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Use of BIOLOG for the Analysis of Microbial Communities from Zinc-Contaminated Soils
Abstract
The BIOLOG procedure, a useful method for assessing differences in community structure between soil microbial communities, could be used to determine the impact of metal contamination of soils and reclamation of these soils; however, high metal levels may affect the results of the BIOLOG assay. This project determined that Zn in soils interfered with extraction of soils with phosphate buffer by forming an insoluble precipitate. High Zn levels were also shown to prevent color formation and to cause false positive readings in the BIOLOG assay. The objectives of this project were developing and validating a modified BIOLOG method for use in the presence of high Zn concentrations. Substitution of 0.1 M Tris buffer for phosphate buffer prevented precipitate formation during the extraction step; however, extraction with Tris buffer could result in high Zn concentrations in the BIOLOG inoculant. Zinc concentrations of >50 mg L-1 in the inoculant were shown to result in false positive readings due to production of a precipitate in the wells. Raising the pH of the extractant to above 7.0 or removal of the Zn with cation exchange resin relieved this problem. Analyses of BIOLOG data showed that neither raising the extractant pH nor using cation exchange resin resulted in observable changes in BIOLOG metabolic profiles. However, use of Tris buffer in place of phosphate buffer did result in a slight change in BIOLOG metabolic profiles. Thus, the nature of the extracting buffer must be considered when analyzing experimental results. The methods developed herein allow use of the BIOLOG procedure for soils with high metal concentrations.
... During incubation at a constant temperature, soil microorganisms oxidize substrates in the plate wells and, simultaneously, reduce colourless tetrazolium dye to a violet formazan. Colour development is measured spectrophotometrically (absorbance or so-called optical density -oD) [13]. The rate of utilization of different substrates by different groups of microorganisms varies, so one can observe high variability in the rate of colour development and its intensity depending on the composition of a microbial community. ...
... Before statistical analyses are done, the absorbance value of a control well (no substrate) is subtracted from the absorbance value of each well containing a substrate. In that way one receives the so-called net absorbance value [13]. Because the absorbance value of the control well can be slightly higher than the absorbance values of some substartes, Hitzl et al. [24] subtracted the absorbance value of the least utilized substrate. ...
... Such results can point out the necessity of reclamation of contaminated soils. Effects of reclamation management on soil microbial communities may also be estimated with the Biolog plates technique [13]. ...
The Biolog technique was introduced into ecological studies to estimate metabolic potential of microbial communities. While utilizing carbon substrates (95 or 31×3, depending on the plate type), microbes reduce a colourless dye to violet formazan. The colour is measured spectrophotometrically. This is a rapid and quite convenient method but it has many drawbacks - for example, only microbes that are cultivable and able to grow in high-nutrient conditions contribute to substrate utilization. The method was used mainly to investigate microbial communities from bulk and rhizosphere soil and to estimate the impact of stressors on soil microbial communities.
... An example of such a problem is that the commonly used phosphate buffer for the extraction of the microorganisms is not an appropriate solution for the extraction of microorganismal communities from a soil with a high zinc content. Phosphate reacts with zinc and influences absorbance measurements, leading to unprecise readings [60,105,106]. Another problem that has to be taken into consideration is that physiological profiles of bacterial communities can provide different results depending on the used buffer type. ...
... Another problem that has to be taken into consideration is that physiological profiles of bacterial communities can provide different results depending on the used buffer type. A source of variation might be the fact that the phosphate buffer is also a source of nutrients for microorganisms [106]. The extraction method is essential when comparing physiological profiles. ...
Understanding ecosystem development of post-mining areas requires observing the development of the plant and microbial communities. It is widely known that mutual interaction is important for both of these groups, and both benefit significantly. The aim of this study was to broaden the knowledge about the relation between the vegetation and functional diversity of bacterial communities in novel ecosystems of post-mining areas and to discuss the potential applicability of methods of studies of bacterial functional diversity in these ecosystems with special attention paid to the BIOLOG method. The functional diversity of microbial communities of five types of microhabitats of post-coal mining heap (Upper Silesia, Poland) was studied using the BIOLOG method. Four of them were covered by spontaneously developed vegetation (two dominated by grasses Calamagrostis epigejos and Poa compressa and two others by dicotyledonous species Daucus carota and Tussilago farfara). The results obtained for vegetated microhabitats were compared with the diversity of microbial communities from non-vegetated types of microhabitat. Our study confirmed that microbial functional diversity measured by the summed area under the curve for all substrates, the richness index, the Shannon-Wiener index and the evenness index mirrors aboveground vegetation diversity. All of these measures differ, especially between non-vegetated patches and grassland patches dominated by C. epigejos and P. compressa.
... The suspension can be prepared in a few ways. The environmental sample may be suspended in sterile water, 0.9% NaCl, peptone water, phosphate buffer, or Tris buffer (Kelly and Tate, 1998;Hitzl et al., 1997). In some cases, this suspension should be filtered to eliminate the larger particles of, e.g., soil or dilute. ...
... The rapidity and range of coloration indicates the dynamics and intensity of microbial metabolism. Spectrophotometric measurement (absorbance or so-called optical density-OD) of color change is carried out at two wavelengths: 560 and 590 nm (Kelly and Tate, 1998). Researchers suggested doing a number of measurements during the incubation and choosing those readings that exhibit approximately the same average well color development (AWCD). ...
The quality of the soil environment and thus its productivity depends to a large extent on the microorganisms present in soil. Their activity, quantity, structural, and functional diversity influence many biochemical processes occurring in the soil. And in recent years, more attention has been focused on the functions of microorganisms and their role in environment. One of the methods used to analysis functional diversity of soil microorganisms is the CLPP (Community Level Physiological Profiling) technique and Biolog EcoPlate method based on it. Numerous references and our results indicate that the EcoPlate is a sensitive method that detects the sensitivity of microorganisms to environmental changes. It provides a wealth of information about the microbial community and can be used for analysis of metabolic diversity in soil microbiome.
... Using Biolog EcoPlates TM (Newark, NJ, USA) which contained three replicates of the 31 most important organic substrates for microbe metabolism, along with a water control, the microbial level physiological profile-defined as the metabolic capacity for degrading various carbon sources after substance exposure-was determined [159]. This approach evaluates the full effect that potentially harmful compounds may have on the metabolism of these communities [122,160,161]. At each concentration of EUG (0.1, 10, 100, and 1000 mg/L), wells of the Biolog EcoPlates TM were filled with 75 mL of soil leachate or processed water and 75 µL of EUG solutions. ...
