Microbial population changes during bioremediation of an experimental oil spill.

Three crude oil bioremediation techniques were applied in a randomized block field experiment simulating a coastal oil spill. Four treatments (no oil control, oil alone, oil plus nutrients, and oil plus nutrients plus an indigenous inoculum) were applied. In situ microbial community structures were monitored by phospholipid fatty acid (PLFA) analysis and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) to (i) identify the bacterial community members responsible for the decontamination of the site and (ii) define an end point for the removal of the hydrocarbon substrate. The results of PLFA analysis demonstrated a community shift in all plots from primarily eukaryotic biomass to gram-negative bacterial biomass with time. PLFA profiles from the oiled plots suggested increased gram-negative biomass and adaptation to metabolic stress compared to unoiled controls. DGGE analysis of untreated control plots revealed a simple, dynamic dominant population structure throughout the experiment. This banding pattern disappeared in all oiled plots, indicating that the structure and diversity of the dominant bacterial community changed substantially. No consistent differences were detected between nutrient-amended and indigenous inoculum-treated plots, but both differed from the oil-only plots. Prominent bands were excised for sequence analysis and indicated that oil treatment encouraged the growth of gram-negative microorganisms within the alpha-proteobacteria and Flexibacter-Cytophaga-Bacteroides phylum. alpha-Proteobacteria were never detected in unoiled controls. PLFA analysis indicated that by week 14 the microbial community structures of the oiled plots were becoming similar to those of the unoiled controls from the same time point, but DGGE analysis suggested that major differences in the bacterial communities remained.     

Microbial Population Analysis as a Measure of Ecosystem Restoration

During a controlled oil spill study in a freshwater wetland, four methods were used to track changes in microbial populations in response to in situ remediation treatments, including nutrient amendments and the removal of surface vegetation. Most probable number (MPN) estimates of alkane and aromatic hydrocarbon degraders showed divergence of the alkane and aromatic degrading populations during the first summer of the experiment. Alkane degraders increased in all plots by 1.5 orders of magnitude and aromatic degraders increased in oiled plots by 3.5 orders of magnitude. Phospholipid fatty acid (PLFA) analysis of biomass and community composition showed no essential differences among treatments. Denaturing gradient gel electrophoresis (DGGE) analysis of the sediment microbial community showed some differences in specific populations of organisms with respect to oiled and unoiled plots. Some organisms were only found in the oiled plots. Sediment toxicity measured against single celled algae showed that the oiled sediments were toxic into the second year of the study, but that nutrient addition relieved the toxicity more rapidly than natural attenuation of the oil.     

Effects of Different Organic Manures on the Biochemical and Microbial Characteristics of Albic Paddy Soil in a Short-Term Experiment

This study aimed to evaluate the effects of chemical fertilizer (NPK), NPK with livestock manure (NPK+M), NPK with straw (NPK+S), and NPK with green manure (NPK+G) on soil enzyme activities and microbial characteristics of albic paddy soil, which is a typical soil with low productivity in China. The responses of extracellular enzyme activities and the microbial community diversity (determined by phospholipid fatty acid analysis [PLFA] and denaturing gradient gel electrophoresis [DGGE]) were measured. The results showed that NPK+M and NPK+S significantly increased rice yield, with NPK+M being approximately 24% greater than NPK. The NPK+M significantly increased soil organic carbon (SOC) and available phosphate (P) and enhanced phosphatase, β-cellobiosidase, L-leucine aminopeptidase and urease activities. The NPK+S significantly increased SOC and available potassium (K) and significantly enhanced N-acetyl-glucosamidase, β-xylosidase, urease, and phenol oxidase activities. The NPK+G significantly improved total nitrogen (N), ammonium N, available P, and N-acetyl-glucosamidase activity. The PLFA biomass was highest under NPK+S, followed by NPK+M and NPK+G treatments. Principal component analysis (PCA) of the PLFA indicated that soils with NPK+M and NPK+S contained higher proportions of unsaturated and cyclopropane fatty acids (biomarkers of fungi and gram-negative bacteria) and soil under NPK+G contained more straight chain saturated fatty acids (representing gram-positive bacteria). PCA of the DGGE patterns showed that organic amendments had a greater influence on fungal community. Cluster analysis of fungal DGGE patterns revealed that NPK+G was clearly separated. Meanwhile, the bacterial community of NPK+M treatment was the most distinct. RDA analysis revealed changes of microbial community composition mostly depended on β-xylosidase, β-cellobiosidase activities, total N and available K contents. The abundances of gram-negative bacterial and fungal PLFAs probably effective in improving fertility of low-yield albic paddy soil because of their significant influence on DGGE profile.     

