A Rapid Microtiter Plate Method To Measure Carbon Dioxide Evolved from Carbon Substrate Amendments so as To Determine the Physiological Profiles of Soil Microbial Communities by Using Whole Soil

Sole-carbon-source tests (Biolog), designed to identify bacteria, have become very popular for metabolically fingerprinting soil microbial communities, despite disadvantages associated with the use of carbon source profiles that primarily select for fast-growing bacteria. In this paper we describe the use of an alternative method that combines the advantages of the Biolog community-level physiological profile (CLPP) method, in which microtiter-based detection plates are used, with the ability to measure carbon dioxide evolution from whole soil. This method facilitates measurement over short periods of time (4 to 6 h) and does not require the extraction and culturing of organisms. Deep-well microtiter plates are used as test wells into which soil is placed. The apparatus to fill the deep-well plates and interface it with a second removable detection plate is described. Two detection systems, a simple colorimetric reaction in absorbent alkali and scintillation counting with radioactive carbon sources, are described. The methods were compared to the Biolog-CLPP system by using soils under different vegetation types and soil treated with wastewater sludge. We aimed to test the hypothesis that using whole soil would have specific advantages over using extracts in that more immediate responses to substrates could be obtained that would reflect activity rather than growth. The whole-soil method was more rapid and gave earlier detection of C source use. Also, the metabolic fingerprints obtained could discriminate between sludge treatments.     

Analysis of the bacterial strains using Biolog plates in the contaminated soil from Riyadh community

Routine manufacture, detonation and disposal of explosives in land and groundwater have resulted in complete pollution. Explosives are xenobiotic compounds, being toxic to biological systems, and their recalcitrance leads to persistence in the environment. The methods currently used for the remediation of explosive contaminated sites are expensive and can result in the formation of toxic products. The present study aimed to investigate the bacterial strains using the Biolog plates in the soil from the Riyadh community. The microbial strains were isolated using the spread plate technique and were identified using the Biolog method. In this study we have analyzed from bacterial families of soil samples, obtained from the different sites in 5 regions at Explosive Institute. Our results conclude that Biolog MicroPlates were developed for the rapid identification of bacterial isolates by sole-carbon source utilization and can be used for the identification of bacteria. Out of five communities, only four families of bacteria indicate that the microbial community lacks significant diversity in region one from the Riyadh community in Saudi Arabia. More studies are needed to be carried out in different regions to validate our results.     

基于梯度稀释法分析细菌多样性对土壤碳代谢的影响

为探究土壤微生物多样性对土壤碳代谢过程的影响,利用梯度稀释法(处理D1、D3和D5分别为稀释10^-1、10^-3和10^-5倍)改变土壤样品中原始土壤微生物群落的多样性,以探究土壤微生物群落多样性减少对土壤碳代谢的影响。进行为期6周的预培养实验,以消除梯度稀释法对土壤样品中微生物群落丰度的影响,并通过Q-PCR和高通量测序测定预培养结束后3种土壤样品中细菌丰度及其基因多样性指数(ACE、Chao1、Shannon),以验证预培养实验结果。后加入等量葡萄糖(0.5g/100g干土)继续培养,并于培养期间测定3种处理土壤的碳矿化速率,进行biolog ECO板实验,分析计算各土壤样品中细菌的功能多样性指数(Shannon指数(H)、Simpson指数(D)、McIntosh指数(U))及碳源代谢强度。结果表明:(1)3种处理土壤样品碳矿化速率及累积碳矿化量大小排序为:D1> D3> D5,且D1与D3、D5处理均有显著差异(P<0.05)。(2)D1处理下土壤样品中微生物群落的孔平均颜色变化率(AWCD)、功能...     