Combining commercial antibiotics with adjuvants to lower their minimum inhibitory concentration (MIC) is vital in combating antimicrobial resistance. Evaluating the ecotoxicity of such compounds is crucial due to environmental and health risks. Here, eugenol was assessed as an adjuvant for 7 commercial antibiotics against 14 pathogenic bacteria in vitro, also examining its acute ecotoxicity on various soil and water organisms (microbiota, Vibrio fischeri, Daphnia magna, Eisenia foetida, and Allium cepa). Using microdilution methods, checkerboard assays, and kinetic studies, the MICs for eugenol were determined together with the nature of its combinations with antibiotics against bacteria, some unexposed to eugenol previously. The lethal dose for the non-target organisms was also determined, as well as the Average Well Color Development and the Community-Level Physiological Profiling for soil and water microbiota. Our findings indicate that eugenol significantly reduces MICs by 75 to 98%, which means that it could be a potent adjuvant. Ecotoxicological assessments showed eugenol to be less harmful to water and soil microbiota compared to studied antibiotics. While Vibrio fischeri and Daphnia magna were susceptible, Allium cepa and Eisenia foetida were minimally affected. Given that only 0.1% of eugenol is excreted by humans without metabolism, its environmental risk when used with antibiotics appears minimal.
... Dilutions with 0.1 M Tris buffer lower than 1:17 resulted in false positive results (oxidation of DOC carried over from the sample). In this case (and whenever metal amendments are anticipated), Tris is recommended (as opposed to phosphate buffer, a common solution for the soil extraction of microbial communities) as the buffer does not form any problematic metal precipitates, which could void the utility of the BIOLOG TM system assay [36]. The remaining curves in figure 3 (SES-based cultures) reflect growth rates and yields that are dramatically affected by one or many limiting nutrients. ...
The article describes a customizable chemically defined aqueous medium paradigm that was developed for the study of microbe - metal interactions. Metal speciation in the medium was predicted and controlled using MINEQL+, a chemical speciation program.
... Wynik może wyrażać gęstość optyczna OD (ang. optical density) bądź absorbancję barwnika wykrytą w studzience (Kelly, Tate 1998). OD odzwierciedla gęstość komórek drobnoustrojów w studzience. ...
... Wynik może wyrażać gęstość optyczna OD (ang. optical density) bądź absorbancję barwnika wykrytą w studzience (Kelly, Tate 1998). OD odzwierciedla gęstość komórek drobnoustrojów w studzience. ...
... Wynik może wyrażać gęstość optyczna OD (ang. optical density) bądź absorbancję barwnika wykrytą w studzience (Kelly, Tate 1998). OD odzwierciedla gęstość komórek drobnoustrojów w studzience. ...
... These include decomposition and nutrient cycling, but also impact of anthropic perturbations and management practices, and resilience (Ovreas, 2000). Garland and Mills (1991) It has since been used to compare different ecological environments (Kaiser et nl.,7998) and different agricultural practices (Fritze et nl.,7997), and to evaluate the impact of chemicals and of introduced microorganisms on ecosystems (Vahjen et al., 1995;Kelly and Tate, 1998 it is necessary to characterize the different species present in a given soil sample. ...
This book contains 18 chapters dealing with management oriented approach by identifying key issues in soil quality and management options to enhance the sustainability of modern agriculture. Topics covered include major plant nutrients (N, P, K), soil acidity, soil organic matter, soil microbial biomass and biodiversity, subsoil compaction, soil structure degradation, erosion, pesticides, industrial and urban waste. In-depth treatments of the soil quality concepts, its history, and its applicability in research and land use planning in Developed and Developing Countries are also discussed. This book will be of significant interest to post-graduate students and researchers in agronomy and in soil, crop and environmental sciences, and to stakeholders involved in issues related to land use and agricultural development.
... Next, 150-µL aliquots of diluted samples were inoculated directly into ECO microplates (BIOLOG, Hayward, CA, USA) and incubated at 25°C in the dark without shaking. Color development was measured spectrophotometrically (Kelly and Tate, 1998), and the plates were read every 24 h at 590 nm for 168 h using the Microlog Rel 4.2 software. Average well color development (AWCD) was calculated for each microplate using the following equation: AWCD = Σ (Aj-Ak) / 31, where Aj is raw absorbance in the well j, and Ak is the absorbance in control well A1 (Garland and Mills, 1991). ...
Glyphosate and glyphosate-containing herbicides have an adverse effect on mammals, humans, and soil microbial ecosystems. Therefore, it is important to develop methods for enhancing glyphosate degradation in soil through bioremediation. We investigated the potential of glyphosate degradation and bioremediation in soil by Bacillus subtilis Bs-15. Bs-15 grew well at high concentrations of glyphosate; the maximum concentration tolerated by Bs-15 reached 40,000 mg/L. The optimal conditions for bacterial growth and glyphosate degradation were less than 10,000 mg/L glyphosate, with a temperature of 35°C and a pH of 8.0. Optimal fermentation occurred at 180 rpm for 60 h with an inoculum ratio of 4%. Bs-15 degraded 17.65% (12 h) to 66.97% (96 h) of glyphosate in sterile soil and 19.01% (12 h) to 71.57% (96 h) in unsterilized soil. Using a BIOLOG ECO plate test, we observed no significant difference in average well color development values between the soil inoculated with Bs-15 and the control soil before 72 h, although there was a significant difference (P < 0.01) after 72 h. In the presence of Bs-15, the 5 functional diversity indices (Shannon index, Shannon uniformity, Simpson index, McIntosh index, and McIntosh uniformity) were greater (P < 0.01) compared with the control soil. These results indicate that Bs-15 could be used to alleviate contamination from glyphosate-containing herbicides, increasing the microbial functional diversity in glyphosate-contaminated soils and thus enhancing the bioremediation of glyphosate-contaminated soils.
... The potential metabolic diversity of soil microbial communities was assessed using BIOLOG plates as described by Kell and Tate [22], which showed the sole carbon source utilization of soil biota. Fresh soil equivalent to 5 g dry weight was added to 100 sterile water in a flask and shaken with a rotary shaker for 10 min, and 10-fold serial dilution was made for this soil suspension. ...
The bioremediation of the simulated lead (Pb)-contaminated soils by incubating with Phanerochaete chrysosporium and straw was studied at laboratory-scale. The soil pH, Pb concentration, soil microbial biomass, microbial metabolic quotient, microbial quotient and microbial biomass C-toN ratios were monitored. The above indicators were to study the stress of Pb on soil and the microbial effects during the bioremediation process. It was found that the soils treated with P. chrysosporium and straw showed a much lower concentration of soluble-exchangeable Pb, lower metabolic quotient and biomass C-toN ratios (0 mg kg −1 dry weight soil, 1.9 mg CO 2-C mg −1 biomass carbon and 4.9 on day 60, respectively) and higher microbial biomass and microbial quotient (2258 mg kg −1 dry weight soil and 7.86% on day 60, respectively) compared with the controls. In addition, the kinetic parameters in the model based on logistic equation were calculated by the BIOLOG data. By analyzing those kinetic parameters some information on the metabolic capacity of the microbial community could be obtained. All the results indicated that the bioavailability of Pb in contaminated soil was reduced so that the potential stress of Pb was alleviated, and also showed that the soil microbial effects and the metabolic capacity of microbial community were improved.
