The diversity of microorganisms associated with <Emphasis Type="Italic">Acromyrmex</Emphasis> leafcutter ants

Background  

Molecular biological techniques are dramatically changing our view of microbial diversity in almost any environment that has so far been investigated. This study presents a systematic survey of the microbial diversity associated with a population of Acromyrmex leafcutter ants. In contrast to previous studies on social insects, which targeted specific groups of symbionts occurring in the gut (termites, Tetraponera ants) or in specialised cells (Camponotus ants) the objective of our present study was to do a total screening of all possible micro-organisms that can be found inside the bodies of these leafcutter ants.     

Microbial Characterization during the Early Habitation of the International Space Station

An evaluation of the microbiota from air, water, and surface samples provided a baseline of microbial characterization onboard the International Space Station (ISS) to gain insight into bacterial and fungal contamination during the initial stages of construction and habitation. Using 16S genetic sequencing and rep-PCR, 63 bacterial strains were isolated for identification and fingerprinted for microbial tracking. Of the bacterial strains that were isolated and fingerprinted, 19 displayed similarity to each other. The use of these molecular tools allowed for the identification of bacteria not previously identified using automated biochemical analysis and provided a clear indication of the source of several ISS contaminants. Strains of Bradyrhizobium and Sphingomonas unable to be identified using sequencing were identified by comparison of rep-PCR DNA fingerprints. Distinct DNA fingerprints for several strains of Methylobacterium provided a clear indication of the source of an ISS water supply contaminant. Fungal and bacterial data acquired during monitoring do not suggest there is a current microbial hazard to the spacecraft, nor does any trend indicate a potential health risk. Previous spacecraft environmental analysis indicated that microbial contamination will increase with time and will require continued surveillance.     

Aspergillus Colonization and Aflatoxin Contamination of Maize and Sesame Kernels in Two Agro‐ecological Zones in Senegal

Aflatoxin contamination of major food crops is a serious problem in Senegal. Maize and sesame samples were collected during a survey in five districts located in two agro‐ecological zones in Senegal to determine levels of aflatoxin contamination and the distribution and toxigenicity potential of members of Aspergillus section Flavi. Maize samples from the Guinea Savannah zone (SG) exhibited lower aflatoxin content and colony‐forming units (cfu) than those collected from the Sudan Savannah (SS) zone. In maize, aflatoxin concentration and cfu of A. flavus varied with cultivars, shelling practices and storage methods. The maize variety ‘Jaune de Bambey’ had high aflatoxin levels in both agro‐ecological zones. Aflatoxin content in machine‐shelled maize (120 ng/g) was more than 10‐fold higher than that in manually shelled (8 ng/g) or unshelled maize. Aflatoxin content (between 0.1 and 1.2 ng/g) and cfu values (between 13 and 42 000 cfu/g) of sesame were low, suggesting a low susceptibility to A. flavus. In both agro‐ecological zones, and in all storage systems, aflatoxin contamination was lower in sesame than in maize. In this study, only three species of Aspergillus section Flavi (A. flavus, A. tamarii and the unnamed taxon S_BG) were observed with the frequency of toxigenic strains remaining below 50% in maize from the SG zone compared with 51% of isolates from samples collected in Sedhiou district in SS zone. The proportion of toxigenic strains isolated from sesame was variable. For both crops, L‐strains were the most prevalent in the two agro‐ecological zones. Some of the atoxigenic strains collected could be valuable microbial resources for the biological control of aflatoxin in Senegal.     

Biofilm growth of individual and dual strains of <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> from the dairy industry on ultrafiltration membranes

Formation of biofilms in dairy membrane plants causes membrane pore blocking, product contamination and subsequent economic loss. To investigate the biofilm growth, two Klebsiella oxytoca strains, K. B006 and TR002, previously isolated from New Zealand dairy membrane plants, were grown both individually and combined on three types of ultrafiltration (UF) membranes in different concentrations of whey medium in biofilm reactors (CBR 90, BioSurface Technologies, Bozeman, USA). Biofilms of both the individual and combined strains grew on the membrane surfaces to levels of 4.9–7.99 log colony-forming units (CFU) cm⁻² measured by standard plate counting after removing the cells by sonication. More biofilm grew on used polyethersulfone (PES) membranes than on new PES and polyvinylidene fluoride (PVDF) membranes. Both strains formed good biofilms, although K. B006 formed a denser biofilm than TR002. This corresponded to our previous study on the attachment of these organisms, where K. B006 attached in greater numbers than K. TR002. The dual strains produced a higher biofilm density than single strains on the new membranes. Biofilm density tended to increase with increased whey concentration. The saturated biofilm was approximately 10⁸ CFU cm⁻². PES membranes appeared to support biofilm growth less readily than did PVDF membranes and therefore may be the preferred material for UF membranes to reduce problems with microbial colonisation. Used membranes were more readily colonised with biofilm than were new membranes. Therefore, selecting a membrane type and monitoring membrane age will help manage biofilm development during UF.     

