Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured 13CO2 respired from [13C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that (13)C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [13C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [13C]phenol, and (iii) 12 daily doses of [13C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted 13CO2 evolution by a factor of 10. Furthermore, imaging of 13C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated 13C into their biomass. PCR amplification and 16S rRNA gene sequencing of 13C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of alpha-, beta-, and gamma-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.     

Fourier transform infrared spectroscopy as a metabolite fingerprinting tool for monitoring the phenotypic changes in complex bacterial communities capable of degrading phenol

The coking process produces great volumes of wastewater contaminated with pollutants such as cyanides, sulfides and phenolics. Chemical and physical remediation of this wastewater removes the majority of these pollutants; however, these processes do not remove phenol and thiocyanate. The removal of these compounds has been effected during bioremediation with activated sludge containing a complex microbial community. In this investigation we acquired activated sludge from an industrial bioreactor capable of degrading phenol. The sludge was incubated in our laboratory and monitored for its ability to degrade phenol over a 48 h period. Multiple samples were taken across the time‐course and analysed by Fourier transform infrared (FT‐IR) spectroscopy. FT‐IR was used as a whole‐organism fingerprinting approach to monitor biochemical changes in the bacterial cells during the degradation of phenol. We also investigated the ability of the activated sludge to degrade phenol following extended periods (2–131 days) of storage in the absence of phenol. A reduction was observed in the ability of the microbial community to degrade phenol and this was accompanied by a detectable biochemical change in the FT‐IR fingerprint related to cellular phenotype of the microbial community. In the absence of phenol a decrease in thiocyanate vibrations was observed, reflecting the ability of these communities to degrade this substrate. Actively degrading communities showed an additional new band in their FT‐IR spectra that could be attributed to phenol degradation products from the ortho‐ and meta‐cleavage of the aromatic ring. This study demonstrates that FT‐IR spectroscopy when combined with chemometric analysis is a very powerful high throughput screening approach for assessing the metabolic capability of complex microbial communities.     

N-acyl-l-homoserine lactones (AHLs) affect microbial community composition and function in activated sludge

The role of intercellular signalling in the regulation of genes and phenotypes in a broad range of bacterial species is now firmly established. In contrast, the impact of intercellular signalling on microbial community parameters, such as species diversity and function, is less well understood. In this study the role of N-acyl-l-homoserine lactones (AHLs) in microbial community dynamics in an industrial wastewater treatment system is addressed. Seven proteobacterial strains producing compounds with AHL-like activity were isolated from the treatment plant. Three of these belong to genera with no previously identified AHL producing species. Addition of AHLs at 2 micro M to sludge samples generated changes in both community function (phenol degradation) and composition as determined by length heterogeneity PCR and denaturing gradient gel electrophoresis. Phenol degradation was more stable as a result of the AHL augmentation. A dominant functional member of the Thauera genus was transiently supplanted by a member of the Comomonas genus in response to AHL addition. This suggests that AHLs can play a role in mediating microbial community parameters and has implications for ecosystem function and industrial wastewater treatment.     

Dynamic changes in microbial community structure and function in phenol-degrading microcosms inoculated with cells from a contaminated aquifer

Contamination of aquifers by organic pollutants threatens groundwater supplies and the environment. In situ biodegradation of organic pollutants by microbial communities is important for the remediation of contaminated sites, but our understanding of the relationship between microbial development and pollutant biodegradation is poor. A particular challenge is understanding the in situ status of microorganisms attached to solid surfaces, but not accessible via conventional sampling of groundwater. We have developed novel flow-through microcosms and examined dynamic changes in microbial community structure and function in a phenol-degrading system. Inoculation of these microcosms with a complex microbial community from a plume in a phenol-contaminated aquifer led to the initial establishment of a population dominated by a few species, most attached to the solid substratum. Initially, phenol biodegradation was incomplete, but as the microbial community structure became more complex, phenol biodegradation was more extensive and complete. These results were replicated between independent microcosms, indicating a deterministic succession of species. This work demonstrates the importance of examining community dynamics when assessing the potential for microbial biodegradation of organic pollutants. It provides a novel system in which such measurements can be made readily and reproducibly to study the temporal development and spatial succession of microbial communities during biodegradation of organic pollutants at interfaces within such environments.     

