Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 +/- 0.07 mol H-2/mol glucose (mean +/- S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 +/- 0.03 mol H-2/mol glucose, 0.17 +/- 0.01 mol H-2/mol glucose, 0.11 +/- 0.01 mol H-2/mol glucose and 0.20 +/- 0.04 mol H-2/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp., However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.

Plankton Community Succession in Artificial Systems Subjected to Cyanobacterial Blooms Removal using Chitosan-Modified Soils

Using artificial systems to simulate natural lake environments with cyanobacterial blooms, we investigated plankton community succession by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting and morphological method. With this approach, we explored potential ecological effects of a newly developed cyanobacterial blooms removal method using chitosan-modified soils. Results of PCR-DGGE and morphological identification showed that plankton communities in the four test systems were nearly identical at the beginning of the experiment. After applying the newly developed and standard removal methods, there was a shift in community composition, but neither chemical conditions nor plankton succession were significantly affected by the cyanobacteria removal process. The planted Vallisneria natans successfully recovered after cyanobacteria removal, whereas that in the box without removal process did not. Additionally, canonical correspondence analysis indicated that other than for zooplankton abundance, total phosphorus was the most important environmental predictor of planktonic composition. The present study and others suggest that dealing with cyanobacteria removal using chitosan-modified soils can play an important role in controlling cyanobacterial blooms in eutrophicated freshwater systems.

Genetic diversity of plankton community as depicted by PCR-DGGE fingerprinting and its relation to morphological composition and environmental factors in lake donghu

To collect information about the genetic diversity of the plankton community and to study how plankton respond to environmental conditions, plankton samples were collected from five stations representing different trophic levels in a shallow, eutrophic lake (Lake Donghu), and investigated by PCR-DGGE fingerprinting. A total of 100 bands (61 of 16S rDNA bands and 39 of 18S rDNA bands) were detected. The DGGE bands unique to any single station accounted for 38% of the total bands, whereas common bands detected at all five stations accounted for only 11%. Using UPGMA clustering and MDS ordination of DGGE fingerprints, stations I and II were found to initially group together into one cluster, which was later joined by station V. Stations III and IV were isolated into two separate groups of one station each. Some differences in grouping relationships were found when analysis was completed on the basis of chemical characteristics and morphological composition, with zooplankton composition showing the greatest variability. However, the most similar stations (I and II) were always initially grouped into one cluster. Moreover, stations that exhibited the same or similar trophic level (stations III and IV), but different concentrations of heavy metals, were further differentiated by the DGGE method. Results of the present study indicated that PCR-DGGE fingerprinting was more sensitive than the traditional methods, as other studies suggested. Additionally, PCR-DGGE appears to be more appropriate for diversity characterization of the plankton community, as it is more canonical, systematic, and effective. Most importantly, fingerprinting results are more convenient for the comparative analyses between different studies. Therefore, the use of the described fingerprinting analysis may provide an operable and sensitive biomonitoring approach to identify critical, and potentially negative, stress within an aquatic ecosystem.

