or extant life, not only on paleolakes, and surface hydrothermalism spring areas, but also all large systems of fossae, which combine hydrologic and volcanic activities, and which provide an energy source, and an underground shelter to prevent surface UV bombardment. Additional information contained in the original.

Grin, E. A.↗

Spatial Distribution of Cyanobacteria in Modern Stromatolites

Living stromatolites consist of complex microbial communities with distinct distribution patterns for different microbial groups. The cyanobacterial populations of Highborne Cay Bahamas exemplify this phenomenon. Field observations reveal distinct distribution patterns for several of these cyanobacterial species. To date 10 different cyanobacterial cultures, including both filamentous and endolithic species, have been isolated from these stromatolites. We will present data on the growth and motility characteristics as well as on the nutritional requirements of these isolates. These data will then be correlated with the field observed distributions for these species. Lastly laboratory simulations of stromatolites grown under various conditions of irradiance, flow and cyanobacterial community composition will be presented. These experiments allow us to evaluate our predictions regarding controls on cyanobacterial distribution.

Prufert-Bebout, Lee↗

How Cyanobacterial Distributions Reveal Flow and Irradiance Conditions of Photosynthetic Biofilm Formation

Microbial life on Earth is enormously abundant at sediment-water interfaces. The fossil record in fact contains abundant evidence of the preservation of life on such surfaces. It is therefore critical to our interpretation of early Earth history, and potentially to history of life on other planets, to be able to recognize life forms at these interfaces. On Earth this life often occurs as organized structures of microbes and their extracellular exudates known as biofilms. When such biofilms occur in areas receiving sunlight photosynthetic biofilms are the dominant form in natural ecosystems due to selective advantage inherent in their ability to utilize solar energy. Cyanobacteria are the dominant phototrophic microbes in most modern and ancient photosynthetic biofilms, microbial mats and stromatolites. Due to their long (3.5 billion year) evolutionary history, this group has extensively diversified resulting in an enormous array of morphologies and physiological abilities. This enormous diversity and specialization results in very specific selection for a particular cyanobacterium in each available photosynthetic niche. Furthermore these organisms can alter their spatial orientation, cell morphology, pigmentation and associations with heterotrophic organisms in order to fine tune their optimization to a given micro-niche. These adaptations can be detected, and if adequate knowledge of the interaction between environmental conditions and organism response is available, the detectable organism response can be used to infer the environmental conditions causing that response. This presentation will detail two specific examples which illustrate this point, Light and water are essential to photosynthesis in cyanobacteria and these organisms have specific detectable behavioural responses to these parameters. We will present cyanobacterial responses to quantified flow and irradiance to demonstrate the interpretative power of distribution and orientation information. This study presents new results, but many such examples are already found in the literature.

Prufert-Bebout, Lee↗

Engineering Strategies for the Design of Plant Nutrient Delivery Systems for Use in Space: Approaches to Countering Microbiological Contamination

Microbiological contamination of crops within space-based crop growth research chambers has been postulated as a potentially significant problem. Microbial infestations; fouling of Nutrient Delivery System (NDS) fluid loops; and the formation of biofilms, have been suggested as the most obvious and important manifestations of the problem. Strict sanitation and quarantine procedures will reduce, but not eliminate, microbial species introduced into plant growing systems in space habitats. Microorganisms transported into space will most likely occur as contaminants on spacecraft components, equipment, the crew, and plant-propagative materials. Illustrations of the potential magnitude of the microbiological contamination issue will be drawn from the literature and from documentation of laboratory and commercial field experience. Engineering strategies for limiting contamination and for the development of countermeasures will be described. Microbiological control technologies and NDS hardware will be discussed. Configurations appropriate for microgravity research facilities, as well as anticipated bio-regenerative life support system implementations, will be explored. An efficiently designed NDS, capable of adequately meeting the environmental needs of crop plants in space, is considered to be critical in both the research and operational domains. Recommended experiments, tests and technology developments, structured to allow the development of prudent engineering solutions, will also be presented.

Gonzales, A. A.↗