Survival of Spacecraft-Associated Microorganisms under Simulated Martian UV Irradiation

Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA+B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three Bacillus spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m⁻² of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, Bacillus subtilis 168. In addition, the exposure time required for UVA+B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the Bacillus species tested, spores of a Bacillus pumilus strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific; B. pumilus SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like B. pumilus SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the sanitation capabilities of UV irradiation are determined.     

UV Resistance of Bacillus anthracis Spores Revisited: Validation of Bacillus subtilis Spores as UV Surrogates for Spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

Evaluation of fluorescence in situ hybridization to detect encapsulated Bacillus pumilus SAFR-032 spores released from poly(methylmethacrylate)

Bacillus pumilus SAFR‐032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H₂O₂, desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR‐032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa‐FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant‐resistant B. pumilus SAFR‐032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa‐FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase‐K, lysozyme, mutanolysin and Triton X‐100 facilitated efficient spore detection by Alexa‐FISH microscopy. Neither of the Alexa‐probes tested gave rise to considerable levels of Lucite‐ or solvent‐associated background autofluorescence, demonstrating the immense potential of Alexa‐FISH for rapid quantification of encapsulated B. pumilus SAFR‐032 spores released from poly(methylmethacrylate).     

Bacillus Endospores Isolated from Granite: Close Molecular Relationships to Globally Distributed Bacillus spp. from Endolithic and Extreme Environments

As part of an ongoing effort to catalog spore-forming bacterial populations in environments conducive to interplanetary transfer by natural impacts or by human spaceflight activities, spores of Bacillus spp. were isolated and characterized from the interior of near-subsurface granite rock collected from the Santa Catalina Mountains, AZ. Granite was found to contain ~500 cultivable Bacillus spores and ~10⁴ total cultivable bacteria per gram. Many of the Bacillus isolates produced a previously unreported diffusible blue fluorescent compound. Two strains of eight tested exhibited increased spore UV resistance relative to a standard Bacillus subtilis UV biodosimetry strain. Fifty-six isolates were identified by repetitive extragenic palindromic PCR (rep-PCR) and 16S rRNA gene analysis as most closely related to B. megaterium (15 isolates), B. simplex (23 isolates), B. drentensis (6 isolates), B. niacini (7 isolates), and, likely, a new species related to B. barbaricus (5 isolates). Granite isolates were very closely related to a limited number of Bacillus spp. previously found to inhabit (i) globally distributed endolithic sites such as biodeteriorated murals, stone tombs, underground caverns, and rock concretions and (ii) extreme environments such as Antarctic soils, deep sea floor sediments, and spacecraft assembly facilities. Thus, it appears that the occurrence of Bacillus spp. in endolithic or extreme environments is not accidental but that these environments create unique niches excluding most Bacillus spp. but to which a limited number of Bacillus spp. are specifically adapted.     

Comparison of UV Inactivation of Spores of Three Encephalitozoon Species with That of Spores of Two DNA Repair-Deficient Bacillus subtilis Biodosimetry Strains

When exposed to 254-nm UV, spores of Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m², respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates. The results indicate that spores of Encephalitozoon spp. are readily inactivated at low UV fluences and that spores of UV-sensitive B. subtilis strains can be useful surrogates in evaluating UV reactor performance.     

Long-Term Survival of <Emphasis Type="Italic">Bacillus</Emphasis> Spores in Alcohol and Identification of 90% Ethanol as Relatively More Spori/Bactericidal