The pollination system of the widely distributed mammal‐pollinated Mucuna macrocarpa (Fabaceae) in the tropics

Although the pollinators of some plant species differ across regions, only a few mammal‐pollinated plant species have regional pollinator differences in Asia. Mucuna macrocarpa (Fabaceae) is pollinated by squirrels, flying foxes, and macaques in subtropical and temperate islands. In this study, the pollination system of M. macrocarpa was identified in tropical Asia, where the genus originally diversified. This species requires “explosive opening” of the flower, where the wing petals must be pressed down and the banner petal pushed upward to fully expose the stamens and pistil. A bagging experiment showed that fruits did not develop in inflorescences (n = 66) with unopened flowers, whereas fruits developed in 68.7% of inflorescences (n = 131) with opened flowers. This indicated that the explosive opening is needed for the species to reproduce. Four potential pollinator mammals were identified by a video camera‐trap survey, and 78.3% and 60.1% of monitored inflorescences (n = 138) were opened by gray‐bellied squirrels (Callosciurus caniceps) and Finlayson's squirrels (C. finlaysonii), respectively, even though more than 10 mammal species visited flowers. Nectar was surrounded by the calyx, and the volume and sugar concentration of secreted nectar did not change during the day. This nectar secretion pattern is similar to those reported by previous studies in other regions. These results showed that the main pollinators of M. macrocarpa in the tropics are squirrels. However, the species' nectar secretion pattern is not specifically adapted to this particular pollinator. Pollinators of M. macrocarpa differ throughout the distribution range based on the fauna present, but there might not have been no distinctive changes in the attractive traits that accompanied these changes in pollinators.     

Yessotoxin detected in mussel (Mytilus californicus) and phytoplankton samples from the U.S. west coast

Yessotoxin (YTX) was detected in an algal sample and two mussel samples (0.07–0.10 μg g⁻¹) collected from Scripps Pier in La Jolla, California during a bloom of Lingulodinium polyedrum. Mussel samples collected from Monterey Bay, California also contained measurable YTX (levels up to 0.06 μg g⁻¹) in samples obtained during a 6-month (weekly) sampling period. Gonyaulax spinifera and L. polyedrum were identified in background concentrations in Monterey Bay during the time of contamination. An algal sample from Washington coastal waters collected during non-bloom conditions also contained YTX, possibly originating from Protoceratium reticulatum.Three strains of L. polyedrum (CCMP1931, CCMP1936, 104A) isolated from southern California coastal waters and one strain of G. spinifera (CCMP409) isolated from Maine were tested for YTX production using two methods, competitive ELISA and liquid chromatography–mass spectrometry (LC–MS). The ELISA method detected YTX in the particulate phase in two of three L. polyedrum strains. The LC–MS method did not detect YTX in the particulate or dissolved phase of any of the strains.To our knowledge, this is the first study to test and confirm YTX in environmental samples from California and Washington coastal waters. It is highly likely that L. polyedrum was responsible for the YTX contamination in the southern California samples. Future research needs to conclusively determine the biological origin(s) of YTX contamination in central California and Washington coastal waters.     

Characterization of an airborne microbial community: A case study in the archive of the University of Coimbra,Portugal

Understanding the structure of indoor airborne microbial communities could be useful in optimizing conservation and disinfection procedures in archive repositories, preventing the biodeterioration of stored collections. In this study we characterized the microbial air community inside the Archive of the University of Coimbra, by identifying different fungal and bacterial organisms retrieved from air samples. The microbial contamination was determined using conventional culture methods, and the isolates were typified using morphological techniques. Results indicated a low microbial air contamination (107 ± 12 CFU/m³), particularly regarding fungal propagules (6 ± 1 CFU/m³). Fungal isolates were identified using ITS-DNA sequencing. Among fungal isolates, Penicillium was the most frequent genus, and Penicillium griseofulvum was the predominant species. Simpson diversity index (1-D) was applied to phenotypic and genotypic results. Total phenotypic diversity varied from 0.4 to 0.8 and regarding fungal species, the diversity was higher than 0.5. These results were compared with previous analyses of the Archive's air, suggesting that short-term changes in atmospheric conditions may influence the indoor air microbial community structure.     