中国东北黑土土壤剖面微生物群落碳源代谢特征

利用Biolog Eco微平板培养法,对中国科学院海伦农田生态系统国家野外科学观测研究站农田(CL)、恢复草地(GL)和人工林地(FL)土壤剖面不同深度土壤中微生物群落碳源代谢特征进行研究。理化性质结果显示,有机碳、全氮和碱解氮含量随深度增加逐渐减少,p H则是上层土壤低于底层。可培养微生物数量从表层(0—20 cm)到底层(180—200 cm)逐渐减少,在表层(0—20 cm)3种可培养微生物数量均为草地农田林地。可培养微生物主要生活在近地表0—60 cm土层中,在60—200cm土层中3种利用方式下可培养细菌、真菌和放线菌数量基本相同。Biolog结果显示,在0—40 cm土层中微生物群落活性最大,底层(180—200 cm)土壤微生物群落活性最小。3种利用方式剖面微生物群落Shannon多样性指数和碳源利用数量从表层到底层逐渐减少,并且与SOC和TN呈极显著正相关关系(P0.05)。与农田相比恢复草地和人工林地剖面20—40 cm土层中微生物群落对各类碳源的利用强度都较高,说明没有农业机械作业的植被自然生长条件下根系会打破原来农田中的犁底层,促进表层(0—20 cm)以下微生物群落活性。碳源利用率和主成分分析结果表明长期不同植被覆盖已经改变了剖面微生物群落碳源代谢特征,而且根系已经影响到100 cm的微生物群落,但还没有影响到180—200cm中的微生物群落。     

A complementary approach to identifying and assessing the remediation potential of hydrocarbonoclastic bacteria

The isolation and assessment of hydrocarbonoclastic bacteria often represents a key strategy in the bioremediation of hydrocarbon-contaminated sites. However the isolation and assessment of such bacteria is often a lengthy and expensive procedure. The aim of this study was to identify potential isolates for use in the remediation of hydrocarbon contaminated sites using a combination of selective isolation plating, the Biolog system and subsequent multivariate analyses. The use of weathered oil as the main C source restricted the number of isolates growing to 5×10(2)CFUg soil(-1). Isolates (n=96) were then assessed individually using Biolog MT2 plates with seven different hydrocarbons (dodecane, tridecane, hexadecane, octadecane, eicosane, naphthalene and phenanthrene). The results indicated that all isolates were able to grow on at least one hydrocarbon from the seven chosen. This confirmed that the isolation media developed was selective in isolating hydrocarbonoclastic bacteria only. Cluster analysis of Biolog data separated the isolates into two discrete clusters with cluster 2 identifying hydrocarbonoclastic bacteria that are effective in degrading a variety of contaminants. Further study on the isolates from cluster 2 was carried out based on their phylogenetic analysis. Phylogenetic analysis of 28 bacterial isolates from cluster 2 based on the 1500bp sequences from 16S rDNA genes using MRBAYES confirmed all isolates as being hydrocarbonoclastic, providing supportive evidence that isolates from cluster 2 have a potential use in bioremediation. This approach could improve both the speed and efficiency of the commercial bioremediation process.     

The effects of pesticides on the diversity of culturable soil bacteria

The numbers of culturable soil bacteria in plots that had received either no pesticides or the full combination (aldicarb, chlorfenvinphos, benomyl, glyphosate, plus chlorotoluron or triadimefon) over a 20 year period were compared. Differences were very small although there were consistently higher numbers on the treated plot, possibly reflecting the greater crop yields which had been reported previously. There was no significant difference in numbers of bacterial colonies with homology to a nif gene probe in soils from the two plots. Genetic fingerprinting of Pseudomonas fluorescens isolates from the plots, using ERIC-PCR, showed that the dominant strains in the two populations were not the same although there was no obvious difference in the degree of diversity. Substrate utilization by microbial populations from the two plots was compared using Biolog plates. The population from the pesticide-treated plot showed a higher rate of substrate utilization which could reflect a slightly higher inoculum of heterotrophic bacteria, but could also indicate greater metabolic diversity in the population.     

