Pyrosequencing-based assessment of bacterial community structure along different management types in German forest and grassland soils

Background

Soil bacteria are important drivers for nearly all biogeochemical cycles in terrestrial ecosystems and participate in most nutrient transformations in soil. In contrast to the importance of soil bacteria for ecosystem functioning, we understand little how different management types affect the soil bacterial community composition.

Methodology/Principal Findings

We used pyrosequencing-based analysis of the V2-V3 16S rRNA gene region to identify changes in bacterial diversity and community structure in nine forest and nine grassland soils from the Schwäbische Alb that covered six different management types. The dataset comprised 598,962 sequences that were affiliated to the domain Bacteria. The number of classified sequences per sample ranged from 23,515 to 39,259. Bacterial diversity was more phylum rich in grassland soils than in forest soils. The dominant taxonomic groups across all samples (>1% of all sequences) were Acidobacteria, Alphaproteobacteria, Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, and Firmicutes. Significant variations in relative abundances of bacterial phyla and proteobacterial classes, including Actinobacteria, Firmicutes, Verrucomicrobia, Cyanobacteria, Gemmatimonadetes and Alphaproteobacteria, between the land use types forest and grassland were observed. At the genus level, significant differences were also recorded for the dominant genera Phenylobacter, Bacillus, Kribbella, Streptomyces, Agromyces, and Defluviicoccus. In addition, soil bacterial community structure showed significant differences between beech and spruce forest soils. The relative abundances of bacterial groups at different taxonomic levels correlated with soil pH, but little or no relationships to management type and other soil properties were found.

Conclusions/Significance

Soil bacterial community composition and diversity of the six analyzed management types showed significant differences between the land use types grassland and forest. Furthermore, bacterial community structure was largely driven by tree species and soil pH.     

Multivariate Analyses of Burkholderia Species in Soil: Effect of Crop and Land Use History

The assessment of Burkholderia diversity in agricultural areas is important considering the potential use of this genus for agronomic and environmental applications. Therefore, the aim of this work was to ascertain how plant species and land use management drive the diversity of the genus Burkholderia. In a greenhouse experiment, different crops, i.e., maize, oat, barley, and grass, were planted in pots containing soils with different land use histories, i.e., maize monoculture, crop rotation, and permanent grassland, for three consecutive growth cycles. The diversity of Burkholderia spp. in the rhizosphere soil was assessed by genus-specific PCR-denaturing gradient gel electrophoresis (DGGE) and analyzed by canonical correspondence analysis (CCA). CCA ordination plots showed that previous land use was the main factor affecting the composition of the Burkholderia community. Although most variation in the Burkholderia community structure was observed between the permanent grassland and agricultural areas, differences between the crop rotation and maize monoculture groups were also observed. Plant species affected Burkholderia community structure to a lesser extent than did land use history. Similarities were observed between Burkholderia populations associated with maize and grass, on the one hand, and between those associated with barley and oat, on the other hand. Additionally, CCA ordination plots demonstrated that these two groups (maize/grass versus barley/oat) had a negative correlation. The identification of bands from the DGGE patterns demonstrated that the species correlated with the environmental variables were mainly affiliated with Burkholderia species that are commonly isolated from soil, in particular Burkholderia glathei, B. caledonica, B. hospita, and B. caribiensis.     

Phylogenetic analysis of the genes for naphthalene and phenanthrene degradation in Burkholderia sp. strains

The genetic systems responsible for naphthalene and phenanthrene catabolism have been analyzed in the five strains of Burkholderia sp. isolated from soil samples (West Siberia) contaminated by heavy residual fuel and in the laboratory collection strain Burkholderia sp. BS3702 isolated from soil samples of the coke gas plant (Vidnoe, Moscow oblast). The results of this work demonstrate that naphthalene and phenanthrene degradation in the above strains is encoded by the sequences not homologous to the classical nah genes of pseudomonades. In the Burkholderia sp. BS3702 strain, the initial stages of phenanthrene degradation and the subsequent stages of salicylate degradation are controlled by the sequences of different evolutionary descent (phn and nag genes).     