辽河干流底质微生物群落结构及芘降解菌研究

辽宁省是以石油化工、煤炭化工和钢铁工业为主的重工业基地。辽河流域近年来经济发展迅速，城市化水平不断提高，但由于产业结构的不合理和污染治理水平的相对滞后，致使辽河流域水体污染严重。对辽河流域水体污染状况、污染物化学与生物学的相互作用、微生物群落结构与功能的关系开展调查研究，对开展污染水体的生物生态修复具有重要的指导意义。 本论文选取辽河流域干流8个水文监测站点的不同时期（丰水期和平水期）底质为研究对象，调查了有机污染物（总油TPHs）和有毒污染物（多环芳烃PAHs）污染程度以及主要来源；采用变性梯度凝胶电泳（DGGE）和磷脂脂肪酸（PLFA）两种分析方法，对其微生物群落结构及多样性进行了分析；并以13C标记的菲和芘为代谢底物，以PLFA为生物标记物，采用气相色谱-稳定同位素比率质谱（GC-c-IRMS）分析技术，鉴定了底质样品中参与菲和芘代谢的主要微生物类群；并利用不同的吸附性载体进行了芘降解菌的富集和筛选。 研究结果表明： 1）平水期总石油烃污染比丰水期严重，TPH 含量分别在276.1～560.6mg/kg（平水期）和157.9～462.2mg/kg（丰水期）之间，辽河入海口TPH污染最重；PAHs含量分别在124.1～270.4ug/kg（平水期）和93.5～209.1ug/kg（丰水期）之间；主要来源于石油类污染物和化石燃料的热解，汽车尾气的污染等。 2) 采用DGGE和PLFA两种方法分析微生物群落结构得到基本一致的结果。微生物多样性与总石油烃含量、总多环芳烃含量无显著相关性，多样性指数是多种污染物和环境因子综合影响的结果。 3) 稳定同位素代谢示踪实验表明，底质中存在菲和芘的降解菌群；参与菲和芘降解的微生物均以G-细菌为主，真菌次之；G+细菌和放线菌也参与代谢；参与菲和芘代谢的菌群有一定的相似性。 4) 利用不同吸附性载体从污染底质样品中筛选到6株降解菌。