长期围封和自由放牧对高寒草甸土壤微生物群落结构及碳源代谢多样性的影响

土壤微生物群落结构和代谢多样性对于维持草地生态系统的功能和稳定性具有重要意义.本研究运用磷脂脂肪酸法(PLFA法)和Biolog微平板技术,研究了青藏高原长期围封和自由放牧2种草地利用方式下的土壤微生物群落结构和群落碳代谢多样性的差异以及影响机制.结果表明: 1)长期围封和自由放牧草地上的土壤微生物群落结构和碳代谢多样性存在显著差异;2)长期围封显著增加了土壤中总PLFA、细菌、真菌和放线菌生物量;3)长期围封草地土壤微生物的碳代谢活性、多样性和丰富度高于自由放牧草地,而均匀度低于放牧草地;4)与自由放牧相比,长期围封显著增加了土壤微生物对聚合物类、糖类、羧酸类、胺类碳源的利用;5)冗余分析表明,植被盖度显著影响了微生物的群落结构和碳代谢多样性.长期围封草地的土壤微生物量、碳代谢多样性指数和丰富度均高于自由放牧土壤,表明长期围封有利于提高草地生态系统中土壤微生物群落及其对碳源代谢的多样性.     

黄土高原石油污染土壤微生物群落结构及其代谢特征

针对污染胁迫下土壤微生物群落变化和代谢变异等问题,基于平板稀释法和Biolog微平板分析方法,研究了陕北黄土高原石油污染土壤微生物群落结构、代谢特征及其功能多样性。结果表明,不同类群的土壤微生物对石油污染胁迫的响应不同,污染土壤细菌和真菌数量高出清洁土壤1个数量级,而污染土壤的放线菌数量极显著减少(P0.01);污染土壤和清洁土壤微生物对糖类和多聚物类碳源较易利用,污染土壤微生物总体上代谢碳源的种类和活性均低于清洁土壤。微生物群落主成分分析(PCA)表明,石油污染土壤和清洁土壤的微生物群落存在显著差异(P0.01),起分异作用的碳源主要为糖类,其次是羧酸类和氨基酸类;随着土壤石油含量增加,典型变量值变异(离散)增大,土壤微生物群落结构稳定性降低。微生物群落多样性分析表明,Shannon丰富度指数(H)、McIntosh均一度指数(U)和Simpson优势度指数(1/D)均达到极显著差异(P0.01),污染土壤微生物群落H和U低于清洁土壤,但是一定浓度的石油污染可以刺激土壤微生物群落中优势种群的生长,1/D增高。研究结果为陕北黄土高原石油污染区土壤微生物修复提供理论基础。     

中国亚热带典型天然次生林土壤微生物碳源代谢功能影响因素

天然次生林地比人工林地不仅土壤肥力较高,且土壤碳代谢功能更强。然而维持天然次生林高碳代谢功能的原因尚不十分清楚。分析天然次生林中土壤微生物碳源代谢功能的影响因素对于调控土壤微生物的功能乃至天然次生林的保护具有重要意义。本文选择中国亚热带地区典型天然次生林,研究了土壤微生物碳源代谢功能与土壤化学和物理因素及植物因素的关系。结果表明,3类因素能显著解释土壤微生物碳源代谢功能54.4% 的变异。乔木层植物多样性、土壤碳氮比、pH值和含水量是导致天然次生林碳代谢功能差异的主要因素,分别显著解释了土壤微生物碳源代谢16.7%、12.4%、10.5%和10.5%的变异。天然次生林较低的土壤碳氮比、较高的土壤含水量和土壤pH值（酸性范围内）,有利于土壤微生物碳源代谢功能的提高,同时天然次生林较高的阔叶树种的比例也能增加土壤微生物碳源代谢功能。     

Desulfurization of dibenzothiophene to 2-hydroxybiphenyl by some newly isolated bacterial strains