Assessing the impact of the biological control agent Bacillus thuringiensis on the indigenous microbial community within the pepper plant phyllosphere

Although biological control agents (BCAs) have been used extensively for controlling insects and pathogens of plants, little is known regarding the effects of such agents on the indigenous microbial communities within the plant phyllosphere. We assessed the effect of the BCA Bacillus thuringiensis (Bt) on the microbial communities within the pepper plant phyllosphere using culture-independent methodologies. Phospholipid fatty acid (PLFA) analysis suggested that the bacterial and fungal biomass were not significantly affected following Bt application. However, principal component analysis of PLFA data indicated that Bt did change the phyllosphere microbial community structure significantly. 16S rRNA gene-directed PCR with denaturing gradient gel electrophoresis (DGGE) also suggested a significant change in the phyllosphere bacterial community structure following Bt inoculation. Phylogenetic analysis of excised DGGE bands suggested a change in bacterial phyla; bands from untreated samples predominantly belonged to the Firmicutes, while Gammaproteobacteria abounded in the treated samples.     

Indigenous Sediment Microbial Activity in Response to Nutrient Enrichment and Plant Growth Following a Controlled Oil Spill on a Freshwater Wetland

The population density and activity of a microbial community associated with the sediment and rhizosphere of an intertidal freshwater wetland dominated by Scirpus pungens was monitored before and following the application of weathered Mesa light crude oil and fertilizers. The influence of nutrient enrichment (fertilizers) and plant growth on oil degradation rates was determined from the resulting data. The study plots (four blocks of replicates) were subjected to five treatments: oil only (natural attenuation); oil plus ammonium nitrate and phosphate, with regular cropping of the plants; oil plus ammonium nitrate and phosphate; oil plus sodium nitrate and phosphate; no oil, ammonium nitrate and phosphate. The plots were regularly monitored in the field for gas production (carbon dioxide and nitrous oxide), and samples were collected for laboratory analysis of denitrification activity, aliphatic and aromatic hydrocarbon degradation activity, and total heteroptrophic bacteria. The viable bacterial population density increased during the first 4 weeks in oiled and unoiled experimental plots that were fertilized. In contrast, population densities in untreated areas remained relatively unchanged throughout the monitoring period. The microbial population demonstrated a rapid and sustained increase in naphthalene mineralization activity in plots that were both fertilized and oiled. Hexadecane mineralization activity increased in response to fertilizer application, with ammonium nitrate causing a larger increase than sodium nitrate. A very significant difference observed in the mineralization of hexadecane was that the surface sediments were much more active than the subsurface sediments. This difference became even more pronounced in the second year of monitoring, even though the treatment regime had been discontinued. This compartmentalization of mineralization activity was not observed for naphthalene. Following fertilizer application, field and laboratory evaluation of nitrogen metabolism in the sediments indicated significant denitrification activity that was not adversely affected by oiling. The results demonstrated that the application of fertilizers stimulated the activities of indigenous hydrocarbon-degrading and denitrifying bacteria, and the presence of oil either enhanced or had no detrimental effect on these activities. As a remediation strategy, the application of fertilizers to a wetland shoreline following an oil spill would promote the growth of indigenous plants and their associated microbial flora, resulting in increased metabolic activity and the potential for increased oil degradation activity.     