UV light‐tunable fluorescent inks and polymer hydrogel films based on carbon nanodots and lanthanide for enhancing anti‐counterfeiting

Novel water‐soluble green fluorescent carbon nanodots (CNs) using methacrylic acid and m‐phenylenediamine as precursors were first synthesized using a one‐pot hydrothermal method. Red fluorescent lanthanide complexes were prepared using lanthanide ion Eu³⁺ and pyridine‐2,6‐dicarboxylic acid. The optical properties of CNs were characterized using ultraviolet visible (UV) spectra and fluorescence spectra, microscopic morphology was characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the elemental composition was characterized using Fourier transform‐infrared spectroscopy (FT‐IR) and X‐ray photoelectron spectra (XPS). The fluorescence spectra of the lanthanide complexes were also measured. A simple strategy was developed to prepare UV light‐tunable fluorescent inks and polymer hydrogels films based on CNs and lanthanide complexes. The fluorescent inks and polymer hydrogels films could be repeatedly switched between green and red fluorescence. The change of color depended on luminescence of the CNs and the lanthanide complexes under 254 and 365 nm UV light, respectively. The UV light‐tunable fluorescent inks and polymer hydrogels films could enhance its anti‐counterfeiting function for data and information.     

Rapid identification of microbial VOCs from tobacco molds using closed-loop stripping and gas chromatography/time-of-flight mass spectrometry

Several microbial volatile organic compounds (MVOCs) that can serve as potential chemical markers for microbial contamination in tobacco have been identified. Four different fungal species, Aspergillus niger (AN), A. ornatus (AO), Pencillium chrysogenum (PC) and Rhizopus stolonifer (RS), commonly reported in moldy tobacco were cultured and screened for MVOCs. Because the MVOCs emitted by a microbial species are substrate specific, the fungal strains were separately grown on potato dextrose agar (PDA) and tobacco products. MVOCs from the mold cultures grown on PDA and tobacco products were extracted using closed-loop stripping analysis (CLSA) and identified by gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). Some of the prominent tobacco mold markers identified by this method include: 1-octen-3-ol; 2-octen-1-ol; 2-methyl-1-butanol; 3-methyl-1-butanol; 1-octene and 2-pentanone. In particular, 1-octen-3-ol was detected in all the mold cultures and moldy tobacco samples analyzed. Olfactory evaluation of 1-octen-3-ol indicated a characteristic musty odor and the odor threshold was determined to be approximately 200 ng/ml. The limits of detection for 1-octen-3-ol using GC/TOF-MS and GC/mass selective detector (MSD) in the full-scan mode and selected ion monitoring (SIM) mode were investigated. The CLSA-GC/TOF-MS demonstrates a fast, sensitive and semi-quantitative analytical technique for screening tobacco materials for the presence of mold via chemical markers of microbial contamination.     

Microbial contamination of traditional medicinal plants sold at the Faraday muthi market,Johannesburg, South Africa

The Faraday traditional healers' trading market is the hub of the medicinal plant trade in Johannesburg, South Africa. Modes of harvesting, transporting, storage and distribution of medicinal plants render them susceptible to microbial attack, and thereby make customers, especially patients with compromised immune systems, vulnerable to infections that could increase morbidity and mortality. This study evaluated the microbial contamination on five frequently used traditional medicinal plant species sold by traders in the Faraday market. Bacterial contamination was determined using serial macro dilutions, spread plate and streak plate techniques. Fifteen bacterial contaminants were identified, the most recurrent being Pantoea sp. and five strains of Bacillus spp. (non-pathogenic). There was little variation between contamination levels of the five different traders, and the mean CFU/g per species ranged from 3.03 × 10⁴ (Hypoxis sp.) to 4.22 × 10⁵ (Hydnora abyssinica). While there was no overall significant levels of contamination, the CFU counts for two plant species purchased from one specific trader (viz. H. abyssinica and Acacia xanthophloea) exceeded maximum acceptable contamination limits set by the World Health Organisation (i.e. ≤ 10⁵ to ≤ 10⁷ CFU/g). The levels of contamination varied greatly between the commercially available over the counter product and the plant samples investigated. The microbial types are predominantly opportunistic pathogens. The implementation of good processing practices therefore clearly influences the quality and safety of medicinal products, especially regarding microbial contamination. It is evident that policies and regulations need to be developed and implemented in order to address possible contamination by opportunistic pathogens.     