溶藻菌群对铜绿微囊藻的溶藻效应及其作用机理研究

水华暴发是一个世界性的问题，近年来在发展中国家显得尤其严重。水华暴发给环境和公众健康带来巨大灾难，一些蓝藻产生的毒素可以造成鱼类、鸟禽和家畜的死亡，而臭名昭著的微囊藻产生的微囊藻毒素更是有强烈致癌效应。因此，寻找控制水华藻类的有效方法非常迫切。在利用物理和化学方法处理不甚理想的情况下，利用溶藻细菌控藻成为一个新的研究方向。溶藻细菌一般直接从富营养化水体中分离，杀藻活力对有害蓝藻具有较强的选择性而不危害其它生物，尤其适合在水华发生初期使用，可以在短时间内达到阻止藻类增殖的效果。本研究富集分离到一个高效溶解铜绿微囊藻的溶藻菌群，对其溶藻效应和溶藻机制进行了探索研究。 1溶藻菌群的富集筛选及其溶微囊藻效果 富集筛选得到一个有明显抑藻效果的菌群，它对铜绿微囊藻有显著溶藻效果。与对照组相比，加入富集的溶藻菌后，第4 d开始出现溶藻现象，6~8 d出现明显的溶藻效果，8 d后测得叶绿素去除率在85%以上。 2 溶藻菌群的作用范围及溶藻特性 富集分离到的溶藻菌群对铜绿微囊藻和念珠藻有显著溶藻作用，对水华微囊藻和其它几株受试微囊藻没有明显溶藻效应。该溶藻菌群不仅可以在液体中溶解铜绿微囊藻，生长在固体平板上的藻苔也有一定的溶藻效应，生成溶藻空斑。保证快速溶藻的最大稀释度可以达到1/100, 000。 3 环境因子对菌群溶藻效力的影响 试验发现，不同的pH、温度、和光照条件下，溶藻菌群溶藻效力明显不同，且不同种类的氮源对其溶藻作用也有一定影响。这些条件对该菌群溶藻作用的影响，在相当的程度上可能取决于它们对藻和细菌两者的生长状况的影响综合。 4 溶藻菌群的溶藻作用机理 溶藻菌液过滤除菌和煮沸灭菌处理后溶藻液，未见明显的溶藻效果，只有原液具有很好的溶藻效果。因此可初步确定，蓝藻细胞的溶解可能是由溶藻菌直接接触藻细胞产生的作用效果。显微镜观察发现，细菌在溶藻的过程中频繁地接触藻细胞并侵入藻细胞，破坏进而裂解杀死藻细胞。这也进一步说明了此溶藻菌是通过直接方式杀藻。 5 溶藻菌群的菌群结构解析 分离有溶藻效果的纯菌的多次尝试都没有成功。结合DGGE和16S rDNA文库综合分析发现：Rubritepida菌，假单胞菌和鞘氨醇单胞菌是存在于铜绿微囊藻中的三种伴生细菌。加入富集的溶藻菌群后，菌群结构发生明显的变化，Rubritepida菌、假单胞菌消失，混合菌群则包含未培养黄杆菌，鞘氨醇单胞菌和噬氢菌，其中黄杆菌是优势菌群，并且细菌种群结构的变化与藻细胞消亡之间有显著的相关性。通过菌种的分离鉴定与DGGE和16S rDNA文库的测序结果比较，一些未培养菌可能在溶藻过程中起重要调控作用。 6 溶藻细菌控藻应用基础 (1) 扩大规模的模拟水华实验进一步确定了细菌对微囊藻的强烈溶解作用。 (2) 铜绿微囊藻(Microcystis aeruginosa 905, zc)、微囊藻(Microcystis spp., zd)和溶藻菌群共培养试验表明，zc可以抑制zd生长，而溶藻菌群可以溶zc。 本研究是第一次报道混合菌群的溶藻效应。该溶藻菌群对带有藻际细菌的铜绿微囊藻具有高效的溶藻效力，表明它对自然界中存在的带菌铜绿微囊藻和其它一些蓝藻的生消具有一定的控制作用。对进一步研究菌藻关系与生态学作用，以及对富营养化湖泊和水库水体中蓝藻暴发的防控，该菌群具有一定的应用潜力。 Cyanobacterial blooms break out frequently all over the world, especially in developing countries. Blooms create enormous disasters to public health and to the environment. Some cyanobacterial blooms produce extremely toxic substances that have killed fish, domestic animals and birds. It has been well known that microcystins, a hepatoxin produced by Microcystis, can promote tumors in humans. So it is very important to find an effective method for controlling the growth of the bloom-forming algae. Measures for controlling such kind of algae include physical, chemic and biologic means, but the former two may damage the aquatic environment and require high-energy inputs. The alternative approach for the elimination of nuisance algae involves the application of algicidal bacteria. The algicidal bacteria, which are nontoxic to other organisms and most of which are isolated from the eutrophic lake in situ, may be potential microbial algaecides. In the initial stages of the water blooms, they are able to restrain the biomass or multiplication of the bloom-forming algae in a short time. In order to use algicidal bacteria to suppress blooms of M. aeruginosa, we isolated a bacterial culture capable of lysing the noxious cyanobacteria M. aeruginosa. In this paper we described some properties of the bacterial culture and its growth-inhibiting or algicidal effects on the growth of M. aeruginosa, and investigated its algicidal mechanisms. 1 Enrichment of a microbial culture that lyses Microcystis aeruginosa A mixed bacterial culture was isolated from a hypereutrophic pond and showed significant algicidal activity against the noxious Microcystis aeruginosa. Algae lysis would be seen obviously 4 days later when the algae culture was killed and became yellow contrast to no-addition controls, and chlorophyll a (chl-a) reduction went beyond 85% 8 days later. 2 The host range and some other algicidal feature of the mixed algicidal culture. Microcystis aeruginosa, Nostoc sp., were susceptible to the mixed algicidal culture, while the lytic effects of this mixed culture on Microcystis flos-aquae and some other tested Microcystis were feeble.The algicidal culture can not only lyse M. aeruginosa in liquid media, but aslo lyse M. aeruginosa lawns on soft agar plates and form plaques. The maximun dilution of the mixed culture required for rapid Microcystis lysis is 1/100, 000. 3 Influences of environmental factors such as pH, temperature, illumination, and the nitrogen source on the lytic activity of the mixed bacterial culture on Microcystis aeruginosa. In our investigations, it was shown that the lytic activity of the mixed bacterial culture on Microcystis aeruginosa was straightly correlated with pH, temperature, illumination, as well as the nitrogen source in the medium. The impacts of these environmental factors on the algicidal activity of the mixed bacterial culture, to a certain extent, may depend on both the algal and the bacterial growth rates under the tested environmental conditions. 4 The mechanisms of algal cell lysis by the algicidal bacteria Death was detected when the mixed bacterial culture was added to the algal culture, but not when only the culture filtrate or autoclaved bacterial culture was added. This indicates that the mixed bacterial culture did not release extracellular products inhibitory to Microcystis aeruginosa. In addition, under the microscope, we observed frequent contacts btween bacteria and algae cells, and some bacteria can even penetrate into target algal cells and destroyed them. These results may suggest that the bacterium kill the alga by direct contact. 5 Molecular Characterization of the algicidal bacterial culture Attempts for isolation of pure bacterium or bacteria from the enrichment culture responsible for Microcystis lysis have so far been failed. Based on PCR-DGGE (denaturing gradient gel electrophoresis) and 16S rDNA clone library analysis, Rubritepida sp., Pseudomonas sp. and Sphingomonas sp., as accompanying bacteria, were existed in M. aeruginosa. The bacterial community in M. aeruginosa showed significant change after adding the enrichment culture, where uncultured Flavorbacterium sp., Sphingomonas sp. and Hydrogenophaga sp. were observed, and the uncultured Flavorbacterium sp. became a dominant species. The obvious correlation can be seen between change of bacterial population and extinction of M. aeruginosa. Compared identification of pure bacterium with sequencing of DGGE bands and the clone distribution of the clone libraries, it was inferred that some uncultured bacteria were probably play an important role in controlling the growth and abundance of M. aeruginosa. This report is the first example of a mixed bacterial culture with the ability to lyse M. aeruginosa. 6 Further study for algae control by applications of algicidal bacteria (1) Algae lysis would be seen obviously 6 days later when the algae culture was killed and became yellow contrast to no-addition controls, and chlorophyll a (chl-a) was reducted to a low level 20 days later in the simulated water bloom experiments. (2) The growth of Microcystis sp. (zd) was restrained by Microcystis aeruginosa 905 (zc) when they were co-cultured together, and zc was lysed by the algicidal bacterial culture. This report is the first example of a mixed bacterial culture with the ability to lyse M. aeruginosa, and its algicidal activity remained high against non-axenic tested M. aeruginosa, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of M. aeruginosa and other cyanobacteria. Such bacteria could also potentially be used as agents to prevent the mass development of cyanobacteria in eutrophic lakes and reservoirs.

Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Soil microbial community analysis using denaturing gradient gel electrophoresis

The most biological diversity on this planet is probably harbored in soils. Understanding the diversity and function of the microbiological component of soil poses great challenges that are being overcome by the application of molecular biological approaches. This review covers one of many approaches being used: separation of polymerase chain reaction (PCR) amplicons using denaturing gradient gel electrophoresis (DGGE). Extraction of nucleic acids directly from soils allows the examination of a community without the limitation posed by cultivation. Polymerase chain reaction provides a means to increase the numbers of a target for its detection on gels. Using the rRNA genes as a target for PCR provides phylogenetic information on populations comprising communities. Fingerprints produced by this method have allowed spatial and temporal comparisons of soil communities within and between locations or among treatments. Numerous samples can be compared because of the rapid high throughput nature of this method. Scientists now have the means to begin addressing complex ecological questions about the spatial, temporal, and nutritional interactions faced by microbes in the soil environment.

Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides

Penaeid shrimp, as an invertebrate, relies on the innate immunity to oppose the microbial invaders. Antimicrobial peptides (AMP) are an integral component of the innate immune system in most organisms and function as an early first line of defense against pathogens, but the knowledge about the pathways to regulate the shrimp AMP gene expression is still absent up to date. In the current study, a Relish homolog (FcRelish) was cloned from Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcRelish consists of 2157 bp, including 1512 bp open reading frame, encoding 504 amino acids. The predicted molecular weight of FcRelish is 57 kDa, and the theoretical PI is 7.00. Spatial expression profiles showed that FcRelish had the highest expression levels in the hemocytes and lymphoid organ. Both Vibrio anguillarium and Micrococcus lysodeikticus stimulation to shrimp can affect the transcription profile of FcRelish. Silencing of FcRelish through DsRNA interference can greatly change the transcription profile of AMP. Therefore, we suggest that FcRelish identified in the present study is closely related to the transcription of AMP, and then we inferred that Imd pathway might exist in shrimp. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of a C-type lectin from the bay scallop Argopecten irradians