... A matéria orgânica do solo é importante para reduzir a fração solúvel dos contaminantes e a biodisponibilidade dos mesmos, diminuindo o efeito de persistência dos poluentes orgânicos. Muitos outros trabalhos também obtiveram um efeito negativo, por testes enzimáticos (urease e desidrogenase ), em solos multicontaminados com hidrocarbonetos e metais pesados (CHANDER & BROOKES, 1991; KELLY & TATE, 1998; KELLY, HAGGBLOM & TATE, 1999; AL-SALEH & OBUEKWE, 2005; SHEN et al., 2005). Com o objetivo de esclarecer a inibição da biodegradação de hidrocarbonetos monoaromáticos pela presença de metais pesados, Amor e colaboradores (2000) testaram vários metais pesados (zinco, níquel e cádmio) na inibição da biodegradação do tolueno em meio de cultura e concluíram que o níquel foi o metal com maior influência negativa, seguido pelo cádmio e por último o zinco. ...
... 1). Zmiana barwy wywołana utlenianiem substratów w studzienkach jest mierzona spektrofotometrycznie jako absorbancja lub gęstość optyczna OD (Optical Density) [38]. ...
Organic wastes are useful in agriculture as organic and nutrients fertilizers. Changes in soil environment after application of organic wastes may be successfully controlled using conventional and molecular methods. Microorganisms play important role in soil organic matter transformations. Evaluation of their enzymatic activity, functional and genetic diversity can be used as sensitive tool to asses soil quality and fertility. It is important to combine conventional microbiological methods with molecular techniques, to obtain more information about population of soil microbiota.
... More research is needed on reclamation of copper mine tailings in the arid south-west. In particular, microbial community populations, structure and activity are important indicators of ecosystem recovery that need to be evaluated during the reclamation process (Seaker and Sopper 1988;Kelly and Tate 1998;Yin et al. 2000). ...
... Recently extensive researches have been focused on the relationship between the microbial community diversity and the processes occurring in the soil, and how they respond to environmental changes [6,7]. Various methods were used to research soil microbial diversity, such as the traditional plate counts of cultivation method, the molecular method, the phospholipids fatty acids analysis method and the BIOLOG method [8][9][10]. ...
There are increasing concerns over facilities cultivation soils of nitrate accumulation in China. Nitrate pollution in secondary salinization soil is regarded as having potential effects on soil microbial communities. Our study was conducted to evaluate effects of secondary salinization on soil microbial functional diversity with the BIOLOG method. The results showed that Average well-color development (AWCD) values declined with the rising of nitrate concentrations to some extent. The results also exhibited that the accumulation of nitrate in soil decreased the carbon sources utilization rates and the microbial species diversity indices. It indicated that nitrate has significantly negative effects on the sole-carbon-source metabolic ability of soil microbial communities. The cluster analysis intuitively demonstrated the distance and relationship between each sample: soil samples with high nitrate content were more close to each other, while soil samples with low were more similar in distances. The principal component analysis (PCA) result further validated that nitrate was inversely correlated to microbial carbon sources utilization intensity and microbial diversity. The four carbon substrates, Carbohydrates, Miscellaneous, Amino acids and Polymers, could reflect most of the information about carbon sources utilization. Microorganisms preferred these four carbon substrates were more vulnerable to nitrate. Thus, these four carbon substrates could be one of the prioritized microbe carbon sources in soil bioremediation.
... These include decomposition and nutrient cycling, but also impact of anthropic perturbations and management practices, and resilience (Ovreas, 2000). Garland and Mills (1991) It has since been used to compare different ecological environments (Kaiser et nl.,7998) and different agricultural practices (Fritze et nl.,7997), and to evaluate the impact of chemicals and of introduced microorganisms on ecosystems (Vahjen et al., 1995;Kelly and Tate, 1998 it is necessary to characterize the different species present in a given soil sample. ...
Plant pathologists have traditionally viewed soil as a hostile environment, harbouring pathogens that have adverse effects on plant health. The emerging concept of soil quality sees pathogens as components of total soil biological diversity, with disease resulting from disturbances to the balance between functional groups in soil. Experimental support for this concept is beginning to be accumulated. Molecular methods based on polymerase chain reaction (PCR) of DNA extracted from soil allow rapid assessment of genetic diversity and will increasingly be used to measure functional diversity as well. This will enable the relationships between diversity and disease suppression to be characterized. Suppressiveness of soils to disease is biological in nature, although modified by abiotic factors, and is of two types. General suppression depends on overall diversity and activity of the soil biota and acts against a broad range of pathogens. Specific suppression is due to particular antagonists or functional groups, and acts against single pathogens. Studies on the effects of management practices on disease suppression are still limited in scope, and are often difficult to interpret because most practices have direct effects on pathogen populations as well as on suppressiveness. Continuous cropping of a plant species selects for microflora adapted to its rhizosphere, which may suppress the activities of some pathogens. Rotations and reduced tillage should increase microbial diversity and increase suppressiveness, but evidence for these causal links is hard to find. Treatments that increase soil organic matter, such as residue retention and application of manure, may increase general suppression, and certain types of manures may also increase specific suppression. Soil biodiversity, disease suppression and management practices are yet to be fully synthesized in models that have general applicability. This means that management thresholds remain speculative in most instances.
... More research is needed on reclamation of copper mine tailings in the arid south-west. In particular, microbial community populations , structure and activity are important indicators of ecosystem recovery that need to be evaluated during the reclamation process (Seaker and Sopper 1988; Kelly and Tate 1998; Yin et al. 2000). The goal of mine tailing reclamation is to ensure selfsustainable microbial and plant communities (Pond et al. 2005; Rosario et al. 2007). ...
This study evaluates the effect of surface application of dried Class A biosolids on microbial populations within copper mine tailings.
Mine tailing sites were established at ASARCO Mission Mine close to Sahuarita Arizona. Site 1 (December 1998) was amended with 248 tons ha(-1) of Class A biosolids. Sites 2 (December 2000) and 3 (April 2006) were amended with 371 and 270 tons ha(-1), respectively. Site D, a neighbouring native desert soil, acted as a control for the evaluation of soil microbial characteristics. Surface amendment of Class A biosolids showed a 4 log(10) increase in heterotrophic plate counts (HPCs) compared to unamended tailings, with the increase being maintained for 10-year period. Microbial activities such as nitrification, sulphur oxidation and dehydrogenase activity were also sustained throughout the study period. 16S rRNA clone libraries obtained from community DNA suggest that mine tailings amended with biosolids achieve diversity and bacterial populations similar to native soil bacterial phyla, 10 years postapplication.
Addition of Class A biosolids to copper mine tailings in the desert south-west increased soil microbial numbers, activity and diversity relative to unamended mine tailings.
The amended tailings resulted in a functional soil with respect to microbial characteristics, which were sustainable over a 10-year period enabling the development of appropriate vegetation.
