【作者】 王强；

【导师】 王仁卿；

【作者基本信息】 山东大学 ， 生态学， 2010， 博士

【摘要】 采用野外调查、实验室分析相结合的研究方法,对山东省两个典型金属矿区内土壤微生物生态特征及其作为环境生物指示指标的可能性进行了研究,并进步考虑了微生物生态研究中对空间因素的纳入。研究结果对矿区生态修复,微生物作为环境质量评价指标的应用提供了理论和技术依据。主要研究结果如下淄博铁矿研究区重金属污染程度属于轻度水平,只在少数几个样品中检出个别元素超标。相对于微生物指标(生物量碳、基础呼吸、BIOLOG指数等)的样品间差异,传统的污染指数方法灵敏度不够,难于检出相应不同。这表明微生物反映土壤环境状况较为灵敏,受除重金属之外的其他因素或因子影响也较大。在pH值升高的前提下,微生物基础呼吸速率表现出了随重金属含量增加而升高的特性。另外,高生理活性类群却没有对应较高的生物量水平,这说明微生物面临着在繁殖扩增和保证稳定之间的权衡,而这种平衡是随实际环境而异的。利用微生物作为环境指示因子必须充分考虑到环境的各方面影响；在此前提下,使用适当的分析方法,仅可以在一定程度上分析特定环境因素对微生物的影响的大小虽然将微生物指标所提供的信息直接用于环境安全指示有一定困难,但用于对传统环境安全指标的修正是一种很有意义的新手段,可提供有效的补充信息。微生物群落不仅受到重金属污染影响,也会反映土壤理化性质的差异,因此是·种很有潜力的环境安全指示指标。大量的微生物指标可以应用主成分分析等数据压缩方法进行简化,而在进一步确认何种环境因素对微生物造成影响时,可以应用环境向量拟合方法,显著性通过置换检验验证。将整个基于排序的微生物特性指标刻画出的样品差异模式应用于对污染因子权重的确定具有较大现实意义。因为这种通过生物刻画所得到得环境因子权重在一定程度上反映了该因子影响生物指标的能力,较为接近客观对生物体的影响。本研究通过计算发现经过微生物特性修正的内梅罗指数与原始内梅罗指数有较大差异。不仅如此,通过微生物确定每种重金属权重后可以直接计算一种新的环境质量指数——BIO指数,我们发现不论基于重金属全量还是有效态,均与原始内梅罗指数有较大差异,这说明基于微生物的权重计算方式考虑到了更多的有价值信息。提供生物体所包含信息的指标需要仔细研究进行确定。本研究中采用生物量碳、基础呼吸以及基于BIOLOG吸光度的几个多样性指标,这种结合涵盖了生物群落大小,生理活性以及碳源利用能力多样性等几个方面,是一种对微生物群落比较综合的刻画。然而这种混合使用也有缺陷,因为很可能有线性关系的作用影响。另外,所选指标难以代表微生物群落结构,只是对其整体表现从不同角度的刻画,较为粗糙。因此,表征群落结构水平的分子方法可能会有更好的效果。招远金矿的研究表明,部分采样点Pb、Cu和Cd的全量含量要高于二级国家环境安全标准,而平均值则均处于安全水平之内。按国家土壤环境技术规范的标准,50%样品内梅罗指数低于0.7,属于洁净水平,约14%低于1,属于尚清洁,位于1-2的轻度污染样品占33%,仅有一个样品超过2(占3%),达到中度污染水平。在因子分析后,无论全量还是有效态重金属,Cd、Zn和Pb都归入了一类中；全量的Cr、Ni、Fe和Mn含量没有相似性,但在有效态含量中,上述几种重金属大体聚成了一类。分布模式暗示了各类别重金属的来源差异。聚于一类的重金属有较大的可能是来源相同的,比如都由于污水的影响、或都出于粉尘的影响所导致,才形成较为类似的分布模式。在重金属主成分与微生物指标的相关分析中,发现这些主成分对微生物产生的影响并不一致。与以往部分矿区微生物研究中重金属会对微生物具有抑制作用的研究结果不同,若干重金属主成分与生物量碳、基础呼吸以及BIOLOG主成分显示了正相关关系,这说明重金属组分对微生物的生长及活性产生了促进作用。微生物指标会受到多种因素的影响,很容易出现环境因子间交互作用的现象。同淄博铁矿类似,在本研究中,pH对微生物群落展现出较强的影响。相关分析表明,在众多微生物指标中,生物量碳是较为敏感的一种指标。BIOLOG方法敏感度较差,难于监测轻微的环境变化。为了实现使用微生物群落指示环境因素的目的,更多的指标及数据分析方法都是必须的。由于pH值与多个微生物指标出现显著相关关系,我们认为本研究中它是对微生物影响对大的环境因素。不仅如此,pH值和可溶性重金属的第三主成分也有显著负相关关系,而第三主成分的最大载荷是来自Cu含量。这是由于低pH有助于Cu的溶解,因此pH较低的样地Cu溶解性较好。另一方面,高pH有助于提高某些类别微生物活性,对微生物生长较为有利,因此pH值和生物量碳以及可培养细菌均呈显著正相关关系。微生物各指标在低水平污染下反应环境变化的能力有较大差异。结果表明,BIOLOG吸光度变化曲线并不随重金属含量变化而变化,而是较难分辨开。在进一步的相关分析中,BIOLOG的第四和第五主成分与金属主成分有显著相关关系。BIOLOG第四主成分在D-xylose, Pyruvic acid methyl ester and Glycogen上有较大的载荷；第五主成分在β-Methyl-D-glucoside, N-acetyl-D-glucosamine andα-Cyclodextrn上有较大的载荷。这说明金属对微生物碳源利用能力的影响是有较大差异的。不过,在把环境因素作为控制变量纳入偏相关分析后,重金属和碳源利用能力的关系发生了较大变化。这说明环境因素对微生物的影响非常复杂,联系环境的解释必须非常小心。总之,BIOLOG方法在揭示环境的微小变化时比较迟钝,较难发现小的差异。生物量碳与若干环境因素均有显著相关关系,这说明生物量碳是一个比较敏感的微生物指标,可以较为迅速的反应环境变化。当然,单一的指标是不安全的,必须联系尽量全面的指标进行综合考察。在金矿区应用微生物指标对传统环境安全指标进行的修正时,使用可培养细菌及真菌数目作为代替BIOLOG指数的生物参数。在对内梅罗指数的修正中,我们发现了与淄博铁矿不同的趋势。在淄博铁矿中的趋势是从没有差异的样品间检出差异,而在招远则表现出了减小相邻样品间差异的特性。我们认为这是由于淄博取样区地势平缓,生物匀质性较强,强于理化性质的空间异质性,因此有弥合重金属指数的表现。在通过微生物生态特性获得独立的重金属生态效应影响权重,进而获得BIO环境质量指数的分析中,我们发现重金属全量和有效态计算出的BIO指数差异极大。这一现象和淄博铁矿中的结果是一致的。原因在于单用化学分析方法测定的土壤重金属有效态,只能称之为提取态,而不应该称为有效态,因为根本没法弄清楚对什么和在哪种层次有效；另外由于提取方法的差异,有效态和全量重金属的差异自然也非常大。不过虽然如此,有效态还是一种比全量更贴近生物体的概念,也有其特定作用,不过在使用之前应该先厘清到底主要对什么有效,以其更加有针对性。总之,我们认为基于微生物生态特性的指数计算方法更多的考虑到生物体特性的指标更加能够体现有害物质对环境安全的影响。因为生命体形成特定的格局即环境各因素综合作用的结果,以这个结果为参考所确定的有害物质的权重更能体现对生物界的直接作用,是一种非常有意义的指标。上述对微生物群落信息的挖掘中仅应用了排序及由回归方法扩展的环境向量拟合。而现代生态学研究中,空间因素的作用是不可忽视的,微生物生态研究也是如此。这样才能揭示客观生态规律,进而提供更为可靠的环境指示。第四章中我们论述了空间方法在微生物生态研究中的作用,从不同研究角度出发,结合空间统计的作用,对空间统计方法在微生物生态研究中的应用的必要性及现状做了简要评述。介绍了空间自相关性的检验,方差图,Mantel检验,Kriging插值等方法在微生物生态研究中的应用,并论述了微生物研究中的尺度问题。空间分析为我们提供了描述现象的手段,所得到插值图或经验方程让我们更好的了解微生物群落在空间的分布模式。基于这些手段,可以结合具体的环境因素进一步分析生态现象及其背后的生态过程。有意识的使用空间分析手段有助于我们对微生物生态以及整个生态系统的理解及解释,对其分布的空间考察定会对更好了解生态系统作用机制有积极推动作用。而对微生物生态更深入的理解对提供环境质量评价的支持信息也是不可或缺的保证。更多还原