This study was taken up with a view to generate basic information on spore hardiness to ethanol in various Bacillus species and related genera, and to assess the effectiveness of different levels of ethanol as a bacterial disinfectant. Predominantly spore-bearing cultures of five Bacillus spp. (B. pumilus, B. subtilis, B. megaterium, B. fusiformis and B. flexus) that were isolated from the spent-alcohol used during plant tissue culture work were challenged with aqueous ethanol (25, 50, 60, 70, 80 and 90% v/v) in 1 ml volumes at 10¹⁰⁻¹¹ CFU ml⁻¹. Monitoring the spore endurance through spotting and plating revealed prolonged tolerance (>12 months) at different alcohol levels depending on the organism except in 90% where no survival was observed beyond 2–12 months. Spores of related genera like Paenibacillus and Lysinibacillus also showed long-term ethanol survival. Alcohol tolerance of spore-forming organisms depended on the extent of spores and spore hardiness, which in turn varied with the organism, strain, age of culture, growing conditions and other factors as authenticated with ATCC strains of B. pumilus and B. subtilis. Aqueous 90% ethanol caused instant inactivation of vegetative cells in different spore formers and twelve other non-sporulating Gram-positive and Gram-negative organisms tested. Taking into account both vegetative cells and spores, the appropriate concentration of ethanol as a disinfectant emerged to be 90% followed by absolute ethanol compared with the generally recommended 70–80% level.     

Molecular microbial diversity of a spacecraft assembly facility

In ongoing investigations to map and archive the microbial footprints in various components of the spacecraft and its accessories, we have examined the microbial populations of the Jet Propulsion Laboratory's Spacecraft Assembly Facility (JPL-SAF). Witness plates made up of spacecraft materials, some painted with spacecraft qualified paints, were exposed for 7 to 9 months at JPL-SAF and examined the particulate materials collected for the incidence of total cultivable aerobic heterotrophs and heat-tolerant (80°C for 15-min.) spore-formers. The results showed that the witness plates coated with spacecraft qualified paints attracted more dust particles than the non-coated stainless steel witness plates. Among the four paints tested, witness plates coated with NS43G accumulated the highest number of particles, and hence attracted more cultivable microbes. The conventional microbiological examination revealed that the JPL-SAF harbors mainly Gram-positive microbes and mostly spore-forming Bacillus species. Most of the isolated microbes were heat resistant to 80°C and proliferate at 60°C. The phylogenetic relationships among 23 cultivable heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. By 16S rDNA sequence analysis, the isolates fell into seven clades:Bacillus licheniformis, B. pumilus, B. cereus, B. circulans, Staphylococcus capitis, Planococcus sp. and Micrococcus lylae. In contrast to the cultivable approach, direct DNA isolation, cloning and 16S rDNA sequencing analysis revealed equal representation of both Gram-positive and Gram-negative microorganisms.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Bacillus endospores isolated from granite: close molecular relationships to globally distributed Bacillus spp. from endolithic and extreme environments

As part of an ongoing effort to catalog spore-forming bacterial populations in environments conducive to interplanetary transfer by natural impacts or by human spaceflight activities, spores of Bacillus spp. were isolated and characterized from the interior of near-subsurface granite rock collected from the Santa Catalina Mountains, AZ. Granite was found to contain approximately 500 cultivable Bacillus spores and approximately 10(4) total cultivable bacteria per gram. Many of the Bacillus isolates produced a previously unreported diffusible blue fluorescent compound. Two strains of eight tested exhibited increased spore UV resistance relative to a standard Bacillus subtilis UV biodosimetry strain. Fifty-six isolates were identified by repetitive extragenic palindromic PCR (rep-PCR) and 16S rRNA gene analysis as most closely related to B. megaterium (15 isolates), B. simplex (23 isolates), B. drentensis (6 isolates), B. niacini (7 isolates), and, likely, a new species related to B. barbaricus (5 isolates). Granite isolates were very closely related to a limited number of Bacillus spp. previously found to inhabit (i) globally distributed endolithic sites such as biodeteriorated murals, stone tombs, underground caverns, and rock concretions and (ii) extreme environments such as Antarctic soils, deep sea floor sediments, and spacecraft assembly facilities. Thus, it appears that the occurrence of Bacillus spp. in endolithic or extreme environments is not accidental but that these environments create unique niches excluding most Bacillus spp. but to which a limited number of Bacillus spp. are specifically adapted.     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Isolation and characterization of polyphenol oxidase- and peroxidase-producing <Emphasis Type="Italic">Bacillus</Emphasis> strains from fully fermented tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Sixteen aerobic endospore-forming Bacillus spp. were isolated from fully fermented tea leaf samples from 10 tea factories in Lahijan and Langrod cities (Gillan province, Iran). Bacillus spp. isolates were characterized using phenotypic characteristics, antibiotic susceptibility and cellular fatty acid (CFA) patterns. Based on the data obtained, five isolates of tea Bacillus spp. (TB): TB2, TB4, TB6, TB10 and TB12 belonged to the species B. subtilis. Two isolates, TB1 and TB14 were recognized as B. licheniformis. Two Bacillus spp. isolates, TB9 and TB 16 were identified as B. sphaericus. Two isolates, TB5 and TB13 were shown to be B. pumilus. Two isolates, TB7 and TB15 belonged to B. cereus. Amongst the isolates, Bacillus sp. TB3, Bacillus sp. TB8 and Bacillus sp. TB11 showed different phenotypic traits, distinct antibiotic sensitivity and fatty acid profiles, and they may represent novel species. The isolates showed polyphenol oxidase (tyrosinase) and peroxidase activities. The highest polyphenol oxidase and peroxidase activities were observed for Bacillus sp. TB3 and B. licheniformis TB14, respectively, where values of 5.48 and 3.73 units mL⁻¹ were observed.     