... Since this was not corrected for, the possibility remains that separation of the soils by the PCA could be attributable to these di¡erences. The e¡ect of the heavy metal in question on the assay also needs to be taken into account since it has been shown that zinc is able to both prevent color formation and cause false-positive readings [34]. Similar investigations should be performed with other heavy metals to determine whether they also in£uence the substrate utilization assay. ...
The effect of long-term exposure to mercury on the soil microbial community was investigated in soil from three different sites along a pollution gradient. The amount of total and bioavailable mercury was negatively correlated to the distance from the center of contamination. The size of the bacterial and protozoan populations was reduced in the most contaminated soil, whereas there was no significant difference in fungal biomass measured as chitinase activity. Based on the number of colony morphotypes, moreover, the culturable bacterial population was structurally less diverse and contained a higher proportion of resistant and fast-growing forms. The profiles of amplified 16S rDNA sequences obtained from community DNA by denaturating gradient gel electrophoresis (DGGE) also reflected the altered community structure and decreased diversity along the mercury gradient as expressed in terms of the number and abundance of bands. The functional potential of the microbial population measured as sole carbon source utilization by Ecoplates® differed between the soils, but there was no change in the number of substrates utilized. The observed changes in the different soil microbial populations are probably a combination of both direct and indirect effects of the mercury contamination.
... Studies about the effect of Zn on the soil respiration using enzymatic methodologies report that the Zn depletes the dehydrogenase activity. This inhibitory effect has been shown in the laboratory [35] and in the field [36,37]. Dehydrogenase activity has been shown to correlate with soil respiratory activity which is also correlated with the heat released by soil metabolism [14,15]. ...
The control on the CO2 coming from soil handling, makes necessary the introduction of new methodologies that inform about the capacity of the soil
as a carbon sink and about the carbon decay. It can be performed through the microbial growth yield efficiency concept by
calorimetry and enthalpy balances. Here it is examined the sensitivity of these indicators to two metal layering phosphates,
AZP [(NH)4Zn2(PO)4(HPO)4] and AIP [(NH)4Fe(PO)4H2O] to assess about their soil impact. Both compounds caused metabolic changes on soil microbial biomass when compared to appropriated
references indicating that the proposed methodology is sensitive to different inorganic sources of microbial growth.
... Techniques have been developed to assess the 20 health of the soil microbial population, including measure of soil function through respiration, N 21 cycling, and ability to utilize added substrates (CELA and SUMNER 2002; CHANG and 22 BROADBENT 1982; MCGRATH 2002; SAUVE 2002). One example is the Biolog extraction 23 (KELLY and TATE 1998), which attempts to evaluate the functionality of the soil microbial 24 population through its ability to utilize a range of carbon sources. The procedure has been 25 criticized for difficulty of interpretation, i.e. organism presence can falsely suggest a robust 26 microbial community, and the ability of different groups of microbes to utilize the same 27 substrates (NATIONAL_RESEARCH_COUNCIL 2003). ...
Bioremediation of metals and radionuclides has had many field tests, demonstrations, and full-scale implementations in recent
years. Field research in this area has occurred for many different metals and radionuclides using a wide array of strategies.
These strategies can be generally characterized in six major categories: biotransformation, bioaccumulation/bisorption, biodegradation
of chelators, volatilization, treatment trains, and natural attenuation. For all field applications there are a number of
critical biogeochemical issues that most be addressed for the successful field application. Monitoring and characterization
parameters that are enabling to bioremediation of metals and radionuclides are presented here. For each of the strategies
a case study is presented to demonstrate a field application that uses this strategy.
... sites seemed to be suppressed compared to RA. In respect to microbial biomass, RS soil showed efficient soil C utilization by microorganisms in August. However, BIOLOG analysis indicated lower C utilization activity of microbial community in RS. We conducted another BIOLOG test using the gradual addition of Cu concentrations to the soil suspension. Kelly and Tate (1998) have reported that Zn concentrations of >50 mg L −1 in the inocula at BIOLOG tests resulted in false positive readings due to production of a precipitate in the well. In the present study, no precipitate was observed in any well inoculated with solution of 25 mg L −1 Cu concentration, corresponding to 1000 μg g −1 dry soil. In this way, ...
The objective of the present study was to increase understanding of the effects of heavy metal pollution and soil properties
on microorganisms in relation to the biomass and microbial functional community. Soil samples were collected from aged polluted
and reference sites on a floodplain. The soil Cu, Zn and Pb total concentrations were much higher at the polluted sites (average
231.6–309.9 mg kg−1, 195.7–233.0 mg kg−1, and 72.4–86.0 mg kg−1, respectively) than at the reference site (average 33.3–44.0 mg kg−1, 76.7–98.0 mg kg−1, and 30.8–41.6 mg kg−1, respectively), while the available heavy metal concentrations in CaCl2 extraction were similar in all sites. Small seasonal variations in the size of microbial biomass were observed. Ambient soil
properties (e.g. total C, N, pH, moisture content, and CEC) affected the soil microbial biomass more than the heavy metal
pollution. However, the aged pollution tended to impact on the composition of the microbial community. PICT (pollution-induced
community tolerance) test using BIOLOG Ecoplates showed enhanced tolerance of the microbial community to Cu stress in the
polluted site. In non polluted but low nutrient, low pH and low moisture soil, the microbial biomass was lower and the microbial
community was more vulnerable to Cu stress. In spite of the low heavy metal availability due to ageing, the BIOLOG technique
provided sensitive detection of microbial community level changes in PICT analysis.
... Pennanen et al. (1998) reported that the BIOLOG w test did not detect change in forest humus microbial communities due to heavy metal or acid rain treatment. On the other hand, several other authors reported that the test was useful in assessing the effect of heavy metals on community-level physiological profiles of microbial communities (Knight et al., 1997; Kelly and Tate III, 1998a,b; Yao et al., 2003). ...
Various parameters of the soil microbial community may be used in soil quality evaluation and environmental risk assessment. The objectives of this study were to assess the effects of different environmental factors on the characteristics of forest humus microbial communities, and to test which environmental factors most affect the gross microbial indices and physiological profiles of these communities. Samples were taken at 71 plots located in a heavily polluted area of the Krakowsko–Cze¸stochowska upland in southern Poland. The samples were analyzed for pH in KCl (pHKCl), organic C (Corg), total N (Nt) and S (St), and for total and soluble Zn, Pb and Cd concentrations. The considered microbial parameters included basal respiration (BAS), microbial biomass (Cmic), Cmic-to-Corg ratio, and community-level physiological profiles (CLPPs) studied using BIOLOG® Ecoplates. Multiple regression analysis was used to estimate the effects of humus properties on the microbial parameters. It indicated that St and Corg-to-Nt ratio were the most important factors positively affecting Cmic (β=0.15 and 0.11, respectively) and BAS (β=0.13 and 0.08, respectively). The Cmic-to-Corg ratio was related positively to St (β=0.12) but negatively to Nt (β=−0.08). The effects of pHKCl and heavy metals on the gross microbial indices were significant but less important. The most important effect on microbial activity on BIOLOG® plates and CLPPs was from pHKCl. The other significant variables included St, Corg-to-Nt and interactions of heavy metals with pHKCl. It was concluded that Cmic, Cmic-to-Corg and BAS might be good indicators of the general status of soil microbial communities, but their use in studying heavy metal effects may entail difficulties in separating the effects of other factors. The sensitivity of the BIOLOG® test to pHKCl suggests that it may be useful for studying the effects of acidification or liming on soil microbial communities. The significant effect of the interactions between heavy metals and other variables on physiological profiles indicated that high heavy metal content affects the metabolic functions of soil microbial populations.