Gram-positive, non-spore-forming, non-acid-fast, rod-shaped aerobic bacteria with the ability to desulfurize dibenzothiophene (DBT) or dibenzosulfone (DBTO₂) were isolated from soil samples contaminated with fossil fuels. Using a bioavailability method, cells with the desired DbtS⁺ phenotype were enriched. Modified fluorescence and colorimetric assays were used for the initial detection of 2-hydroxybiphenyl (OH-BP) in microtiter plates; subsequently, isolates were grown in wells of microtiter plates and screened for the production of desulfurization product. Fluorescence under UV light and the production of colored product in the phenol assay were used as presumptive indications of production of OH-BP. Confirmation of the presence of OH-BP was achieved with HPLC, UV-absorbance, and mass spectrometry. Nutrient utilization and fatty acid composition (as discerned with Biolog plates and gas chromatography, respectively) were used to identify presumptively the strains as Rhodococcus erythropolis; colony and cell morphology may not be consistent with the identification achieved by nutrient utilization and fatty acid composition. The desulfurization end product, OH-BP, can not be used as carbon source by the tested strain, N1-36.     

Metabolic and phylogenetic analysis of microbial communities during phytoremediation of soil contaminated with weathered hydrocarbons and heavy metals

In the current study, the microbial ecology of weathered hydrocarbon and heavy metal contaminated soil undergoing phytoremediation was studied. The relationship of functional diversity, measured as carbon source utilisation in Biolog plates and extracellular enzymatic activities, and genetic diversity of bacteria was evaluated. Denaturing gradient gel electrophoresis was used for community analyses at the species level. Bulk soil and rhizosphere soil from pine and poplar plantations were analysed separately to determine if the plant rhizosphere impacted hydrocarbon degradation. Prevailing microbial communities in the field site were both genetically and metabolically diverse. Furthermore, both tree rhizosphere and fertilisation affected the compositions of these communities and increased activities of extracellular aminopeptidases. In addition, the abundance of alkane hydroxylase and naphthalene dioxygenase genes in the communities was low, but the prevalence of these genes was increased by the addition of bioavailable hydrocarbons. Tree rhizosphere communities had greater hydrocarbon degradation potential than those of bulk soil. Hydrocarbon utilising communities were dominated generally by the species Ralstonia eutropha and bacteria belonging to the genus Burkholderia. Despite the presence of viable hydrocarbon-degrading microbiota, decomposition of hydrocarbons from weathered hydrocarbon contaminated soil over four years, regardless of the presence of vegetation, was low in unfertilised soil. Compost addition enhanced the removal of hydrocarbons.     

Alternative method for rapidly screening microbial isolates for their potential to degrade volatile contaminants

Summary A method is described for rapidly screening the metabolic potential of bacteria to oxidize semivolatile and volatile compounds as a sole carbon source. The method is based on automated system that utilizes Microplates^TM manufactured by Biolog, Inc. (Hayward, CA, USA). This system detects bacterial respiratory activity from the oxidation of a carbon source introduced in volatile form. This is in contrast to the original design, which is based on inoculating a carbon source directly into each well. The 96-well (MT) microtiter plates contain nutrients and a tetrazolium dye. When a bacterial species is capable of oxidizing a volatile carbon substrate, the dye turns purple, and a spectrophotometric plate reader quantifies the response. As a test of this method 150 isolates, including isolates known to degrade some of the test compounds and negative controls were evaluated for their potential to oxidize carbon tetrachloride, toluene, ando-xylene. Thirty-seven isolates (25%) were qualitatively identified as contaminant oxidizers, and thirteen of these (35%) showed significant degradation capabilities for both toluene ando-xylene.Environmental Sciences Division Publication Number 4277.     

农田土壤固碳措施的温室气体泄漏和净减排潜力

农田土壤固碳措施作为京都议定书认可的大气CO2减排途径受到了广泛关注.研究表明,农田土壤固碳措施在主要农业国家和全球都具有很大的固碳潜力.但是,实施农田土壤固碳措施有可能影响农业中化石燃料消耗和其他农业投入的CO2排放和非CO2温室气体排放.这些土壤碳库以外的温室气体排放变化可能抵消部分甚至全部土壤固碳效果,构成了农田土壤固碳措施的温室气体泄漏.因此,将土壤固碳和温室气体泄漏综合计算的净减排潜力成为了判定土壤固碳措施可行性的首要标准.综述总结了目前较受重视的一些农田措施(包括施用化学氮肥、免耕和保护性耕作、灌溉、秸秆还田、施用禽畜粪便以及污灌)的土壤固碳潜力,温室气体泄漏和净减排潜力研究成果.结果表明,温室气体泄漏可抵消以上措施土壤固碳效益的-241%～660%.建议在今后的研究中,应该关注土壤碳饱和、气候变化及土地利用变化对农田固碳措施温室气体泄漏和净减排潜力的评估结果的影响.     