Biomonitoring of continuous microbial community adaptation towards more efficient phenol-degradation in a fed-batch bioreactor

The anaerobic degradation of phenol was studied in a fed-batch culture. Nitrate was added as electron acceptor and phenol was provided three times, to a final concentration of 200 mg/l. Randomly amplified polymorphic DNA (RAPD) and terminal fraction fragment length polymorphism (T-RFLP) were used and compared in order to monitor the microbial succession in the reactor. Phenol degradation started after an initial lag phase of 14 days and was then completed within a few days. In addition, the duration of the lag phase was shortened and the degradation rate was increased after each phenol amendment. Nitrate reduction correlated with microbial growth and phenol depletion, confirming that the degradation was carried out anaerobically. Results from the DNA analysis showed that the structure of the microbial community changed after each phenol amendment. This study confirms the potential for anaerobic degradation of environmental pollutants and also confirms that microbial acclimation towards faster degradation rates occurred upon repeated substrate amendments. Furthermore, both of the DNA-based techniques described the phenol degradation-linked community shifts with similar general results. RAPD is a faster, simpler technique that gives a higher resolution and consequently reflects the shifts in the microbial community structure better, whereas T-RFLP is more suitable for phylogenetic studies.     

Microbial community utilization of recalcitrant and simple carbon compounds: impact of oak-woodland plant communities

Little is known about how the structure of microbial communities impacts carbon cycling or how soil microbial community composition mediates plant effects on C-decomposition processes. We examined the degradation of four ¹³C-labeled compounds (starch, xylose, vanillin, and pine litter), quantified rates of associated enzyme activities, and identified microbial groups utilizing the ¹³C-labeled substrates in soils under oaks and in adjacent open grasslands. By quantifying increases in non-¹³C-labeled carbon in microbial biomarkers, we were also able to identify functional groups responsible for the metabolism of indigenous soil organic matter. Although microbial community composition differed between oak and grassland soils, the microbial groups responsible for starch, xylose, and vanillin degradation, as defined by ¹³C-PLFA, did not differ significantly between oak and grassland soils. Microbial groups responsible for pine litter and SOM-C degradation did differ between the two soils. Enhanced degradation of SOM resulting from substrate addition (priming) was greater in grassland soils, particularly in response to pine litter addition; under these conditions, fungal and Gram + biomarkers showed more incorporation of SOM-C than did Gram – biomarkers. In contrast, the oak soil microbial community primarily incorporated C from the added substrates. More ¹³C (from both simple and recalcitrant sources) was incorporated into the Gram – biomarkers than Gram + biomarkers despite the fact that the Gram + group generally comprised a greater portion of the bacterial biomass than did markers for the Gram – group. These experiments begin to identify components of the soil microbial community responsible for decomposition of different types of C-substrates. The results demonstrate that the presence of distinctly different plant communities did not alter the microbial community profile responsible for decomposition of relatively labile C-substrates but did alter the profiles of microbial communities responsible for decomposition of the more recalcitrant substrates, pine litter and indigenous soil organic matter.     

Isolation of RNA from bacterial samples of the human gastrointestinal tract

The human gastrointestinal (GI) tract contains a complex microbial community that consists of numerous uncultured microbes. Therefore, nucleic-acid-based approaches have been introduced to study microbial diversity and activity, and these depend on the proper isolation of DNA, rRNA and mRNA. Here, we present an RNA isolation protocol that is suitable for a wide variety of GI tract samples. The procedure for isolating DNA from GI tract samples is described in another Nature Protocols article. One of the benefits of our RNA isolation protocol is that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The RNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction and removal of DNA. In our laboratory, this protocol has resulted in the isolation of rRNA and mRNA of sufficient quality and quantity for microbial diversity and activity studies. Depending on the number of samples, the sample type and the quenching procedure chosen, the whole procedure can be performed within 2.5-4 h.     