Bacterial community of very wet and acidic subalpine forest and fire-induced grassland soils

The subalpine forest and grassland ecosystems at Tatajia in Yushan National Park, Taiwan, at an elevation of 2,700 m, mean annual precipitation of 4,100 mm, mean annual temperature of 9.5°C, and soil pH near 3.5, represent land types whose bacterial communities have not been previously characterized. To this end, small subunit (SSU) rRNA libraries were prepared from environmental DNA, and 319 clones were sequenced and characterized. Despite differences in vegetation, Acidobacteria, Proteobacteria and Firmicutes were the most abundant phyla in soil communities from the forest and grassland. Although not significantly different, on the basis of Chao1, Shannon and other indices and rarefaction analyses, the diversity of the bacterial community of grassland appeared higher than that of the forest. The composition of the most abundant operational taxonomic units (OTUs) also differed between the grassland and forest communities. Because the grassland was formed by fire 30 years ago from forest, these results indicated a different bacterial community could form within that time. Moreover, most of the OTUs abundant in Tatajia soils had been previously detected in other studies, but in lower numbers. Therefore, the bacterial communities in Tatajia differed in relative abundance but not in types of bacteria present. However, one acidobacterial OTU abundant in Tatajia had previously been found to be abundant in soils from around the world. Thus, this OTU may represent a particularly abundant and cosmopolitan bacterial phylotype.     

Shifts in soil bacterial community after eight years of land-use change

The interaction between plants, soil and microorganisms is considered to be the major driver of ecosystem functions and any modification of plant cover and/or soil properties might affect the microbial structure, which, in turn, will influence ecological processes. Assuming that soil properties are the major drivers of soil bacterial diversity and structure within the same soil type, it can be postulated whether plant cover causes significant shifts in soil bacterial community composition. To address this question, this study used 16S rRNA pyrosequencing to detect differences in diversity, composition and/or relative abundance of bacterial taxa from an area covered by pristine forest, as well as eight-year-old grassland surrounded by the same forest. It was shown that a total of 69% of the operational taxonomic units (OTUs) were shared between environments. Overall, forest and grassland samples presented the same diversity and the clustering analysis did not show the occurrence of very distinctive bacterial communities between environments. However, 11 OTUs were detected in statistically significant higher abundance in the forest samples but in lower abundance in the grassland samples, whereas 12 OTUs occurred in statistically significant higher abundance in the grassland samples but in lower abundance in the forest samples. The results suggested the prevalence of a resilient core microbial community that did not suffer any change related to land use, soil type or edaphic conditions. The results illustrated that the history of land use might influence present-day community structure.     

东北丘陵区林地转型耕地对土壤编码碱性磷酸酶基因的细菌群落的影响

【目的】通过研究林地转型耕地对土壤编码碱性磷酸酶基因的细菌群落丰度、多样性和结构的影响,为丘陵区耕地长期施肥下农田土壤微生物多样性丧失的影响机制以及未来的退耕还林过程中土壤微生物多样性的提升和土地可持续利用研究提供一些基础数据和技术支撑。【方法】采用实时荧光定量PCR (real-time quantitative PCR,qPCR)和高通量测序技术解析土壤编码碱性磷酸酶基因的细菌群落的丰度、多样性和结构变化,并耦合土壤化学性质分析,明确土壤编码碱性磷酸酶基因的细菌群落丰度和多样性与土壤化学性质的关系以及关键的驱动因子。【结果】林地垦殖为农田后,长期施肥导致土壤酸化,pH从5.58降至4.72,而土壤速效磷则从2.49 mg/kg增至49.3 mg/kg。相应地,耕地土壤编码碱性磷酸酶基因的细菌群落的丰度和Shannon指数均显著低于林地。基于编码碱性磷酸酶的phoD基因(alkaline phosphatase-encoding gene)序列的物种分类表明,丘陵区土壤编码碱性磷酸酶基因的细菌群落的优势门为变形菌门(Proteobacteria)、蓝藻门(Cyanobacteria)、浮霉菌门(Planctomycetes)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)和疣微菌门(Verrucomicrobia),其中林地土壤的蓝藻门的相对丰度显著高于耕地。耕地土壤的慢生根瘤菌属(Bradyrhizobium)和芽孢杆菌属(Bacillus)的相对丰度显著高于林地,而中慢生根瘤菌属(Mesorhizobium)、假单胞菌属(Pseudomonas)、Chlorogloea属、Gemmata属、Phormidesmis属和Pseudolabrys属的相对丰度显著低于林地。土壤编码碱性磷酸酶基因的细菌群落结构因林地转型耕地而发生显著改变。phoD基因丰度和Shannon指数与pH显著正相关,而与总磷、速效磷、硝态氮和铵态氮均显著负相关,其中土壤速效磷是这些影响因素中影响最强烈的,长期施用无机磷肥导致含碱性磷酸酶的土壤细菌群落对有机磷分解的能力退化。【结论】林地转型耕地加之长期施肥改变了土壤pH和速效磷,并在其他理化因子的协同驱动下,导致土壤编码碱性磷酸酶基因的细菌群落丰度、多样性和结构的显著变化。     