Indigenous Sediment Microbial Activity in Response to Nutrient Enrichment and Plant Growth Following a Controlled Oil Spill on a Freshwater Wetland

The population density and activity of a microbial community associated with the sediment and rhizosphere of an intertidal freshwater wetland dominated by Scirpus pungens was monitored before and following the application of weathered Mesa light crude oil and fertilizers. The influence of nutrient enrichment (fertilizers) and plant growth on oil degradation rates was determined from the resulting data. The study plots (four blocks of replicates) were subjected to five treatments: oil only (natural attenuation); oil plus ammonium nitrate and phosphate, with regular cropping of the plants; oil plus ammonium nitrate and phosphate; oil plus sodium nitrate and phosphate; no oil, ammonium nitrate and phosphate. The plots were regularly monitored in the field for gas production (carbon dioxide and nitrous oxide), and samples were collected for laboratory analysis of denitrification activity, aliphatic and aromatic hydrocarbon degradation activity, and total heteroptrophic bacteria.

The viable bacterial population density increased during the first 4 weeks in oiled and unoiled experimental plots that were fertilized. In contrast, population densities in untreated areas remained relatively unchanged throughout the monitoring period. The microbial population demonstrated a rapid and sustained increase in naphthalene mineralization activity in plots that were both fertilized and oiled. Hexadecane mineralization activity increased in response to fertilizer application, with ammonium nitrate causing a larger increase than sodium nitrate. A very significant difference observed in the mineralization of hexadecane was that the surface sediments were much more active than the subsurface sediments. This difference became even more pronounced in the second year of monitoring, even though the treatment regime had been discontinued. This compartmentalization of mineralization activity was not observed for naphthalene. Following fertilizer application, field and laboratory evaluation of nitrogen metabolism in the sediments indicated significant denitrification activity that was not adversely affected by oiling. The results demonstrated that the application of fertilizers stimulated the activities of indigenous hydrocarbon-degrading and denitrifying bacteria, and the presence of oil either enhanced or had no detrimental effect on these activities. As a remediation strategy, the application of fertilizers to a wetland shoreline following an oil spill would promote the growth of indigenous plants and their associated microbial flora, resulting in increased metabolic activity and the potential for increased oil degradation activity.     

Sheep-urine-induced changes in soil microbial community structure

Soil microbial communities play an important role in nutrient cycling and nutrient availability, especially in unimproved soils. In grazed pastures, sheep urine causes local changes in nutrient concentration which may be a source of heterogeneity in microbial community structure. In the present study, we investigated the effects of synthetic urine on soil microbial community structure, using physiological (community level physiological profiling, CLPP), biochemical (phospholipid fatty acid analysis, PLFA) and molecular (denaturing gradient gel electrophoresis, DGGE) fingerprinting methods. PLFA data suggested that synthetic urine treatment had no significant effect on total microbial (total PLFA), total bacterial or fungal biomass; however, significant changes in microbial community structure were observed with both PLFA and DGGE data. PLFA data suggested that synthetic urine induced a shift towards communities with higher concentrations of branched fatty acids. DGGE banding patterns derived from control and treated soils differed, due to a higher proportion of DNA sequences migrating only to the upper regions of the gel in synthetic urine-treated samples. The shifts in community structure measured by PLFA and DGGE were significantly correlated with one another, suggesting that both datasets reflected the same changes in microbial communities. Synthetic urine treatment preferentially stimulated the use of rhizosphere-C in sole-carbon-source utilisation profiles. The changes caused by synthetic urine addition accounted for only 10-15% of the total variability in community structure, suggesting that overall microbial community structure was reasonably stable and that changes were confined to a small proportion of the communities.     

Characterization of Humus Microbial Communities in Adjacent Forest Types That Differ in Nitrogen Availability