Seasonal Dynamics of Microbial Community Composition and Function in Oak Canopy and Open Grassland Soils

Soil microbial communities are closely associated with aboveground plant communities, with multiple potential drivers of this relationship. Plants can affect available soil carbon, temperature, and water content, which each have the potential to affect microbial community composition and function. These same variables change seasonally, and thus plant control on microbial community composition may be modulated or overshadowed by annual climatic patterns. We examined microbial community composition, C cycling processes, and environmental data in California annual grassland soils from beneath oak canopies and in open grassland areas to distinguish factors controlling microbial community composition and function seasonally and in association with the two plant overstory communities. Every 3 months for up to 2 years, we monitored microbial community composition using phospholipid fatty acid (PLFA) analysis, microbial biomass, respiration rates, microbial enzyme activities, and the activity of microbial groups using isotope labeling of PLFA biomarkers (¹³C-PLFA). Distinct microbial communities were associated with oak canopy soils and open grassland soils and microbial communities displayed seasonal patterns from year to year. The effects of plant species and seasonal climate on microbial community composition were similar in magnitude. In this Mediterranean ecosystem, plant control of microbial community composition was primarily due to effects on soil water content, whereas the changes in microbial community composition seasonally appeared to be due, in large part, to soil temperature. Available soil carbon was not a significant control on microbial community composition. Microbial community composition (PLFA) and ¹³C-PLFA ordination values were strongly related to intra-annual variability in soil enzyme activities and soil respiration, but microbial biomass was not. In this Mediterranean climate, soil microclimate appeared to be the master variable controlling microbial community composition and function.     

Insights into the fate of a 13C labelled phenol pulse for stable isotope probing (SIP) experiments

Stable isotope probing (SIP) using DNA or RNA as a biomarker has proven to be a useful method for attributing substrate utilisation to specific microbial taxa. In this study we followed the transfer of a (13)C(6)-phenol pulse in an activated sludge micro-reactor to examine the resulting distribution of labelled carbon in the context of SIP. Most of the added phenol was metabolically converted within the first 100 min after (13)C(6)-phenol addition, with 49% incorporated into microbial biomass and 6% respired as CO(2). Less than 1% of the total (13)C labelled carbon supplied was incorporated into microbial RNA and DNA, with RNA labelling 6.5 times faster than DNA. The remainder of the added (13)C was adsorbed and/or complexed to suspended solids within the sludge. The (13)C content of nucleic acids increased beyond the initial consumption of the (13)C-phenol pulse. This study confirms that RNA labels more efficiently than DNA and reveals that only a small proportion of a pulse is incorporated into nucleic acids. Evidence of continued (13)C incorporation into nucleic acids suggests that cross-feeding of the SIP substrate was rapid. This highlights both the benefits of using a biomarker that is rapidly labelled and the importance of sampling within appropriate timescales to avoid or capture the effects of cross-feeding, depending on the goal of the study.     

Use of Field-Based Stable Isotope Probing To Identify Adapted Populations and Track Carbon Flow through a Phenol-Degrading Soil Microbial Community

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured ¹³CO₂ respired from [¹³C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that ¹³C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [¹³C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [¹³C]phenol, and (iii) 12 daily doses of [¹³C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted ¹³CO₂ evolution by a factor of 10. Furthermore, imaging of ¹³C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated ¹³C into their biomass. PCR amplification and 16S rRNA gene sequencing of ¹³C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of α-, β-, and γ-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.     