Molecular Method To Assess the Diversity of Burkholderia Species in Environmental Samples

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.     

Soil Bacterial Community Response to Differences in Agricultural Management along with Seasonal Changes in a Mediterranean Region

Land-use change is considered likely to be one of main drivers of biodiversity changes in grassland ecosystems. To gain insight into the impact of land use on the underlying soil bacterial communities, we aimed at determining the effects of agricultural management, along with seasonal variations, on soil bacterial community in a Mediterranean ecosystem where different land-use and plant cover types led to the creation of a soil and vegetation gradient. A set of soils subjected to different anthropogenic impact in a typical Mediterranean landscape, dominated by Quercus suber L., was examined in spring and autumn: a natural cork-oak forest, a pasture, a managed meadow, and two vineyards (ploughed and grass covered). Land uses affected the chemical and structural composition of the most stabilised fractions of soil organic matter and reduced soil C stocks and labile organic matter at both sampling season. A significant effect of land uses on bacterial community structure as well as an interaction effect between land uses and season was revealed by the EP index. Cluster analysis of culture-dependent DGGE patterns showed a different seasonal distribution of soil bacterial populations with subgroups associated to different land uses, in agreement with culture-independent T-RFLP results. Soils subjected to low human inputs (cork-oak forest and pasture) showed a more stable bacterial community than those with high human input (vineyards and managed meadow). Phylogenetic analysis revealed the predominance of Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes phyla with differences in class composition across the site, suggesting that the microbial composition changes in response to land uses. Taken altogether, our data suggest that soil bacterial communities were seasonally distinct and exhibited compositional shifts that tracked with changes in land use and soil management. These findings may contribute to future searches for bacterial bio-indicators of soil health and sustainable productivity.     

Effect of Agricultural Management Regime on Burkholderia Community Structure in Soil

The main objective of this study was to determine the Burkholderia community structure associated with areas under different agricultural management and to evaluate to which extent this community structure is affected by changes in agricultural management. Two fields with distinct soil history (arable land and permanent grassland) were exposed to three agricultural management regimes (crop rotation, maize monoculture, and grassland). By using a culture-independent approach, based on a Burkholderia-specific polymerase chain reaction–denaturing gradient gel electrophoresis system, it was possible to observe the conversion of Burkholderia communities typical for permanent grassland to those of arable land after four consecutive years. However, the time needed to achieve the reverse transition, i.e., converting the Burkholderia community associated with arable land to that of grassland, was beyond the duration of the field experiment. In addition, by applying principal response curves, the direction and extent of the conversion from grassland to arable land (maize monoculture and to crop rotation) were determined. Hence, the results suggested that agricultural practices, such as fertilization and tillage, were more effective in changing the Burkholderia community structure than agricultural management regime. To determine the effect of agricultural management on the Burkholderia population with biocontrol abilities, the culturable fraction of the Burkholderia community was assessed. The areas under permanent grassland and grassland converted to maize monoculture had the highest percentages of Burkholderia strains with antagonistic activity against Rhizoctonia solani AG-3, mainly Burkholderia pyrrocinia and Burkholderia sp. LMG 22929. The isolation frequency of antagonistic isolates from arable land was extremely low. Our results indicate that (changes in) agricultural management, mainly crop rotation, affect the frequency of isolation of antagonistic Burkholderia strains and that grassland represents a reservoir of Burkholderia species with great potential for agricultural applications.     