... Subsequently, for each well the area under the absorbance curve was calculated. Since the addition of metals may result in colour development (Kelly and Tate, 1998), additional Biolog 1 plates were inoculated with sterile Cu and Zn solutions containing 5, 20, 80 or 320 mg l À1 Zn or Cu. The areas under curve calculated for the wells inoculated with solutions containing microorganisms were corrected for metal effects by subtracting the values obtained for the wells inoculated with appropriate sterile metal solutions. ...
The long-term accumulation of heavy metals in forest soil organic layers may adversely affect soil microorganisms. The objectives of the study were to assess the effects of high contents of Cu and Zn on the soil microbial communities and their tolerance to metal pollution. Several microbial indices such as microbial biomass (Cmic), the basal soil respiration (BAS), the community level physiological profiles (CLPP) and the pollution-induced community tolerance (PICT) approach, based on Biolog® EcoPlate assay, were used. The soil organic layer samples were taken in the Cu-polluted (CuP), Zn-polluted (ZnP) and unpolluted (UP) Scots pine forests. Despite very high total heavy metal concentrations in soils Cmic, BAS and CLPPs at the polluted sites did not differ from those at the UP sites. A damaging effect of heavy metals was detected only by the tolerance measurements with Biolog® plates. PICT to Cu at the CuP sites was found for five substrates. At the ZnP sites significant PICT to Zn was found for 10 out of 31 substrates. Furthermore, the CuP samples exhibited lower resistance to Zn and the ZnP samples to Cu compared with the UP samples, indicating that at the polluted sites microbial resistance to additional stress caused by another metal was decreased. Since the tolerance measurements using Biolog® assay appeared to be more sensitive than other methods used, we concluded that this approach could be useful in environmental risk assessment.
... They play key roles in food webs and nutrient cycles (Bouwman et al., 1994; Bloem et al., 1997; Schouten et al., 1997). Effects of metals on bacterial communities have been demonstrated under laboratory conditions (Bååth et al., 1998; Dıàz-Raviña and Bååth, 1996; Doelman et al., 1994), in spiked-fields and under natural conditions (Gong et al., 2000; Kandeler et al., 2000; Kelly and Tate, 1998; Brookes and Mcgrath, 1984; Jordan and Lechevalier, 1975; Davis et al., 2004). Bacterial communities exposed to a high level of metals showed different physiological traits and bacterial species composition as well as a development of tolerance to metals. ...
This study aimed to assess effects of metals on bacterial communities in grassland soil and to discriminate these effects from natural variability in soil properties. Changes in gross parameters of bacterial communities were investigated by the determination of 14C-leucine and 3H-thymidine incorporation rates, CO2 evolution, biomass indicators and N mineralization rates. 14C-leucine incorporation rate and CO2 evolution showed correlations with all metals tested as well as with organic matter content. 3H-thymidine incorporation rates, biomass indicators and N mineralization rates did not strongly correlate with any physical or chemical parameters. Further, community-level physiological profiles (CLPP) and polymerase chain reaction (PCR)-amplified denaturing gradient gel electrophoresis (DGGE) of 16S rDNA were performed. Monte-Carlo permutation testing was performed to analyse CLPP and DGGE data to allow for a stringent discrimination between sources of effects. CLPP changes correlated with the Pb concentration and pH in the soil. DGGE changes correlated with Pb and Cu concentrations and organic matter content. Pollution-induced community tolerance (PICT) was not found for any of the metals assessed. A causal relation between the effects observed on bacterial communities and the presence of metals was not established with PICT. The negative outcome of PICT can probably be attributed to indirect effects or to methodological problems. We concluded that the observed shifts in CLPP and DGGE patterns are a strong indication of metals effects on bacterial communities in this grassland. These effects could only be filtered from the total variation by multivariate analyses.
... However, an In terms of assay suitability, CDM was compatible with a number of commonly performed assays on the soil solution and the soil microbial community. In this study, the compatibility with metabolic diversity profiling (BIOLOG™ system) precipitates, which could void the utility of the BIOLOG™ system assay (Kelly and Tate, 1998). ...
"Graduate Program in Environmental Sciences." Thesis (Ph. D.)--Rutgers University, 2005. Includes abstract. Vita. Includes bibliographical references.
... Ecotoxicological effects of heavy metals on soil microbes have been recently widely investigated at the ecosystem level using culture-independent methods including BIOLOG microplate techniques [1][2][3], DNAbased approaches [4][5][6], and phospholipid fatty acid analyses (PLFA) [7,8]. The majority of these studies are concerned with the microbiological differences at the community level between non-polluted and contami-nated sites. ...
The phylogenetic positions of 60 bacterial strains isolated from three tailing piles were determined by analyzing their partial 16S rRNA gene sequences. These strains were divided into three phylogenetically distinct groups of Arthrobacter and likely represented several non-described species. The physiological diversities of these phylogenetically and geographically distinct populations were evaluated based on their resistance to five heavy metals (Zn, Pb, Cd, Cu and Co) and four antibiotics (Kan, Rif, Str and Amp), and differences in utilization of 49 carbon sources. Genetic differentiations were demonstrated with randomly amplified polymorphic DNA (RAPD) analysis. These biological parameters were significantly different among three phylogenetically distinct groups. Notably, detectable differences were also observed among three geographically distinct subdivisions with similar taxonomic position. These results indicate that mine tailings are an ideal site for investigating the differentiation of natural bacterial populations in response to extreme metal contamination. Additionally, these environments appear to harbor many previously not characterized bacterial species, which are potentially important candidates for application in bioremediation due to their remarkable resistance to multiple metals.
The quality of the soil environment and thus, its productivity depend to a large extent on the microorganisms present in soil. Their activity, quantity, structural, and functional diversity influence many biochemical processes occurring in the soil. And in recent years, more attention has been focused on the functions of microorganisms and their role in environment. One of the methods used to analysis functional diversity of soil microorganisms is the Community Level Physiological Profiling technique and Biolog EcoPlate method based on it. Numerous references and our results indicate that the EcoPlate is a sensitive method that detects the sensitivity of microorganisms to environmental changes. It provides a wealth of information about the microbial community and can be used for analysis of metabolic diversity in soil microbiome.