不同富集和分离培养条件下的含酚废水处理生物膜微生物群落结构与活性比较分析

采用Biolog和变性梯度凝胶电泳(DGGE)技术研究了不同苯酚浓度培养对焦化废水处理厂反硝化池生物膜样品中微生物群落结构和代谢类型的影响。DGGE结果表明, 不同浓度苯酚和不同培养方式富集培养后, 细菌16S rDNA的部分条带分布谱形发生改变, 还有部分条带只受到了苯酚浓度变化的影响; 富集培养过程中由于碳源组成相对焦化废水简单, DGGE条带所代表的优势微生物多样性有所降低。Biolog试验结果表明, 生物膜样本的细菌群落代谢能力最强; 低浓度苯酚富集后的样品能利用的底物碳源类型最丰富。对Biolog试验结果的主成分分析显示, 相同浓度苯酚富集培养后的细菌群落代谢功能多样性相似, 但从DGGE结果看出其结构组成产生了变化。富集培养使样品微生物群落的代谢功能发生改变, 低浓度的苯酚富集增加了群落中微生物的代谢类型。而不同条件获得的分离物其苯酚降解能力的初步分析也表明, 富集与分离条件对苯酚降解菌的分离能力和得到的菌株特性具有差别。     

Influence of easily degradable naturally occurring carbon substrates on biodegradation of monosubstituted phenols by aquatic bacteria

The influence of readily degradable, naturally occurring carbon substrates on the biodegradation of several monosubstitued phenols (m-cresol, m-aminophenol, p-chlorophenol) was examined. The natural substrate classes used were amino acids, carbohydrates, and fatty acids. Samples of the microbial community from Lake Michie, a mesotrophic reservoir, were adapted to different levels of representatives from each natural substrate class in chemostats. After an extended adaptation period, the ability of the microbial community to degrade the monosubstituted phenols was determined by using a radiolabeled substrate uptake and mineralization method. Several microbiological characteristics of the communities were also measured. Adaptation to increasing concentrations of amino acids, carbohydrates, or fatty acids enhanced the ability of the microbial community to degrade all three phenols. The stimulation was largest for m-cresol and m-aminophenol. The mechanism responsible for the enhancement of monosubstituted phenol metabolism was not clearly identified, but the observation that adaptation to amino acids also increased the biodegradation of glucose and, to a lesser extent, naphthalene suggests a general stimulation of microbial metabolism. This study demonstrates that prior exposure to labile, natural substrates can significantly enhance the ability of aquatic microbial communities to respond to xenobiotics.     

Effect of adaptation to phenol on biodegradation of monosubstituted phenols by aquatic microbial communities.

The adaptation of a mixed aquatic microbial community to phenol was examined in microcosms receiving phenol as a sole carbon source. Extended exposure (adaptation) to phenol resulted in adaptation of the microbial community to the structurally related aromatic compounds m-cresol, m-aminophenol, and p-chlorophenol. The increased biodegradation potential of the phenol-adapted microbial community was accompanied by a concurrent increase in the number of microorganisms able to degrade the three test compounds. Thus, adaptation to the three test chemicals was likely a growth-related result of extended exposure to phenol. The results indicate that adaptation to a single chemical may increase the assimilative capacity of an aquatic environment for other related chemicals even in the absence of adaptation-inducing levels of those materials.     

DNA stable-isotope probing

Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4-5 days, with much of the time being committed to the ultracentrifugation step.     

Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a 13CO2 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of 13C-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded 13C compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from 12C and 13C RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.     

Air-stripping effects on cell growth with volatile substrates

The removal of substate molecules from aerobic microbial cultures is due to both consumption by microorganisms and stripping by the air stream. The air stripping component can be described by a constant parameter for low concentrations of volatile substrates. This air stripping parameter was found to have a value of 0.0033 h(-1) for phenol molecules in a typical fermentation situation. The determination and inclusion of this constant is important for modeling microbial growth. For Pseudomonas putida growing on phenol, it is shown that air stripping is responsible for all of the original decline in phenol concentration. Further, the kinetic inhibition constant is sensitive to both the value of the air stripping parameter and the value of the initial concentration of bacteria. The experimental data for Pseudomonas putida growing on phenol was fit by a non-linear, least squares technique to isolate the inhibition constant between 100 and 600 ppm.     