Minerals Affect the Specific Diversity of Forest Soil Bacterial Communities

Minerals constitute an ecological niche poorly investigated in the soil, in spite of their important role in biogeochemical cycles and plant nutrition. To evaluate the impact of minerals on the structure of the soil bacterial communities, we compared the bacterial diversity on mineral surfaces and in the surrounding soil. Three pure and calibrated minerals (apatite, plagioclase and a mix of phlogopite-quartz) were buried into the organo-mineral layer of a forest soil. After a 4-year incubation in soil conditions, mineral weathering and microbial colonization were evaluated. Apatite and plagioclase were the only two significantly weathered minerals. The analysis of the 16S rRNA gene sequences generated by the cloning-sequencing procedure revealed that bacterial diversity was higher in the surrounding soil and on the unweathered phlogopite-quartz samples compared with the other minerals. Moreover, a multivariate analysis based on the relative abundance of the main taxonomic groups in each compartments of origin demonstrated that the bacterial communities from the bulk soil differed from that colonizing the minerals. A significant correlation was obtained between the dissolution rate of the minerals and the relative abundance of Beta-proteobacteria detected. Notably, many sequences coming from bacteria colonizing the mineral surfaces, whatever the mineral, harbored high similarity with efficient mineral weathering bacteria belonging to Burkholderia and Collimonas genera, previously isolated on the same experimental site. Taken together, the present results provide new highlights concerning the bacterial communities colonizing minerals surfaces in the soil and suggests that the minerals create true ecological niches: the mineralosphere.     

Soil Bacterial Community Shift Correlated with Change from Forest to Pasture Vegetation in a Tropical Soil

The change in vegetative cover of a Hawaiian soil from forest to pasture led to significant changes in the composition of the soil bacterial community. DNAs were extracted from both soil habitats and compared for the abundance of guanine-plus-cytosine (G+C) content, by analysis of abundance of phylotypes of small-subunit ribosomal DNA (SSU rDNA) amplified from fractions with 63 and 35% G+C contents, and by phylogenetic analysis of the dominant rDNA clones in the 63% G+C content fraction. All three methods showed differences between the forest and pasture habitats, providing evidence that vegetation had a strong influence on microbial community composition at three levels of taxon resolution. The forest soil DNA had a peak in G+C content of 61%, while the DNA of the pasture soil had a peak in G+C content of 67%. None of the dominant phylotypes found in the forest soil were detected in the pasture soil. For the 63% G+C fraction SSU rDNA sequence analysis of the three most dominant members revealed that their phyla changed from Fibrobacter and Syntrophomonas assemblages in the forest soil to Burkholderia and Rhizobium–Agrobacterium assemblages in the pasture soil.     

Pyrosequence analyses of bacterial communities during simulated in situ bioremediation of polycyclic aromatic hydrocarbon-contaminated soil

Barcoded amplicon pyrosequencing was used to generate libraries of partial 16S rRNA genes from two columns designed to simulate in situ bioremediation of polycyclic aromatic hydrocarbons (PAHs) in weathered, contaminated soil. Both columns received a continuous flow of artificial groundwater but one of the columns additionally tested the impact of biostimulation with oxygen and inorganic nutrients on indigenous soil bacterial communities. The penetration of oxygen to previously anoxic regions of the columns resulted in the most significant community changes. PAH-degrading bacteria previously determined by stable-isotope probing (SIP) of the untreated soil generally responded negatively to the treatment conditions, with only members of the Acidovorax and a group of uncharacterized PAH-degrading Gammaproteobacteria maintaining a significant presence in the columns. Additional groups of sequences associated with the Betaproteobacterial family Rhodocyclaceae (including those associated with PAH degradation in other soils), and the Thiobacillus, Thermomonas, and Bradyrhizobium genera were also present in high abundance in the biostimulated column. Similar community responses were previously observed during biostimulated ex situ treatment of the same soil in aerobic, slurry-phase bioreactors. While the low relative abundance of many SIP-determined groups in the column libraries may be a reflection of the slow removal of PAHs in that system, the similar response of known PAH degraders in a higher-rate bioreactor system suggests that alternative PAH-degrading bacteria, unidentified by SIP of the untreated soil, may also be enriched in engineered systems.     