The distribution pattern of the microbial community in mountains is an important component of biodiversity research. Many environmental factors vary significantly with elevation on a relatively small scale in subalpine and alpine environments. These factors may markedly affect microbial community composition and function. In this study, we analyzed phospholipid fatty acid (PLFA) profiles and phosphorus (P) fractions in soils from 9 sites along an elevation gradient (3500–4100 m above sea level (a.s.l.)) of the Shergyla Mountain, Tibet in China. Many biomarker PLFAs indicated that there were biogeochemical trends of the microbial distribution patterns of some soil microorganisms, which were most often increasing, U-shaped and unimodal trends along the elevation gradient. A redundancy analysis (RDA) and correlations indicated that P factors (e.g., Resin-Pi, NaHCO3-Pi and NaHCO3-Po) were more important in controlling the microbial PLFA distribution pattern than other factors (e.g., MAT, MAP, pH, TOC, TN and soil moisture) in this study area. Microorganisms are strongly associated with P fractions. Our results suggested that microbial communities were subjected to P stresses and that the distribution patterns of microbial communities were shaped by bioavailable P along the elevation gradient. Our work also hints that P geochemical processes drive the microbial diversity of the Shergyla Mountains.
Antibiotics’ (ATBs) occurrence in soil ecosystems has a relevant effect in the structure and functionality of edaphic microbial communities, mainly because of their amendment with manure and biosolids that alter their key ecological functions.
In this study, the impact of eight widely consumed ATBs on a natural soil microbial community, characterized through 16 S rRNA gene sequencing, was evaluated. Changes induced by the ATBs in the growth of the soil microbiota and in the community-level physiological profiling (CLPP), using Biolog EcoPlates™, were measured as endpoint.
The eight assayed ATBs lead to a significant decrease in the growth of soil microbial communities in a dose-dependent way, ordered by its effect as follows: chloramphenicol > gentamycin > erythromycin > ampicillin > penicillin > amoxicillin > tetracycline > streptomycin. Chloramphenicol, gentamycin, and erythromycin adversely affected the physiological profile of the soil community, especially its ability to metabolize amino acids, carboxylic and ketonic acids and polymers. The analysis of the relationship between the physico-chemical properties of ATBs, as well as their mechanism of action, revealed that, except for the aminoglycosides, each ATB is influenced by a different physico-chemical parameters, even for ATBs of the same family.
Significant effects were detected from 100 μg mL to 1, concentrations that can be found in digested sludge, biosolids and even in fertilized soils after repeated application of manure, so cumulative and long-term effects of these antibiotics on soil environment cannot be ruled out.
Współczesne badania różnorodności mikroorganizmów dotyczą także różnorodności funkcjonalnej. Firma Biolog® stworzyła metodę badawczą pozwalającą na uzyskanie bardzo dużej liczby wyników w oparciu o zdolność drobnoustrojów do rozkładu różnych źródeł węgla (CLPP). Ich produkty, a w szczególności płytki ECO, służą do analizy całych społeczności mikroorganizmów pochodzących z próbek środowiskowych. Profilowanie populacji odbywa się dzięki spektrofotometrycznym pomiarom barwnej reakcji redoks. Zachodzi ona na 96-dołkowych płytkach zawierających różne substraty węglowe
i barwnik, którym jest fiolet krystaliczny, w momencie gdy zainokulowane do studzienki mikroorganizmy metabolizują dane źródło węgla. Metoda ta ma zarówno wady, jak i zalety, jednak przyczynia się do lepszego poznania współpracy występującej między mikroorganizmami w środowisku.
Variation of soil-microbial communities are good bioindicators of human impacts in soils, such as different soils management or contamination. Considering that traditional methods of isolation and taxonomic analysis do not consider the functionality of the microbial community, Community-Level Physiological Profiles (CLPP) emerged as a complementary methodology to study microbial communities. Several studies have shown that Biolog® EcoPlates® are very useful for determining physiological differences between communities from different samples. However, commercial microplates have some disadvantages which led us to the idea of replacing them by microplates prepared in the laboratory (Laboratory's). Here, we compared both types of microplates using soil samples from a bioremediation assay. We compared a) the average well color development for each sample, b) the averages of absorbance values for each type of microplate, c) Principal Components, and d) Shannon-Weaver's diversity index (H). Although Laboratory's showed significantly lower Average absorbance values than EcoPlates®, the principal component analysis and diversity index did not differ between types of microplates. In conclusion, both types of microplates showed a relatively similar ability to detect differences in the CLPP of the treatments studied. Consequently, microplates prepared in laboratory are a reliable and economical tool to study the physiology of soil microbial communities.
The phylogenetic positions of 60 bacterial strains isolated from three tailing piles were determined by analyzing their partial 16S rRNA gene sequences. These strains were divided into three phylogenetically distinct groups of Arthrobacter and likely represented several non-described species. The physiological diversities of these phylogenetically and geographically distinct populations were evaluated based on their resistance to five heavy metals (Zn, Pb, Cd, Cu and Co) and four antibiotics (Kan, Rif, Str and Amp), and differences in utilization of 49 carbon sources. Genetic differentiations were demonstrated with randomly amplified polymorphic DNA (RAPD) analysis. These biological parameters were significantly different among three phylogenetically distinct groups. Notably, detectable differences were also observed among three geographically distinct subdivisions with similar taxonomic position. These results indicate that mine tailings are an ideal site for investigating the differentiation of natural bacterial populations in response to extreme metal contamination. Additionally, these environments appear to harbor many previously not characterized bacterial species, which are potentially important candidates for application in bioremediation due to their remarkable resistance to multiple metals.
Se evaluó la dinámica de la actividad de la enzima deshidrogenasa (ADH) en un suelo Calciorthids de la Península de Paraguaná (estado Falcón) enmendado con tres residuos orgánicos: lodo residual proveniente del tratamiento de aguas servidas, estiércol de chivo, y residuo del procesamiento industrial de la sábila, Aloe vera, a dosis de 1 y 2%. Suelo y tratamientos fueron incubados aeróbicamente, en condiciones de laboratorio, durante 64 días. La cuantificación de la ADH es uno de los métodos más usados para determinar la actividad de los microorganismos en el suelo, y se basa en la determinación colorimétrica del producto liberado 2,3,5-trifenilformazan (TFF) que se origina después de incubar la muestra con cloruro de 2,3,5-trifeniltetrazolio (CTT) a 37 ºC por 24 horas. La incorporación de materiales orgánicos al suelo incrementó significativamente (P=0,05) la actividad de esta enzima con respecto al control. Este incremento se mantuvo hasta el final del período de incubación, alcanzando valores de 474 mg TFF g-1 suelo 24 h-1 sobre base seca para el tratamiento con el residuo vegetal a dosis de 1% y 466 mg TFF g-1 suelo 24 h-1 para el tratamiento con estiércol de chivo al 2%. Para los tratamientos orgánicos el incremento inicial en la actividad de la enzima fue mayor con la dosis más alta, observándose una clara tendencia a disminuir con el transcurso del tiempo.