To address the link between soil microbial community composition and soil processes, we investigated the microbial communities in forest floors of two forest types that differ substantially in nitrogen availability. Cedar-hemlock (CH) and hemlock-amabilis fir (HA) forests are both common on northern Vancouver Island, B.C., occurring adjacently across the landscape. CH forest floors have low nitrogen availability and HA high nitrogen availability. Total microbial biomass was assessed using chloroform fumigation-extraction and community composition was assessed using several cultivation-independent approaches: denaturing gradient gel electrophoresis (DGGE) of the bacterial communities, ribosomal intergenic spacer analysis (RISA) of the bacterial and fungal communities, and phospholipid fatty acid (PLFA) profiles of the whole microbial community. We did not detect differences in the bacterial communities of each forest type using DGGE and RISA, but differences in the fungal communities were detected using RISA. PLFA analysis detected subtle differences in overall composition of the microbial community between the forest types, as well as in particular groups of organisms. Fungal PLFAs were more abundant in the nitrogen-poor CH forests. Bacteria were proportionally more abundant in HA forests than CH in the lower humus layer, and Gram-positive bacteria were proportionally more abundant in HA forests irrespective of layer. Bacterial and fungal communities were distinct in the F, upper humus, and lower humus layers of the forest floor and total biomass decreased in deeper layers. These results indicate that there are distinct patterns in forest floor microbial community composition at the landscape scale, which may be important for understanding nutrient availability to forest vegetation.     

南亚热带3种人工林土壤微生物生物量和微生物群落结构特征

以我国南亚热带格木、红椎和马尾松人工林为对象,采用氯仿熏蒸浸提法和磷脂脂肪酸法(PLFA)分析了林地土壤微生物生物量和微生物群落结构组成.结果表明: 林分和季节因素均显著影响土壤微生物生物量、总PLFAs量、细菌PLFAs量和真菌PLFAs量,且干季林分下的土壤微生物生物量、总PLFAs量、单个PLFA量均大于雨季.红椎人工林土壤微生物生物量碳(MBC)和总PLFAs量最高,而格木人工林土壤微生物生物量氮(MBN)最高.土壤pH值对土壤丛枝菌根真菌(16:1ω5c)的影响达到极显著正相关水平.土壤总PLFAs量、革兰氏阳性菌(G⁺)以及腐生真菌(18:2ω6,9c)、革兰氏阳性菌/革兰氏阴性菌(G⁺/G^-)与土壤有机碳、全氮和全磷显著相关,表明土壤有机碳、全氮、全磷含量是影响该地区土壤微生物数量和种类的重要因素.外生菌根真菌(18:1ω9c)和丛枝菌根真菌与土壤碳氮比值呈极显著相关.     

Nutrient and Carbon Additions to the Microbial Soil Community and its Impact on Tree Seedlings in a Boreal Spruce Forest

We have added glucose and nutrients to manipulate soil microbial activity and nutrient availability in a boreal spruce forest to study the performance of birch and spruce seedlings in relation to the soil microbial community. The proportion of aboveground biomass in the seedlings was largest in plots amended with extra nutrients, while ectomycorrhizal (ECM) colonisation was low in these plots. ECM appeared beneficial for growth of both species, but only at low levels of colonisation (<25% ECM colonised root-tips). The soil microbial biomass, as determined by total PLFA, was largest in plots treated with glucose and there was a significant negative relationship between birch seedling size and levels of total PLFA in soil. This could be taken to suggest that poor seedling growth was due to nutrient limitation caused by microbial assimilation. However, the treatment response of the birch seedlings was generally weak, and spruce often showed no response at all to the addition of nutrients and glucose. The most consistent parameter for the variation in plant performance, as well as for the microbial soil community, was the block-effect. This suggests a strong spatial structure in the soil microbial community, and that this structure was robust with respect to our treatments even though they continued over a 3-year period.     

Bioavailability to Fish of Sediment PAH as an Indicator of the Success of In Situ Remediation Treatments at an Experimental Oil Spill

A weathered medium crude oil was applied to experimental plots of Scirpus pungens (Three-square Bulrush) in a freshwater wetland to determine the efficacy of strategies for shoreline oil spill bioremediation based on nutrient enrichment (bioremediation) and plant growth (phytoremediation). Plots were unoiled, oiled with no added nutrients, or oiled with repeated applications of phosphate and nitrate fertilizers. Following initial treatments, the experimental plots were raked to simulate the activity of wave action on oil penetration, and plants in one fertilized plot were cut repeatedly. The sediments were sampled at regular intervals for 15 months after oiling, and the loss of oil was assessed by 4-day laboratory tests of polynuclear aromatic hydrocarbon (PAH) bioaccumulation by trout, as demonstrated by increases in activity of liver cytochrome P450 (CYP1A) enzymes. Oil alone, oil mixed with sediments in the lab, and oiled sediments from treated plots all induced CYP1A activity relative to untreated controls, indicating the presence and bioavailability of PAH. Induction did not vary with nutrient treatments, but declined by 80% within 15 months of oiling, and chemical analyses indicated equivalent losses of hydrocarbons in sediment. These results demonstrate that bioavailable PAHs persisted in measurable quantities for at least 1.25 years following oiling, and that stimulation of plant growth did not affect the rate of oil disappearance. The controlling factors were likely weathering and sediment movement.     