亚高山/高山森林土壤有机层氨氧化细菌和氨氧化古菌丰度特征

为了解季节性冻融作用对川西亚高山/高山地区土壤氨氧化微生物群落的影响,采用qPCR技术,以氨单加氧酶基因的α亚基(amoA)为标记,在生长阶段、冻结阶段、融化阶段中的9个关键时期调查了该地区不同森林群落：岷江冷杉(Abies faxoniana)原始林(PF)、岷江冷杉(A. faxoniana)和红桦(Betula albosinensis)混交林(MF)、岷江冷杉次生林(SF)土壤有机层的氨氧化细菌(ammonia-oxidizing bacteria, AOB)和氨氧化古菌(ammonia-oxidizing archaea, AOA)丰度的特征。结果表明,三个森林群落土壤有机层中都具有相当数量的氨氧化细菌和古菌,均表现出从生长阶段至冻结阶段显著降低,在冻结阶段最低,但冻结阶段后显著增加,在融化阶段为全年最高的趋势。土壤氨氧化微生物类群结构(AOA/AOB)受负积温影响明显。冻结后期三个森林群落土壤负积温最大时,AOA数量明显高于AOB,但其他关键时期土壤氨氧化微生物类群结构与群落类型密切相关。高海拔的PF群落土壤有机层表现为AOA>AOB(冻结初期除外),低海拔的SF群落中表现为AOB>AOA(冻结后期除外),而MF群落则仅在融冻期和生长季节末期表现为AOB>AOA。这些结果为认识亚高山/高山森林及其相似区域的生态过程提供了一定的科学依据。     

Distinct Bacterial Communities Dominate Tropical and Temperate Zone Leaf Litter

Little is known of the bacterial community of tropical rainforest leaf litter and how it might differ from temperate forest leaf litter and from the soils underneath. We sampled leaf litter in a similarly advanced stage of decay, and for comparison, we also sampled the surface layer of soil, at three tropical forest sites in Malaysia and four temperate forest sites in South Korea. Illumina sequencing targeting partial bacterial 16S ribosomal ribonucleic acid (rRNA) gene revealed that the bacterial community composition of both temperate and tropical litter is quite distinct from the soils underneath. Litter in both temperate and tropical forest was dominated by Proteobacteria and Actinobacteria, while soil is dominated by Acidobacteria and, to a lesser extent, Proteobacteria. However, bacterial communities of temperate and tropical litter clustered separately from one another on an ordination. The soil bacterial community structures were also distinctive to each climatic zone, suggesting that there must be a climate-specific biogeographical pattern in bacterial community composition. The differences were also found in the level of diversity. The temperate litter has a higher operational taxonomic unit (OTU) diversity than the tropical litter, paralleling the trend in soil diversity. Overall, it is striking that the difference in community composition between the leaf litter and the soil a few centimeters underneath is about the same as that between leaf litter in tropical and temperate climates, thousands of kilometers apart. However, one substantial difference was that the leaf litter of two tropical forest sites, Meranti and Forest Research Institute Malaysia (FRIM), was overwhelmingly dominated by the single genus Burkholderia, at 37 and 23 % of reads, respectively. The 454 sequencing result showed that most Burkholderia species in tropical leaf litter belong to nonpathogenic “plant beneficial” lineages. The differences from the temperate zone in the bacterial community of tropical forest litter may be partly a product of its differing chemistry, although the unvarying climate might also play a role, as might interactions with other organisms such as fungi. The single genus Burkholderia may be seen as potentially playing a major role in decomposition and nutrient cycling in tropical forests, but apparently not in temperate forests.     