Interactive effects of wildfire and permafrost on microbial communities and soil processes in an Alaskan black spruce forest

Boreal forests contain significant quantities of soil carbon that may be oxidized to CO₂ given future increases in climate warming and wildfire behavior. At the ecosystem scale, decomposition and heterotrophic respiration are strongly controlled by temperature and moisture, but we questioned whether changes in microbial biomass, activity, or community structure induced by fire might also affect these processes. We particularly wanted to understand whether postfire reductions in microbial biomass could affect rates of decomposition. Additionally, we compared the short‐term effects of wildfire to the long‐term effects of climate warming and permafrost decline. We compared soil microbial communities between control and recently burned soils that were located in areas with and without permafrost near Delta Junction, AK. In addition to soil physical variables, we quantified changes in microbial biomass, fungal biomass, fungal community composition, and C cycling processes (phenol oxidase enzyme activity, lignin decomposition, and microbial respiration). Five years following fire, organic surface horizons had lower microbial biomass, fungal biomass, and dissolved organic carbon (DOC) concentrations compared with control soils. Reductions in soil fungi were associated with reductions in phenol oxidase activity and lignin decomposition. Effects of wildfire on microbial biomass and activity in the mineral soil were minor. Microbial community composition was affected by wildfire, but the effect was greater in nonpermafrost soils. Although the presence of permafrost increased soil moisture contents, effects on microbial biomass and activity were limited to mineral soils that showed lower fungal biomass but higher activity compared with soils without permafrost. Fungal abundance and moisture were strong predictors of phenol oxidase enzyme activity in soil. Phenol oxidase enzyme activity, in turn, was linearly related to both ¹³C lignin decomposition and microbial respiration in incubation studies. Taken together, these results indicate that reductions in fungal biomass in postfire soils and lower soil moisture in nonpermafrost soils reduced the potential of soil heterotrophs to decompose soil carbon. Although in the field increased rates of microbial respiration can be observed in postfire soils due to warmer soil conditions, reductions in fungal biomass and activity may limit rates of decomposition.     

Structure of a Natural Microbial Community in a Nitroaromatic Contaminated Groundwater Is Altered during Biodegradation of Extrinsic,but Not Intrinsic Substrates

A BSTRACTThis study demonstrates microbial community changes over time in a nitroaromatic-contaminated groundwater upon amendment with hydrocarbons previously unknown to the microbial community (extrinsic) and hydrocarbons previously known to the microbial community (intrinsic). Sealed flasks, shaken and incubated at 25 degrees C, containing contaminated groundwater and salts were amended twice with extrinsic hydrocarbons including phenol, benzoic acid, and naphthalene, and intrinsic hydrocarbons including 2,4-dinitrotoluene (2,4-DNT) and para-nitrotoluene ( p-NT). Microbial growth, biodegradation, and community structure changes measured by random amplified polymorphic DNA (RAPD) and quantitative PCR (qPCR) targeting catechol-2,3-dioxygenase (C23O) genes were monitored over time. All amended substrates were biodegraded after both substrate amendments except for 2,4-DNT, which was only partially degraded after the second amendment. Unique microbial communities were developed in flasks amended with phenol, benzoic acid, and naphthalene. However, in the flasks amended with intrinsic hydrocarbons the microbial community remained similar to the unamended control flasks. The relative amount of C23O genes detected by qPCR correlated with the biodegradation of phenol and naphthalene but not with 2,4-DNT. The results showed that a selection for microorganisms capable of catabolizing extrinsic hydrocarbons naturally and initially present in the nitroaromatic-contaminated groundwater occurred. However, growth-linked biodegradation of added intrinsic hydrocarbons was not selective.