The study focused on the change of microbial characteristics affected by Plumbum pollution with purple paddy soil in an incubation experiment. The results showed that low concentration of Plumbum had little effect on most of microbial amounts, biological activity and enzymatic activity. However, denitrifying activity was inhibited severely, and inhibition rate was up to 98%. Medium and high concentration of Plumbum significantly reduced the amounts and activity of all microorganisms and enzymatic activity, which increased with incubation time. Negative correlations were found between Plumbum concentrations and microbial amounts, biological activity and enzymatic activities except fungi and actinomyces. Thus they can be used to indicate the Plumbum pollution levels to some extent. LD(50) of denitrifying bacteria (DB) and ED50 of denitrifying activity were 852mg/kg and 33.5mg/kg. Across all test soil microbes, denitrifying bacteria was most sensitive to Plumbum pollution in purple paddy soil. Value of early warning showed that anaerobic cellulose-decomposing bacteria (ACDB) and actinomyces were also sensitive to Plumbum pollution. We concluded that denitrifying activity, actinomyces, ACDB or DB can be chosen as predictor of Plumbum contamination in purple paddy soil.
Impact of Cr(3+) pollution on soil microbial quantity, enzyme activity and biological activity in purple paddy soil were studied under incubation conditions. The results showed that amounts of all tested microbes and enzyme activities in soil were inhibited by low Cr(3+) concentration (200mg/kg). After 7-day incubation, sulfate-reducing activity, methanogen activity, denitrifying activity and anaerobic nitrogen-fixing activity in soil were reduced by 34%, 66%, 98% and 65% respectively. Amounts of soil microbes were remarkably inhibited with medium Cr(3+) concentration (400mg/kg), all with reduction of more than 50%; and all tested soil biological activity was almost recovered in the fourth week except soil denitrifying activity. Activities of urease, invertase, neutral phosphatase and catalase were decreased by 60%, 21%, 59% and 42%, respectively. With high Cr(3+) concentration (1600mg/kg), amounts and activities of tested microbes had only about 1% of that with control. As calculated from the regression equation, the ED50 (ecological dose) values of activities of soil urease, invertase and catalase were around 800mg/kg; the ED50 values of soil sulfate-reducing activity, methanogen activity and anaerobic nitrogen-fixing activity were also around 800mg/kg with an exception of soil denitrifying activity which ranged 35 to 39 mg/kg. According to the Standards of National Soil Environmental Quality in China and their sensitivities to 400mg/kg Cr(3+) concentration, quantity of denitrifying bacteria, urease activity and denitrifying activity could be selected as indicators of early warning for Cr(3+) pollution in purple paddy soil.
Analyses of soil samples revealed that the level of lead (total or bioavailable) was three-fold greater in crude oil contaminated than in uncontaminated Kuwaiti soils. Investigation of the possible inhibitory effect of lead on hydrocarbon degradation by the soil microbiota showed that the number of hydrocarbon-degrading bacteria decreased with increased levels of lead nitrate added to soil samples, whether oil polluted or not. At 1.0mg lead nitrate g−1 dry soil, the number of degraders of hexadecane, naphthalene and crude oil declined by 14%, 23% and 53%, respectively. In a similar manner, the degradation and mineralization of different hydrocarbons decreased with increased lead content in cultures, although the decreases were not significantly different (P>0.05). The dehydrogenase activities of soil samples containing hydrocarbons as substrates also declined with an increase in the lead content of soil samples.
Substrate utilization tests with Biolog® plates were used to obtain information on differences in community composition and
on changes in the metabolic diversity and activity of microorganisms in samples of various soils polluted with heavy metals
in a region of a former mine in Hungary. Differences in population and metabolic diversity were observed depending on heavy
metal contamination and low pH. Both factors caused a lower metabolic activity and a loss in diversity. The BIOLOG assay was
found to be a useful tool to evaluate the microbiological fitness of soil in polluted areas. Principal component analysis
of GN and ECO plates resulted in a different description and classification of the present bacterial community.
For over a decade, community level physiological profile (CLPP) assays, which assess a microbial community's capacity to metabolize specific sole carbon sources under defined laboratory conditions, have been popular for study of environmental soil samples. One such assay, BiOLOG™ allows for the colorimetric measurement of metabolism through the reduction of a tetrazolium dye, which yields optical density (OD) data for each substrate. Bacterial communities are extracted from soil and 150 μL of this extract is inoculated directly into each well of the microtitre plate. The combined metabolic data obtained are most often analyzed with multivariate statistical analyses, such as principal component analysis (PCA). The objectives of this study were (1) to develop a simple, visual, statistically valid method of determining community capabilities to utilize specific substrates in CLPP studies and (2) to test the number of samples needed for such discrimination to be reliable. This was done by direct comparison of the OD values obtained for two closely related microbial communities (surface and subsurface soil), plotted against a one-to-one (y=x) line. Due to variability in the portion of the soil microbial community inoculated into the individual test wells, the accuracy of the method was dependent on the number of replicates analyzed. A variety of data set sizes were tested, from n=3 samples/soil depth to n=40 samples/depth. The method was statistically valid for all data sets tested. Those substrates that deviated from the one-to-one line consistently had F values greater than 1. Additionally, data sets of n=30,35 and 40 samples/depth consistently allowed identification of the eight substrates whose metabolism varied significantly between the two test soil communities. In conclusion, this one-to-one comparison has been shown to be a statistically valid analytical method to compare individual substrate usage between soils.
This paper summarises our recent field studies on the microbial communities of boreal coniferous forest humus exposed to environmental stress, heavy metals and changes in humus pH. The microbial community was measured using the phospholipid fatty acid (PLFA) and Biolog® analyses, and the actual factor in the environment exerting the selective pressure on the bacterial community was estimated with the help of tolerance determinations using the 3H-thymidine incorporation technique. The field experiments showed that the structure of the microbial community inhabiting the boreal coniferous forest humus was influenced by changes in humus pH and heavy metal concentrations at levels where no, or only small, effects on the microbial biomass or carbon mineralization rate were seen. The alterations in the humus PLFA patterns were related to the abundance of the major groups of microorganisms, bacteria and fungi. Changes in the relative proportions of Gram-negative and Gram-positive bacteria, including actinomycetes, were also shown. With the help of the 3H-thymidine incorporation technique, it was demonstrated that forest humus bacterial communities exposed to heavy metals or alterations in humus pH were able to adapt to the environmental disturbance in question. When combining the results from the PLFA and 3H-thymidine analyses, it was revealed that the increased tolerance of the humus bacterial community to heavy metals or to altered pH resulted at least partly from a change in microbial species composition. Coniferous forest humus seemed to contain a bacterial group, consisting mainly of Gram-positive bacteria, which were adapted more easily to the acidifying environment and a group of bacteria, mainly Gram-negative ones, which were more easily adapted to the humus with a higher pH. The Biolog® technique, which determines the community level physiological profile (CLPP) of the bacterial community was less sensitive and less suitable than the PLFA analysis to detect the characteristics of the forest humus microbial community. The 3H-thymidine incorporation technique was the most sensitive of the techniques used in this study to detect the influence of environmental disturbances on the microbial community. In addition, a gradient of coniferous forest stands having naturally different humus pH because of the different site properties was studied to compare these natural microbial communities with the communities subjected to anthropogenic change in humus pH. In order to reveal the similarity of the humus samples with respect to their community structure, the PLFA patterns from all the field studies were subjected to multivariate cluster analysis. The structure of the forest humus microbial community was shown to be strongly influenced by the indigenous fertility of the coniferous forest site type, which was in turn related to humus nutritional status, pH, moisture, tree species and ground vegetation. Thus, a prerequisite for successful determination of the impacts of environmental stress on forest humus microbial community is the homogeneity of the forest site types between the experimental plots.