Bacterial Community Diversity in the Brazilian Atlantic Forest Soils

The aim of this study was to characterize the bacterial community diversity of the Brazilian Atlantic forest soil by means of both cultivation and 16S rRNA clone libraries. A collection of 86 representative isolates, obtained from six samples of Atlantic forest soils from the National Park of Serra dos Órgãos (PARNASO), belonged to the genera Arthrobacter, Bacillus, Burkholderia, Leifsonia, Paenibacillus, Pseudomonas, Ralstonia, Serratia, and Streptomyces according to the 16S rRNA sequences. Representative isolates from the different genera degraded cellulose and lignin. The culture-independent analysis based on 894 partial 16S rRNA gene sequences revealed that the most frequently retrieved groups belonged to the phyla Acidobacteria (29–54%), Proteobacteria (16–38%), and Verrucomicrobia (0.6–14%). The majority of the sequences (82.6%) were unidentified singletons and doubletons, indicating a high diversity of rare unique sequences. Chao1 estimator disclosed a high number of phyla (41–152) and species (263–446). This is the first survey on the Atlantic Forest soils using a combination of cultivation and culture-independent approaches. We conclude that the Brazilian Atlantic Forest soil represents a vast source of novel bacteria.     

黑河中上游地区2000-2010年土地利用变化及水土保持服务功能

基于遥感影像数据,分析了黑河中上游地区2000年到2010年土地利用变化,在此基础上,从上游、中游以及整个区域的角度综合评价了该区域的水源涵养和土壤保持生态系统服务功能物质量变化。研究结果表明:1研究区60%以上为未利用地,2000—2010年,耕地、林地和未利用地面积大量增加,草地和水域面积减少。2耕地增加以荒漠和草地转入为主;林地转入表现为草地和荒漠造林,转出主要为林地荒漠化;草地转出主要去向为造林和荒漠化,而增加主要来源于冰雪覆盖地和荒漠;未利用地增加主要来源于草地的退化。3研究区土地利用程度较低,由东南向西北递减,2000—2010年,上游土地利用程度有所降低,中游大部分地区呈上升趋势。4由于上游山区林地面积的增加,植被覆盖下土壤的水源涵养量显著增加,土壤贮水量增加了0.764×108t。5土壤保持量由1.860×108t增加到1.899×108t,主要原因为草地转化为林地。在气候变化背景下,黑河流域应控制上游地区人工林面积,减少林地蒸腾量,以增加下游可用水量;适当控制中游地区耕地面积的扩张趋势,保证湿地、草地等自然生态系统生态用水量,维持流域山地-绿洲-荒漠系统的稳定,进而持续发挥生态系统土壤保持等功能。     

Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders

31 different bacterial strains isolated using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, were investigated for their ability to mineralize 2,4-D and the related herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Most of the strains mineralize 2,4-D considerably faster than MCPA. Three novel primer sets were developed enabling amplification of full-length coding sequences (CDS) of the three known tfdA gene classes known to be involved in phenoxy acid degradation. 16S rRNA genes were also sequenced; and in order to investigate possible linkage between tfdA gene classes and bacterial species, tfdA and 16S rRNA gene phylogeny was compared. Three distinctly different classes of tfdA genes were observed, with class I tfdA sequences further partitioned into the two sub-classes I-a and I-b based on more subtle differences. Comparison of phylogenies derived from 16S rRNA gene sequences and tfdA gene sequences revealed that most class II tfdA genes were encoded by Burkholderia sp., while class I-a, I-b and III genes were found in a more diverse array of bacteria.     

A simple strategy for investigating the diversity and hydrocarbon degradation abilities of cultivable bacteria from contaminated soil