Effect of different carbon sources on community composition of bacterial enrichments from soil

Soil is a highly heterogeneous matrix, which can contain thousands of different bacterial species per gram. Only a small component of this diversity (maybe <1%) is commonly captured using standard isolation techniques, although indications are that a larger proportion of the soil community is in fact culturable. Better isolation techniques yielding greater bacterial diversity would be of benefit for understanding the metabolic activity and capability of many soil microorganisms. We studied the response of soil bacterial communities to carbon source enrichment in small matrices by means of terminal restriction fragment length polymorphism (TRFLP) analysis. The community composition of replicate enrichments from soil displayed high variability, likely attributable to soil heterogeneity. An analysis of TRFLP data indicated that enrichment on structurally similar carbon sources selected for similar bacterial communities. The same analysis indicated that communities first enriched on glucose or benzoate and subsequently transferred into medium containing an alternate carbon source retained a distinct community signature induced by the carbon source used in the primary enrichment. Enrichment on leucine presented a selective challenge that was able to override the imprint left by primary enrichment on acetate. In a time series experiment community change was most rapid 18 hours after inoculation, corresponding to exponential growth. Community composition did not stabilize even 4 days after secondary enrichment. Four different soil types were enriched on four different carbon sources. TRFLP analysis indicated that in three out of four cases communities enriched on the same carbon source were more similar regardless of which soil type was used. Conversely, the garden soil samples yielded similar enrichment communities regardless of the enrichment carbon source. Our results indicate that in order to maximize the diversity of bacteria recovered from the environment, multiple enrichments should be performed using a chemically diverse set of carbon sources.     

Application of carbon source utilization patterns to measure the metabolic similarity of complex dental plaque biofilm microcosms

Biolog technology was applied to measure the metabolic similarity of plaque biofilm microcosms, which model the complex properties of dental plaque in vivo. The choice of Biolog plate, incubation time, and incubation conditions strongly influenced utilization profiles. For plaque biofilm microcosms, Biolog GP2 plates incubated anaerobically in an H2-free atmosphere gave the clearest profile. To test the application of the Biolog GP2 assay, plaque microcosms were developed under different nutrient conditions in which the frequency of sucrose application was varied. Cluster analysis of Biolog GP2 data from 10 microcosm biofilms correlated with sucrose frequency. Aciduric bacteria (Streptococcus mutans plus lactobacilli) predominated in the plaques receiving high-frequency sucrose applications. Agreement between the Biolog GP2 groupings with nutrient and compositional changes suggests that Biolog analysis is a valuable technique for analyzing the metabolic similarity of dental plaque biofilm microcosms and other high-nutrient or predominantly anaerobic ecosystems.     

Physiological diversity of bacterial communities from different soil locations on Livingston Island, South Shetland archipelago, Antarctica

Terrestrial food webs of Antarctica are simple and dominated by microorganisms. Soil bacteria play an important role in nutrient cycling, yet little is known about their capacity to utilize different carbon sources and to participate in site nutrient turnover. Biolog EcoPlate? was applied to study the catabolic activity and physiological diversity of bacteria inhabiting the soil of moss, vascular plants, and fell field habitats from Livingston Island, Antarctica. Additionally, the number of oligotrophic and copiotrophic bacteria was counted by the agar plate method. Results indicated a lack of site-specific distribution of bacterial abundance, in contrast to bacterial catabolic activity and community level physiological profiles. Community level physiological profiles revealed a common capacity of soil bacteria to intensively utilize polyols, which are cryoprotectants widely produced by Antarctic organisms, as well as site-specific phenolic compounds (vegetated habitats), amino acids/amines (moss habitats), carbohydrates and carboxylic acids (fell field habitat). It was concluded that the physiology of soil bacteria is habitat specific concerning both the rate of catabolic activity and pattern of carbon source utilization.     