【Abstract】 The dissertation presented studies of two typical metal deposits in ShanDong province. With field investigation and laboratory studies,we made surveys of microbial ecology in these two deposits area and explored the potentiality of them to be complemental environmental quality indicators. Spatial methods in microbial ecology were also well summarized for further study. The results provided theoretical and practical foundation to deposits’ ecological restoration,microbial community behavior and the application of environmental quality assessment.The main results are presented as follows:The Zibo iron deposits were slightly contaminated and traditional environmental quality indices were hard to detect sensitive pollutions,not mention their biological effects.Biological indices were sensitive to environmental changes and can be use as complemental environmental safety indicators.In mild contaminated area where traditional pollution indices can hardly detect changes,microbial behaviors changed significantly according to the environmental conditions.The property thus accomplished the microbes’ potentiality to be used as complemental safety indicators of environment.The effects of heavy metals and environmental parameters could be detected by environmental fitting methods and their effects as could be estimated by random permutation test.In this mild degree of contamination as Zibo iron deposit area, while Nemerow indices kept still in most of samples, microbial behaviors exhibited a dazzling diversity. With the increase of pH,BAS of microbial community could appear stimulated by certain kinds of metals.Higher physiological activities may not lead to greater biomass,the trade-off strategy between sustaining and developing may change with particular environments. Microbial indices could be affected by various environmental factors; therefore, it should be very careful to use the function of microbes to monitor specific contaminations.However, with proper analysis method, the effects from different environment factors could be evaluated.On the whole,the submission of microbial behaviors in detecting holistic qualities shows great promise for the future,however, the development of new relative methods and more broad data collection should also be required.Also it is difficult to use microbial parameters as environmental quality indices independently, but their application to supply complemental information to traditional environmental quality index is feasible and meaningful.The ordination patterns base on microbial parameters are valuable to determine the weighting of environmental factors,including toxic substances, in the influencing of environment, emphasizing on the biological aspects.We can modify traditional environmental quality indices and establish completely new environmental quality index, and here we named it BIO Index.In our study, the modified Nemerow Index had great differences with the origin one and that indicated the ignorance of traditional environmental index to biological influences. Furthermore, we calculated the BIO index based on total metal contents and available contents separated. The results were all different from the Nemerow index which suggested the effect of considering microbial behavior by imposing their weighting to toxic parameters.Of course, the selection of microbial parameters to information supplement must be of great consideration. In this study, we used microbial carbon, soil basic respiration, average well color development and Shannon index developed from BIOLOG panels. The collection represented the microbial community size, physiological activity and the ability in carbon utilization, which represented a comprehensive description of microbial activities. However, the mix up of parameters of different aspect can lead to confusion in data analysis for the co linearity in the origin data can not be detected primarily. In addition, the microbial parameters in our study were too rough to represent the subtle community structures.In the study of Zhaoyuan gold deposit, a broad rang of sample collection were applied. In this area, average concentrations of some kinds of metals (Pb, Cu and Cd) were higher than the safe level according to Chinese national environmental quality standard for soils.Single metal effects on microbes can be determined in laboratory but