C-type lectins are Ca2+ dependent carbohydrate-recognition proteins that play crucial roles in the invertebrate innate immunity, such as nonself recognition, activation of proPO system, antibacterial activity, promotion of phagocytosis and nodule formation. In this study, a novel C-type lectin of bay scallops Argopecten irradians (Ai Lec) was identified using expressed sequence tag (EST) and RACE techniques. The Ai Lec cDNA encoded a polypeptide of 171 amino acids with a putative signal peptide of 21 amino acid residues and a mature protein of 150 amino acids. The deduced amino acid sequence of Ai Lec was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 131 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. The expression of Ai Lec transcript was dominantly detected in the hepatopancreas and slightly detected in the haemocytes of normal scallops. 6 h after Vibrio anguillarum-challenge and 8 h after Micrococcus luteus-challenge, the temporal expression of Ai Lec mRNA in hemocytes was increased by 4.4- and 3.6-folds, respectively. The results suggested that Ai Lec was a constitutive and inducible acute-phase protein and might be involved in immune response to Gram-negative and Gram-positive microbial infection in bay scallop A. irradians.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Identification of nonlinear dynamic systems: Time-frequency filtering and skeleton curves

The nonlinear behavior varying with the instantaneous response was analyzed through the joint time-frequency analysis method for a class of S. D. O. F nonlinear system. A masking operator an definite regions is defined and two theorems are presented. Based on these, the nonlinear system is modeled with a special time-varying linear one, called the generalized skeleton linear system (GSLS). The frequency skeleton curve and the damping skeleton curve are defined to describe the main feature of the non-linearity as well. Moreover, an identification method is proposed through the skeleton curves and the time-frequency filtering technique.

An Amino Acid Substitution Matrix for Protein Conformation Identification

Amino acid substitution matrices play an essential role in protein sequence alignment, a fundamental task in bioinformatics. Most widely used matrices, such as PAM matrices derived from homologous sequences and BLOSUM matrices derived from aligned segments of PROSITE, did not integrate conformation information in their construction. There are a few structure-based matrices, which are derived from limited data of structure alignment. Using databases PDB_SELECT and DSSP, we create a database of sequence-conformation blocks which explicitly represent sequence-structure relationship. Members in a block are identical in conformation and are highly similar in sequence. From this block database, we derive a conformation-specific amino acid substitution matrix CBSM60. The matrix shows an improved performance in conformational segment search and homolog detection.

Identification of Nonlinear Systems Through Time-Frequency Filtering Technique

In the previous paper, a class of nonlinear system is mapped to a so-called skeleton linear model (SLM) based on the joint time-frequency analysis method. Behavior of the nonlinear system may be indicated quantitatively by the variance of the coefficients of SLM versus its response. Using this model we propose an identification method for nonlinear systems based on nonstationary vibration data in this paper. The key technique in the identification procedure is a time-frequency filtering method by which solution of the SLM is extracted from the response data of the corresponding nonlinear system. Two time-frequency filtering methods are discussed here. One is based on the quadratic time-frequency distribution and its inverse transform, the other is based on the quadratic time-frequency distribution and the wavelet transform. Both numerical examples and an experimental application are given to illustrate the validity of the technique.

Some advances in study of crack identification problems

In the present paper, the crack identification problems are investigated. This kind of problems belong to the scope of inverse problems and are usually ill-posed on their solutions. The paper includes two parts: (1) Based on the dynamic BIEM and the optimization method and using the measured dynamic information on outer boundary, the identification of crack in a finite domain is investigated and a method for choosing the high sensitive frequency region is proposed successfully to improve the precision. (2) Based on 3-D static BIEM and hypersingular integral equation theory, the penny crack identification in a finite body is reduced to an optimization problem. The investigation gives us some initial understanding on the 3-D inverse problems.