Antimicrobial Activities and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of <Emphasis Type="Italic">Bacillus</Emphasis> Isolates from the Marine Sponge <Emphasis Type="Italic">Aplysina aerophoba</Emphasis>

The aim of this study was to isolate bacteria that are resistant to the strong antimicrobial metabolites characteristic of Aplysina aerophoba. For this purpose, bacterial isolation was performed on agar plates to which sponge tissue extract had been added. Following screening for antifungal and antimicrobial activities, 5 strains were chosen for more detailed analyses. 16S ribosomal DNA sequencing revealed that all isolates belonged to the genus Bacillus, specifically B. subtilis and B. pumilus. Using a combination of matrix-assisted laser desorption/ ionization mass spectrometry typing of whole cells and antimicrobial bioassays against selected reference strains, the bioactive metabolites were identified as lipopeptides.     

Phenotypic persistence and external shielding ultraviolet radiation inactivation kinetic model

Aim: To develop an inactivation kinetic model to describe ultraviolet (UV) dose‐response behaviour for micro‐organisms that exhibit tailing using two commonly referenced causes for tailing: physical shielding of micro‐organisms and phenotypic persistence. Methods and Results: Dose‐response data for Escherichia coli, Mycobacterium terrae and Bacillus subtilis spores exposed to UV radiation were fit to the phenotypic persistence and external shielding (PPES) model. The fraction of persistent micro‐organisms in the original population (N_persistent/N_total) that exhibited reduced sensitivity to UV radiation was estimated by the PPES model as approx. 10^?7, 10^?5 and 10^?4 for E. coli, B. subtilis spores and Myco. terrae, respectively. Particle shielding effects were evaluated for Myco. terrae and resulted in additional reduction in UV sensitivity. Conclusions: Tailing occurred in laboratory experiments even when clumping and shielding were eliminated as major factors in UV resistance, suggesting that phenotypic persistence in addition to shielding may be important to consider when evaluating dose‐response curves for disinfection applications. Significance and Impact of the Study: The PPES model provides a mechanistically plausible tool for estimating the dose–response behaviour for micro‐organisms that exhibit tailing in dispersed and aggregated settings. Accurate dose‐response behaviour (including the tailing region) is critical to the analysis and validation of all UV disinfection systems.     

Biochemical characterisation of grain mould resistant and susceptible genotypes and PGPR-induced resistance in the host to Curvularia lunata and Fusarium proliferatum