The use of indigenous bacterial strains is a valuable bioremediation strategy for cleaning the environment from hydrocarbon pollutants. The isolation and selection of hydrocarbon-degrading bacteria is therefore crucial for obtaining the most promising strains for site decontamination. Two different media, a minimal medium supplemented with a mixture of polycyclic aromatic hydrocarbons and a MS medium supplemented with triphenyltetrazolium chloride, were used for the isolation of bacterial strains from two hydrocarbon contaminated soils and from their enrichment phases. The hydrocarbon degradation abilities of these bacterial isolates were easily and rapidly assessed using the 2,6-dichlorophenol indophenol assay. The diversity of the bacterial communities isolated from these two soil samples and from their enrichment phases was evaluated by the combination of a bacterial clustering method, fluorescence ITS-PCR, and bacterial identification by 16S rRNA sequencing. Different PCR-based assays were performed in order to detect the genes responsible for hydrocarbon degradation. The best hydrocarbon-degrading bacteria, including Arthrobacter sp., Enterobacter sp., Sphingomonas sp., Pseudomonas koreensis, Pseudomonas putida and Pseudomonas plecoglossicida, were isolated directly from the soil samples on minimal medium. The nahAc gene was detected only in 13 Gram-negative isolates and the sequences of nahAc-like genes were obtained from Enterobacter, Stenotrophomonas, Pseudomonas brenneri, Pseudomonas entomophila and P. koreensis strains. The combination of isolation on minimal medium with the 2,6-dichlorophenol indophenol assay was effective in selecting different hydrocarbon-degrading strains from 353 isolates.     

Identification of Benzo[a]pyrene-Metabolizing Bacteria in Forest Soils by Using DNA-Based Stable-Isotope Probing

DNA-based stable-isotope probing (DNA-SIP) was used in this study to investigate the uncultivated bacteria with benzo[a]pyrene (BaP) metabolism capacities in two Chinese forest soils (Mt. Maoer in Heilongjiang Province and Mt. Baicaowa in Hubei Province). We characterized three different phylotypes with responsibility for BaP degradation, none of which were previously reported as BaP-degrading microorganisms by SIP. In Mt. Maoer soil microcosms, the putative BaP degraders were classified as belonging to the genus Terrimonas (family Chitinophagaceae, order Sphingobacteriales), whereas Burkholderia spp. were the key BaP degraders in Mt. Baicaowa soils. The addition of metabolic salicylate significantly increased BaP degradation efficiency in Mt. Maoer soils, and the BaP-metabolizing bacteria shifted to the microorganisms in the family Oxalobacteraceae (genus unclassified). Meanwhile, salicylate addition did not change either BaP degradation or putative BaP degraders in Mt. Baicaowa. Polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) genes were amplified, sequenced, and quantified in the DNA-SIP ¹³C heavy fraction to further confirm the BaP metabolism. By illuminating the microbial diversity and salicylate additive effects on BaP degradation across different soils, the results increased our understanding of BaP natural attenuation and provided a possible approach to enhance the bioremediation of BaP-contaminated soils.     

Effect of the Mycorrhizosphere on the Genotypic and Metabolic Diversity of the Bacterial Communities Involved in Mineral Weathering in a Forest Soil

To date, several bacterial species have been described as mineral-weathering agents which improve plant nutrition and growth. However, the possible relationships between mineral-weathering potential, taxonomic identity, and metabolic ability have not been investigated thus far. In this study, we characterized a collection of 61 bacterial strains isolated from Scleroderma citrinum mycorrhizae, the mycorrhizosphere, and the adjacent bulk soil in an oak forest. The ability of bacteria to weather biotite was assessed with a new microplate bioassay that measures the pH and the quantity of iron released from this mineral. We showed that weathering bacteria occurred more frequently in the vicinity of S. citrinum than in the bulk soil. Moreover, the weathering efficacy of the mycorrhizosphere bacterial isolates was significantly greater than that of the bulk soil isolates. All the bacterial isolates were identified by partial 16S rRNA gene sequence analysis as members of the genera Burkholderia, Collimonas, Pseudomonas, and Sphingomonas, and their carbon metabolism was characterized by the BIOLOG method. The most efficient isolates belonged to the genera Burkholderia and Collimonas. Multivariate analysis resulted in identification of three metabolic groups, one of which contained mainly bacterial isolates associated with S. citrinum and exhibiting high mineral-weathering potential. Therefore, our results support the hypothesis that by its carbon metabolism this fungus selects in the bulk soil reservoir a bacterial community with high weathering potential, and they also address the question of functional complementation between mycorrhizal fungi and bacteria in the ectomycorrhizal complex for the promotion of tree nutrition.