Using Syndromic Surveillance to Investigate Tattoo-Related Skin Infections in New York City

In response to two isolated cases of Mycobacterium chelonae infections in tattoo recipients where tap water was used to dilute ink, the New York City (NYC) Department of Health and Mental Hygiene conducted an investigation using Emergency Department (ED) syndromic surveillance to assess whether an outbreak was occuring. ED visits with chief complaints containing the key word “tattoo” from November 1, 2012 to March 18, 2013 were selected for study. NYC laboratories were also contacted and asked to report skin or soft tissue cultures in tattoo recipients that were positive for non-tuberculosis mycobacterial infection (NTM). Thirty-one TREDV were identified and 14 (45%) were interviewed to determine if a NTM was the cause for the visit. One ED visit met the case definition and was referred to a dermatologist. This individual was negative for NTM. No tattoo-associated NTM cases were reported by NYC laboratories. ED syndromic surveillance was utilized to investigate a non-reportable condition for which no other data source existed. The results were reassuring that an outbreak of NTM in tattoo recipients was not occurring. In response to concerns about potential NTM infections, the department sent a letter to all licensed tattoo artists advising them not to dilute tattoo ink with tap water.     

Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae

Background  

Mycoplasma hyopneumoniae is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP). Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, Mycoplasma hyopneumoniae, we performed comparative protein profiling of three M. hyopneumoniae strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422.     

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices^1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.Direct-write assembly^4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 μm). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (< 1 μm) can be patterned and assembled into larger arrays and multidimensional architectures.In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.     

A novel case of autogamy and cleistogamy in Dendrobium wangliangii: A rare orchid distributed in the dry‐hot valley

Dendrobium wangliangii is an epiphytic orchid distributed in the Jinshajiang dry‐hot valley in Luquan County, Yunnan Province, China. Most Dendrobium spp. typically have a low fruit set, but this orchid shows a higher fruit set under natural conditions despite the lack of effective pollinators. The pollination biology of the critically endangered D. wangliangii was investigated in this study. A fruit set rate of 33.33 ± 4.71% was observed after bagging treatment in 2017 and a high fruit set rate (65.72 ± 4.44% in 2011; 50.79 ± 5.44% in 2017) was observed under natural conditions, indicating that D. wangliangii is characterized by spontaneous self‐pollination. The anther cap blocked the growing pollinium; thus, the pollinium slid down and reached the stigmatic cavity, leading to autogamous self‐pollination. Specifically, 51.50% of 162 unopened flowers (total 257 flowers) of this Dendrobium species under extreme water‐deficit conditions developed into fruits, suggesting the presence of cleistogamy in D. wangliangii. Here, cleistogamy may represent the primary mode of pollination for this orchid. Spontaneous self‐pollination and specific cleistogamous autogamy could represent major adaptions to the drought and pollinator‐scarce habitat in the Jinshajiang dry‐hot valley.     

Identification of Actinomycetes Producing Phospholipase D with High Transphosphatidylation Activity

Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA–DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.     

Microbiological characterization of a coke oven contaminated site and evaluation of its potential for bioremediation

The soil microbial population of a coke oven site was investigated in order to evaluate its potential for bioremediation. The study was carried out in soil samples with distinct polynuclear aromatic hydrocarbon (PAH) contamination levels, comparing the population profiles constituted by total heterotrophic and PAH-utilizing strains. Isolation of degrading strains was performed with phenanthrene or pyrene as sole carbon sources. The ability to degrade other PAHs, such as anthracene, fluorene and fluoranthene was also investigated. The results showed a reduction of 30% in species diversity and microbial density drops one order of magnitude in contaminated samples. Furthermore, the number of PAH-utilizing colonies was higher in the contaminated area and about 20% of the isolates were able to degrade phenanthrene and pyrene, while this value decreased to 0.15% in uncontaminated samples. Three PAH-degrader strains were identified as: CDC gr. IV C-2, Aeromonas sp. and Pseudomonas vesicularis. The ability of these strains to degrade other PAHs was also investigated.     