Comparison of characterization and microbial communities in rice straw- and wheat straw-based compost for <Emphasis Type="Italic">Agaricus bisporus</Emphasis> production

Rice straw (RS) is an important raw material for the preparation of Agaricus bisporus compost in China. In this study, the characterization of composting process from RS and wheat straw (WS) was compared for mushroom production. The results showed that the temperature in RS compost increased rapidly compared with WS compost, and the carbon (C)/nitrogen (N) ratio decreased quickly. The microbial changes during the Phase I and Phase II composting process were monitored using denaturing gradient gel electrophoresis (DGGE) and phospholipid fatty acid (PLFA) analysis. Bacteria were the dominant species during the process of composting and the bacterial community structure dramatically changed during heap composting according to the DGGE results. The bacterial community diversity of RS compost was abundant compared with WS compost at stages 4–5, but no distinct difference was observed after the controlled tunnel Phase II process. The total amount of PLFAs of RS compost, as an indicator of microbial biomass, was higher than that of WS. Clustering by DGGE and principal component analysis of the PLFA compositions revealed that there were differences in both the microbial population and community structure between RS- and WS-based composts. Our data indicated that composting of RS resulted in improved degradation and assimilation of breakdown products by A. bisporus, and suggested that the RS compost was effective for sustaining A. bisporus mushroom growth as well as conventional WS compost.     

Population trends and reproductive success of the European shag Phalacrocorax aristotelis on the Iberian Peninsula following the Prestige oil spill

In 2003, immediately following the Prestige oil spill in Galicia, Spain, we studied the population trends and reproductive performance of European shags (Phalacrocorax aristotelis) at oiled and unoiled colonies. This bird is an important member of the nearshore marine community, breeding in the area affected by the Prestige oil spill. The European shag feeds around the breeding colonies throughout the year, making it a useful indicator of environmental change. Before the oil spill, population trends were similar between oiled and unoiled colonies. Nevertheless, colonies located within the path of the oil suffered greater declines (ca. 10%) compared with pre-spill trends and with population trends at unoiled colonies. In 2003, the breeding success was 50% lower in oiled colonies compared with unoiled colonies. The data available from pre-spill years suggest that the annual reproductive success did not differ among colonies before the impact. European shags breeding at colonies affected by oil showed a negative initial impact from the Prestige oil spill. The reduction in reproductive success at oiled colonies may be due to sub-lethal effects of oil exposure or low food availability after the oil spill.     

Soil Microbial Community Structure and Metabolic Activity of Pinus elliottii Plantations across Different Stand Ages in a Subtropical Area

Soil microbes play an essential role in the forest ecosystem as an active component. This study examined the hypothesis that soil microbial community structure and metabolic activity would vary with the increasing stand ages in long-term pure plantations of Pinus elliottii. The phospholipid fatty acids (PLFA) combined with community level physiological profiles (CLPP) method was used to assess these characteristics in the rhizospheric soils of P. elliottii. We found that the soil microbial communities were significantly different among different stand ages of P. elliottii plantations. The PLFA analysis indicated that the bacterial biomass was higher than the actinomycic and fungal biomass in all stand ages. However, the bacterial biomass decreased with the increasing stand ages, while the fungal biomass increased. The four maximum biomarker concentrations in rhizospheric soils of P. elliottii for all stand ages were 18:1ω9c, 16:1ω7c, 18:3ω6c (6,9,12) and cy19:0, representing measures of fungal and gram negative bacterial biomass. In addition, CLPP analysis revealed that the utilization rate of amino acids, polymers, phenolic acids, and carbohydrates of soil microbial community gradually decreased with increasing stand ages, though this pattern was not observed for carboxylic acids and amines. Microbial community diversity, as determined by the Simpson index, Shannon-Wiener index, Richness index and McIntosh index, significantly decreased as stand age increased. Overall, both the PLFA and CLPP illustrated that the long-term pure plantation pattern exacerbated the microecological imbalance previously described in the rhizospheric soils of P. elliottii, and markedly decreased the soil microbial community diversity and metabolic activity. Based on the correlation analysis, we concluded that the soil nutrient and C/N ratio most significantly contributed to the variation of soil microbial community structure and metabolic activity in different stand ages of P. elliottii plantations.     