Resistance to biotic stresses in plants is either due to the presence of preformed biochemical compounds or induced in response to external stimulus. In this study, 13 grain mould resistant and seven susceptible lines of sorghum were analysed for biochemical defence mechanism. The levels of total phenols and phenylalanine ammonia lyase were almost same in the resistant and susceptible genotypes. However, two additional isoforms of peroxidase were found in the three of the 13 resistant genotypes. The isoform peroxidase corresponding to the R _f value of 0.25 was found in the resistant genotypes IS 13969, ICSB 377 and IS 8219-1, and two genotypes IS 13969 and ICSB 377 had an additional isoform corresponding to the R _f value of 0.32. The results indicated the genotype specific association of peroxidases with grain mould resistance in sorghum. Nine bacterial strains (Bacillus pumilus SB 21, Bacillus megaterium HiB 9, Bacillus subtilis BCB 19, Pseudomonas plecoglossicida SRI 156, Brevibacterium antiquum SRI 158, B. pumilus INR 7, P. fluorescens UOM SAR 80, P. fluorescens UOM SAR 14, B. pumilus SE 34) were tested to induce systemic resistance in sorghum cultivars 296B and Bulk Y against the highly pathogenic grain mould pathogens Curvularia lunata and Fusarium proliferatum, respectively. The bacterial isolates were effective in inducing resistance in sorghum. Among the strains tested, SRI 158 was found highly effective in reducing grain mould severity in both the genotypes.     

Characterization of Bacillus Probiotics Available for Human Use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Microbial survival in the stratosphere and implications for global dispersal

Spores of Bacillus subtilis were exposed to a series of stratosphere simulations. In total, five distinct treatments measured the effect of reduced pressure, low temperature, high desiccation, and intense ultraviolet (UV) irradiation on stratosphere-isolated and ground-isolated B. subtilis strains. Environmental conditions were based on springtime data from a mid-latitude region of the lower stratosphere (20 km). Experimentally, each treatment consisted of the following independent or combined conditions: −70°C, 56 mb, 10–12% relative humidity and 0.00421, 5.11, and 54.64 W/m² of UVC (200–280 nm), UVB (280–315 nm), UVA (315–400 nm), respectively. Bacteria were deposited on metal coupon surfaces in monolayers of ~1 × 10⁶ spores and prepared with palagonite (particle size < 20 μm). After 6 h of exposure to the stratosphere environment, 99.9% of B. subtilis spores were killed due to UV irradiation. In contrast, temperature, desiccation, and pressure simulations without UV had no effect on spore viability up through 96 h. There were no differences in survival between the stratosphere-isolated versus ground-isolated B. subtilis strains. Inactivation of most bacteria in our simulation indicates that the stratosphere can be a critical barrier to long-distance microbial dispersal and that survival in the upper atmosphere may be constrained by UV irradiation.     

Role of Dipicolinic Acid in Survival of Bacillus subtilis Spores Exposed to Artificial and Solar UV Radiation

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of Bacillus subtilis spore dry weight and has been shown to play a significant role in the survival of B. subtilis spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon dpaAB were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the gerA, gerB, and gerK nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.     

Isolation,evaluation and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> from cotton rhizospheric soil with biocontrol activity against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Present investigation is based on the isolation of Bacillus subtilis from cotton rhizosphere and their evaluation as biocontrol agent against Fusarium oxysporum. The production of extracellular hydrolytic enzyme was studied for determining the antagonism. 43% of 21 isolates were identified under the B. subtilis group on the basis of biochemical characterization. 38% isolates showed competitive activity against Fusarium oxysporum and exhibit more than 50% mycelial inhibition in dual culture bioassay. The pot assay of cotton by seed treatment and soil amendment technique under green house condition showed the competent activity of the isolates in preventing the wilting of cotton seedlings due to F. oxysporum infection. SVI values of 30 day old seedlings indicated that the soil inoculation with B. subtilis BP-2 and seed treatment with B. subtilis BP-9 significantly promoted the growth of cotton seedlings. RAPD profiling revealed the diversity in the Bacillus subtilis group, ranging from 10 to 32%. The discriminative pattern among the isolates belonging to the same species was validated by 16S rDNA partial sequencing which identified them into four different strains of B. subtilis.     

枯草芽孢杆菌表面展示技术用于黏膜疫苗的研究进展

枯草杆菌全名枯草芽孢杆菌(Bacillus subtilis),因其优秀的益生特性及芽孢良好的抗逆性而备受研究者青睐,由于芽孢的特殊结构及独特的生理特性,是酶和免疫原等外源蛋白的理想锚定点。采用枯草杆菌进行芽孢表面展示被认为是表达高活性和高稳定性的外源蛋白的方法之一。本文主要对枯草杆菌芽孢表面展示抗原蛋白以生产黏膜疫苗的策略和应用前景进行综述。