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.     

Influence of co-inoculation of bacteria-cyanobacteria on crop yield and C–N sequestration in soil under rice crop

The performance of three selected bacterial strains—PR3, PR7 and PR10 (Providencia sp., Brevundimonas sp., Ochrobacterium sp.) and three cyanobacterial strains CR1, CR2 and CR3 (Anabaena sp., Calothrix sp., Anabaena sp.), and their combinations was evaluated in a pot experiment with rice variety Pusa-1460, comprising 51 treatments along with recommended fertilizer controls. Highest yield enhancement of 19.02% was recorded in T12 (CR2), over control, while significant enhancement in nitrogen fixing potential was recorded in treatments involving combination of bacterial-cyanobacterial strains—T37 (PR3 + CR1 + CR3) and T21 (PR7 + CR1). Organic carbon was significantly increased in all microbe-inoculated treatments, which could be correlated with microbial biomass carbon values and activities of all the enzymes tested in our study. Also, panicle weight and plant biomass were highly correlated with soil microbial carbon. Comparative evaluation revealed the superior performance of strains CR2, CR1 (both Anabaena sp.) and PR10 (Ochrobacterium sp.) in increasing the growth and grain yield of rice and improving soil health, besides N (nitrogen) savings of 40–80 kg ha⁻¹. The study for the first time illustrated the positive effects of co-inoculation of bacterial and cyanobacterial strains for integrated nutrient management of rice crop.     

Microbial quality of equine frozen semen

Bacteriological surveillance is little applied in management of equine frozen semen but it is quite important to verify the microbial contamination in order to find out the chance of transmission of pathology to the mare in AI. Authors describe a qualitative and quantitative analysis for bacterial contamination on long time (3–17 years) equine frozen semen stored in liquid nitrogen. The semen checked, produced in Italy and in another Europe country, was cryopreserved in liquid nitrogen inside sealed plastic straws. One hundred and ten straws were checked out for pathogenic and no pathogenic bacteria, aerobes and anaerobes and fungi (moulds and yeasts). The Total Microbial Charge was quite variable with an average of about 1.4 × 10⁵ CFU/ml. Mostly the microbial agents identified were fungi (17.5%), Enterobacter-coccus spp. (15%), Pseudomonas spp. (6.25%), Stenothophomonas maltophila (6.25%) and anaerobic bacteria like Propionibacterium granulosum (7.5%) and Clostridium spp. (3.75%). 3.75% were unidentified Gram-negative rod and cocci. Streptococcus spp., Staph. aureus, E. coli, Th. equigenitalis and Mycoplasma spp. were not detected. The most represented species were Enterobacter-coccus spp. (1.1 × 10⁵ CFU/ml), St. maltophila (8 × 10⁴ CFU/ml) and Pr. granulosum (7 × 10⁴ CFU/ml) while yeast and even more moulds were little abundant (4.7 × 10⁴ and 3.4 × 10⁴ CFU/ml respectively). The knowledge of equine frozen semen microbial quality is essential to check out transmission of venereal disease and improve the quality of cryopreserved germplasm.     

降解1,3-二甲基-2-咪唑烷酮细菌的分离及功能评价

[背景] 1,3-二甲基-2-咪唑烷酮（1,3-Dimethyl-2-Imidazolidinone,DMI）作为一种强极性非质子溶剂,在生产和应用过程的环境中有稳定残留问题,存在安全隐患。[目的] 分离筛选具有降解DMI能力的微生物菌株,为清除环境中残留的DMI提供优良的微生物菌种资源。[方法] 从DMI生产区域土壤采集样品分离DMI抗性微生物,采用形态学及分子生物学鉴定确定其分类地位,并对DMI降解能力进行测定。[结果] 分离到最高能够耐受5%（体积分数） DMI的微生物菌株,形态学及分子生物学鉴定初步表明获得的菌株DT-1和DT-2为贝莱斯芽孢杆菌（Bacillus velezensis）;全细胞及细胞提取液均具有降解DMI的能力;其中菌株DT-1及其细胞提取液对1%（体积分数） DMI的降解率分别达到48%和68%。[结论] 从DMI生产区域土壤中分离到具有DMI降解能力的芽孢杆菌,不但可为DMI污染的微生物治理提供优良微生物资源,而且扩展了人们对芽孢杆菌生物学功能的认识。