Prokaryotic metabolic activity and community structure in Antarctic continental shelf sediments

The prokaryote community activity and structural characteristics within marine sediment sampled across a continental shelf area located off eastern Antarctica (66 degrees S, 143 degrees E; depth range, 709 to 964 m) were studied. Correlations were found between microbial biomass and aminopeptidase and chitinase rates, which were used as proxies for microbial activity. Biomass and activity were maximal within the 0- to 3-cm depth range and declined rapidly with sediment depths below 5 cm. Most-probable-number counting using a dilute carbohydrate-containing medium recovered 1.7 to 3.8% of the sediment total bacterial count, with mostly facultatively anaerobic psychrophiles cultured. The median optimal growth temperature for the sediment isolates was 15 degrees C. Many of the isolates identified belonged to genera characteristic of deep-sea habitats, although most appear to be novel species. Phospholipid fatty acid (PLFA) and isoprenoid glycerol dialkyl glycerol tetraether analyses indicated that the samples contained lipid components typical of marine sediments, with profiles varying little between samples at the same depth; however, significant differences in PLFA profiles were found between depths of 0 to 1 cm and 13 to 15 cm, reflecting the presence of a different microbial community. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified bacterial 16S rRNA genes revealed that between samples and across sediment core depths of 1 to 4 cm, the community structure appeared homogenous; however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. Sequencing of DGGE bands and rRNA probe hybridization analysis revealed that the major community members belonged to delta proteobacteria, putative sulfide oxidizers of the gamma proteobacteria, Flavobacteria, Planctomycetales, and Archaea. rRNA hybridization analyses also indicated that these groups were present at similar levels in the top layer across the shelf region.     

改变碳输入对亚热带人工林土壤微生物生物量和群落组成的影响

通过在亚热带杉木(Cunninghamia lanceolata)和米老排(Mytilaria laosensis)人工林中设置互换凋落物、去除凋落物、去除凋落物+去除根系和对照处理来分析改变地上、地下碳输入对人工林土壤微生物生物量和群落组成的影响。结果显示,改变地上、地下碳输入对土壤微生物生物量碳、氮的影响因树种而异。在米老排林中,土壤微生物生物量不受碳源的限制。而在杉木林中,加入米老排凋落物、去除凋落物和去除凋落物+去除根系3种处理中土壤微生物生物量碳、氮具有明显增加的趋势。磷脂脂肪酸分析结果显示,杉木林中,添加高质量的米老排凋落物后,革兰氏阳性细菌、阴性细菌、丛枝菌根真菌、放线菌和真菌群落生物量分别显著增加了24%、24%、53%、25%、28%,革兰氏阴性细菌和丛枝菌根真菌的相对丰度均有显著增加。与对照相比,杉木林中去除凋落物后革兰氏阳性细菌、阴性细菌、丛枝菌根真菌、放线菌和真菌群落生物量分别显著增加了22%、29%、44%、25%、52%,真菌与细菌比值显著增加了21%。但是,去除凋落物+去除根系处理对两个树种人工林土壤微生物群落组成均无显著影响。米老排和杉木林土壤微生物生物量碳、氮的季节变化格局不同,土壤养分有效性可能是驱动土壤微生物生物量季节变化的主要因子。未来研究需要关注凋落物和根系在不同树种人工林中对土壤微生物群落的相对贡献。     

Impact of fumigants on soil microbial communities.

Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity, H, was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, H increased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to Bacillus spp. and Heliothrix oregonensis. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact.     