combined effects in nature fields were hard to distinguish. Statistic analyses were applied to determine the combing effects of heavy metals. Metal data was first explained by factor analysis so the original distribute structure of these metals could be detected and their combing effects can be determined. We found that combing effects of heavy metals in Zhaoyuan gold deposit can be detected by factor analysis and so the weighing of different metals can be estimated. Cd, Zn, and Pb were categorized together either in total and soluble metals. Cr, Ni, Fe, Mn did not show similarity in total metal contents but strongly assembled in soluble metals. Such a separation of metals showed a common source/origin of those which were clustered together, and implied their similar distribution patterns.However, PCs extracted with such a method did not correlate with microbial parameters in an accordant way; most of them even had positive correlation with Cmic BAS, BIOLOG PCs. That means a higher concentration of heavy metals promote microbes’growth, which is in violent contrast with previous studies. However, these studies were carried out in conditions where study areas were deeply polluted and so metal concentration played a major role in influencing microbial behavior. In our study, as all samples were collected from farm filed, metal concentration did not go to such a high level that it could influence microbes in an obvious mode.In addition, a higher concentration of heavy metals did not always reduce microbial activities.It had been found that certain microbial species, asα-subdivision of Proteobacteria and the genus Burkholderia includes species may even prosperous under heavy metal polluted conditions,so the role of heavy metals should be carefully considered according to practical situations.Microbial indices could be affected by various environmental factors and the function of microbes to monitor slight processing metal contaminations was limited. Just as in Zibo deposit, in this case study, pH showed more influence to microbial communities and among those indices Cmic appeared to be the most sensitive parameter. There were congenitally deficient in BIOLOG methods and explanations of the data should be careful,with fully consideration of the situation in the study area.Development of new methods and more broad data collection should be necessary. We regard the dominate factor of influence here was the pH values, for it had the most close relationship with many parameters. Significant negative correlation was found between pH and PC3 of soluble metals, which had the largest loading on Cu. That is because lower pH could be helpful to solubilize Cu. In fact, soluble metal contents were easier show higher value as pH declined for the lower pH would increase the availability of heavy metal. A relatively higher pH value could also promote microbial activities,that was why significant positive correlations were found between pH value and microbial carbon and culturable aerobic heterotrophic bacteria numbers.Also microbial parameters could hardly reflect metal contaminations on the whole, their differences of sensitivities still could be observed. In our study, the incubation curve of AWCD calculated from BIOLOG data did not separate well according to heavy metal contents. In further analysis, the fourth and fifth components were influenced by metal components significantly.The fourth component had high loading on D-xylose, Pyruvic acid methyl ester and Glycogen; the fifth component had high loading onβ-Methyl-D-glucoside, N-acetyl-D-glucosamine andα-Cyclodextrn. That proved an uneven importance of the carbon source utilization.However, the relation between carbon source utilization and heavy metals changed when other soil properties were included as control variables. That suggests the influence to microbes of soil were complicated and the interpretation