基于数字土壤制图技术的土壤有机碳储量估算

精准的土壤属性空间分布信息有助于提升土壤有机碳储量估算的精度.本研究以河南省济源市南山林场为研究区,以地形因子为预测因子,利用模糊C均值(FCM)聚类方法对土壤有机碳含量、土壤容重、土壤厚度和土壤砾石含量进行数字土壤预测制图,基于数字制图结果实现土壤有机碳密度预测制图和土壤有机碳储量估算.结果表明:基于数字土壤制图方法...     

Analysis of fungal communities by sole carbon source utilization profiles

A simple method for characterization of fungal communities in environmental samples was developed. Dilute suspensions of samples in 0.2% agar containing three different antibiotics were pipetted into 96-well plates (Biolog SF-N) containing a diverse collection of 95 different carbon sources. The plates were incubated for 4-12 days at 22 degrees C and the absorbance measured at 650 nm. Canonical variates analysis was then used to analyze the multivariate data. This method allowed fungal communities in rhizosphere soil of corn and soybean to be distinguished according to soil and plant type. Data taken at a single time-point, which varied greatly in total absorbance of the plate, separated rhizosphere samples primarily by soil type. When multiple time-points were combined to keep the total absorbance constant, differences in substrate utilization patterns due to different plant types could be distinguished. The method was also applicable to analysis of phylloplane and compost fungal communities. This method is readily applied to large numbers of samples and should be useful for community analysis in a variety of agricultural and ecological studies.     

松嫩草地4种植物功能群土壤微生物碳源利用的差异

为了探讨草地不同植物功能群土壤微生物碳源利用差异,利用Biolog-ECO微平板检测法,研究了松嫩放牧草地禾草(Grass)、羊草(Leymus chinensis)、杂类草(Forb)和豆科牧草(Legume)4种植物功能群土壤微生物碳源代谢的多样性变化特征。结果表明,在培养的240h内,4种不同植物功能群的土壤微生物对碳源的利用程度均随着时间的延长而升高,表明微生物代谢活性随着时间而增强;不同植物功能群土壤微生物总体活性(AWCD)(P0.001)、Shannon-Wiener指数(H)(P0.001)、物种丰富度(R)(P=0.005)、Pielou均匀度指数(E)(P0.001)差异显著;其中禾草的各项指数明显高于其他3种(P0.01),杂类草的各指标均最低。禾草、羊草对糖类、氨基酸类、羧酸类、多聚物类有较好的利用,豆科牧草除羧酸类外对其他碳源都有更好的利用,杂类草只对酚类碳源利用率最高,而对其他碳源利用率极低。总体得出4种功能群土壤微生物的碳源利用率顺序为:禾草羊草豆科牧草杂类草。     

Modelling the long-term response to positive and negative priming of soil organic carbon by black carbon

Observed increases in the mineralization rate of labile organic carbon (LOC) in the presence of black carbon (BC) have led to speculation that corresponding decreases in non-pyrogenic (i.e. non-BC) soil organic carbon (npSOC) could significantly reduce or negate the C sequestration benefit of BC in soils. Here we show that the potential effect of an increased LOC decomposition rate on long-term npSOC stocks is negligible, even when using assumptions that would favour large losses, potentially causing no more than 3–4 % loss of npSOC over 100 years if 50 % of above-ground crop residues were converted to BC annually. Conversely, if the BC-stimulated enhanced stabilization of npSOC that has been observed in laboratory trials is extrapolated to the long-term, it would greatly increase soil carbon stocks by 30–60 %. Annual additions of BC derived from crop residues would increase total SOC (including both BC and npSOC) by an amount five times greater than the potential increase from enhanced stabilization and an order of magnitude greater than losses of npSOC caused by annual removals of biomass to provide BC feedstock.