Prokaryotic Metabolic Activity and Community Structure in Antarctic Continental Shelf Sediments

The prokaryote community activity and structural characteristics within marine sediment sampled across a continental shelf area located off eastern Antarctica (66°S, 143°E; depth range, 709 to 964 m) were studied. Correlations were found between microbial biomass and aminopeptidase and chitinase rates, which were used as proxies for microbial activity. Biomass and activity were maximal within the 0- to 3-cm depth range and declined rapidly with sediment depths below 5 cm. Most-probable-number counting using a dilute carbohydrate-containing medium recovered 1.7 to 3.8% of the sediment total bacterial count, with mostly facultatively anaerobic psychrophiles cultured. The median optimal growth temperature for the sediment isolates was 15°C. Many of the isolates identified belonged to genera characteristic of deep-sea habitats, although most appear to be novel species. Phospholipid fatty acid (PLFA) and isoprenoid glycerol dialkyl glycerol tetraether analyses indicated that the samples contained lipid components typical of marine sediments, with profiles varying little between samples at the same depth; however, significant differences in PLFA profiles were found between depths of 0 to 1 cm and 13 to 15 cm, reflecting the presence of a different microbial community. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified bacterial 16S rRNA genes revealed that between samples and across sediment core depths of 1 to 4 cm, the community structure appeared homogenous; however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. Sequencing of DGGE bands and rRNA probe hybridization analysis revealed that the major community members belonged to delta proteobacteria, putative sulfide oxidizers of the gamma proteobacteria, Flavobacteria, Planctomycetales, and Archaea. rRNA hybridization analyses also indicated that these groups were present at similar levels in the top layer across the shelf region.     

Characterization of metal-resistant soil eubacteria by polymerase chain reaction--denaturing gradient gel electrophoresis with isolation of resistant strains.

Contamination of soils with heavy metal ions is a major problem on industrial and defense-related sites worldwide. The bioavailability and mobility of these contaminants is partially determined by the microbial biomass present at these sites. In this study, we have assessed the effect of the addition of a mixture of toxic metal salts on the prokaryotic community of microcosms consisting of sandy-loam soil using direct molecular analysis of the recoverable eubacterial 16S rDNA molecules by polymerase chain reaction--denaturing gradient gel electrophoresis (PCR-DGGE) and limited phospholipid fatty acid analysis (PLFA). Addition of toxic metals (nonradioactive surrogates of Sr, Co, Cs, Cd) resulted in rapid (ca. 1 week) changes in the DGGE profile of the indigenous eubacterial community when compared with pristine controls. These changes were stable over the course of the experiment (8 weeks). No changes in the eubacterial population of control microcosms were detected. The major changes in community structure in metal-contaminated microcosms consisted of the appearance of four novel bands not detected in controls. Sequence analysis of these bands suggested that two organisms related to the genus Acinetobacter and two related to the genus Burkholderia carried a selective advantage over other indigenous eubacteria under heavy metal induced stress. The Burkholderia spp. were then cultured and further characterized using lipid analysis.     

Effect of starvation length upon microbial activity in a biomass recycle reactor

The kinetics of substrate degradation and bacterial growth was determined in a microbial community from a biomass recycle reactor that had been deprived of substrate feed for 0–32 days. Starvation caused changes in bacterial numbers, community composition, and physiological state. Substrate starvation for less than 1 day resulted in modest (less than threefold) changes in endogenous respiration rate, ATP content, and biomass level. During a starvation period of 32 days, there were substantial changes in microbial community composition, as assessed by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR amplicons of a portion of the 16S rDNA or by phospholipid fatty acid (PLFA) analysis. When the starved communities were stimulated with organic nutrients, the growth kinetics was a function of the length of the starvation period. For starvation periods of 2–8 days prior to nutrient addition, there was a phase of suboptimal exponential growth (S-phase) in which the exponential growth rate was about 30% of the ultimate unrestricted growth rate. S-phase lasted for 2–8 h and then unrestricted growth occurred at rates of 0.3–0.4 h⁻¹. At starvation times of 12 and 20 days, a lag phase preceded S-phase and the unrestricted growth phase. Received 04 January 2002/ Accepted in revised form 08 August 2002