should be careful. On the whole, BIOLOG methods were incapable to reveal subtle changes of environment. Cmic showed significant correlations with many environmental parameters and positive correlations were found between Cmic and metal PCs. It may be regarded as also Cmic acted sensitive to environment but the interpretation of it must be careful. Culturable microbes can be used as a complement and reference to the uncultured methods.The application of microbial parameters as complemental environmental quality indices showed significant results.In this case,we used culturable microbes to replace BIOLOG indices in the ordination analysis to get another aspect of description.We found the modification of Nemerow index shown different trends compared to Zibo deposit for the samples.While in Zibo deposit Nemerow index became more different in adjacent samples,Zhaoyuan samples near each other turned to be more similar. We deemed it was caused by the relative consistence in farm land so microbes in the same area may be liable to have similar activities.After the calculation of BIO index,we found totally different trends based on total and available metal contents, as the same in the first study.The reason came from the definition and extraction methods of available metals. Metals soluble in certain liquid were called available form but it is hard to find out how available they are to organisms. Further more, driven by the difference of different extraction method, available contents were for sure would have no determinate relationship to total metal concentrations, let along linear relationship. That is why available metal contents showed different environmental quality indices to total metal contents. Of course, the applications of the concept of "available concentration" were still helpful for they are after all more close to the survival of organisms. We can use BIO index to define their environmental influences and make careful survey of to what extent they are available.Based on the two cases, we believe the BIO index give more thoughts to biological conditions and is valid in reflect environmental safety. The theory is biological indices are honor to environmental influence and their behaviors is the results of different environmental portrays. In a simple way we integrated the most reliable information to calculate environmental quality index,and we deem it is a significative indicator.The biological informations integrated in environmental quality index were mainly based on ordination method in this study. However, descriptions of biological patterns without spatial consideration were somehow incomplete.The applications of spatial analysis methods are becoming hot of the press in microbial ecology studies.However, due to subject barriers and the minuteness of microbes,spatial concepts did not receive adequate attention in China.So we made a summary of spatial methods in microbial ecology studies.Of course,due to the research technique limitations and the special characteristic of microbes, most studies are only at the exploratory stage while the relative methods are also developing. This chapter focuses on the intents of spatial analysis,combined with the purpose of microbial ecological studies, and gives a presentation of the submission of spatial methods in the microbial ecology. We introduced the descriptions of spatial autocorrelation in microbial ecology studies, including Moran’I and Geary’s c, Mantel test and the use of variogram analysis. Kriging was refereed as a tool to predict microbial patterns and the scale in microbial studies were discussed too. Our work will be helpful in the understanding of applying spatial method in microbial ecology and give rise to the attention of this kind of method. Certainly, biological information with spatial consideration will be helpful to give references to environmental quality indices.更多还原