Long-term diversity-disturbance responses of soil bacterial communities to copper were determined from field-soils (Spalding; South Australia) exposed to Cu in doses ranging from 0 through to 4012 mg Cu kg−1 soil. Nearly 6 years after application of Cu, the structure of the total bacterial community showed change over the Cu gradient (PCR-DGGE profiling). 16S rRNA clone libraries, generated from unexposed and exposed (1003 mg Cu added kg−1 soil) treatments, had significantly different taxa composition. In particular, Acidobacteria were abundant in unexposed soil but were nearly absent from the Cu-exposed sample (P<0.05), which was dominated by Firmicute bacteria (P<0.05). Analysis of community profiles of Acidobacteria, Bacillus, Pseudomonas and Sphingomonas showed significant changes in structural composition with increasing soil Cu. The diversity (Simpsons index) of the Acidobacteria community was more sensitive to increasing concentrations of CaCl-extractable soil Cu (CuExt) than other groups, with decline in diversity occurring at 0.13 CuExt mg kg−1 soil. In contrast, diversity in the Bacillus community increased until 10.4 CuExt mg kg−1 soil, showing that this group was 2 orders of magnitude more resistant to Cu than Acidobacteria. Sphingomonas was the most resistant to Cu; however, this group along with Pseudomonas represented only a small percentage of total soil bacteria. Changes in bacterial community structure, but not diversity, were concomitant with a decrease in catabolic function (BioLog). Reduction in function followed a dose-response pattern with CuExt levels (R2=0.86). The EC50 for functional loss was 0.21 CuExt mg kg−1 soil, which coincided with loss of Acidobacteria diversity. The microbial responses were confirmed as being due to Cu and not shifts in soil pH (from use of CuSO4) as parallel Zn-based field plots (ZnSO4) were dissimilar. Changes in the diversity of most bacterial groups with soil Cu followed a unimodal response – i.e. diversity initially increased with Cu addition until a critical value was reached, whereupon it sharply decreased. These responses are indicative of the intermediate-disturbance-hypothesis, a macroecological theory that has not been widely tested in environmental microbial ecosystems.
Soil microcosms each consisting of approximately 20 kg soil were set up in the laboratory. Three microcosms were amended with 6000 mg zinc kg−1, a concentration chosen to approximate the quantities of total zinc found at a contaminated field site. Three control microcosms received no zinc amendment. Three pH control microcosms received H2SO4, adequate to result in the same pH decrease as occurred in the zinc amended soil. Mean soluble zinc contents were 4660 mg kg−1 (amended soils), 1.08 mg kg−1 (unamended soils), and 2.10 mg kg−1 (pH adjusted soils). Biological community assessments included culturable bacteria (plate counts), microbial biomass (chloroform fumigation), dehydrogenase activity, proportion of zinc resistant bacteria (plate counts on zinc amended agar), and changes in community structure (BIOLOG and phospholipid fatty acid (PLFA) analysis). After 15 d of incubation, zinc amendment resulted in an 87% decrease in viable counts and a 47% decrease in microbial biomass. Dehydrogenase activity decreased by 95%, and the proportion of zinc resistant bacteria increased from 0.08% to 0.75%. Microbial community structure alteration resulting from zinc amendment was indicated by shifts in both BIOLOG metabolic profiles and PLFA profiles. Effects of zinc on PLFA profiles included relative decreases in indicator fatty acids for arbuscular mycorrhizal fungi and actinomycetes. Some adaptation of the microbial communities to the zinc amendment was suggested by the fact that after 420 d incubation there were no significant differences in culturable bacterial populations or microbial biomass of the control and zinc amended systems. However, other microbial properties measured in this study showed that zinc still affected microbial community structure and activity after 420 d. Dehydrogenase activity of the zinc treated communities remained 93% lower than the controls. The percentage of zinc resistant populations was still significantly higher for the zinc treated communities. Similarly, the BIOLOG and PLFA profiles continued to show differences in the structure of the zinc treated and control microbial communities. Effects of zinc on PLFA profiles at day 420 included a relative decrease in an indicator fatty acid for arbuscular mycorrhizal fungi and a relative increase in an indicator fatty acid for fungi. The comparison of PLFA profiles also showed mixed results for several fatty acid indicators for actinomycetes. The pH control treatment showed similar effects on population size and activity as the zinc treatment, although in most cases there was a much stronger effect with the zinc treatment. In addition, the pH treatment caused changes in BIOLOG and PLFA profiles which were different than the changes seen with zinc treatment.
A simple method for assessing the effects of a chemical on the degradation of -labeled grass tissue was developed for future ecotoxicology studies. Grass was grown for several weeks in a simple soil microcosm amended with -glucose to generate a atmosphere. The resulting grass tissue (4 μCi g−1) was placed in two soils (Cohoctah loam from a grassy field and Pipestone sand from a pine woods) within specialized biometer flasks. Production of was 47.1% (s = 2.3%) after 4 weeks, and was similar for both soils sampled in April and July. Production of in Cohoctah soil amended with 3,5-dichlorophenol showed a hormesis response curve with slight stimulation at 80 mg kg−1, followed by inhibition at 400 mg kg−1. Finally, when -grass tissue stored frozen for a year was added to soil stored refrigerated for 10 months, production was nearly identical to experiments with fresh materials. Our technique combines the sensitivity and accuracy of 14C methods with simplicity, low cost, and a minimum of 14C waste to allow evaluation of a chemical on the soil's litter degradation system.
Potential toxic metal (Zn, Cd, Pb, Cu) contamination of groundwater at the periodically flooded DePue Dredged Sediment Disposal Area (DSDA), was assessed through the collection of groundwater and soil samples and water level data, geochemical modeling, and leaching and microcosm experiments. The median Zn concentrations in groundwater at three locations were between 2 and 5 mg/L. There was no evidence that contaminated groundwater has migrated offsite. Based on lake-sediment and soil data, the order of decreasing mobility in the site groundwater was Zn > Cu > Cd > Pb. Small amounts of Zn and Cd (< 2%) were leached from soil samples inside the DSDA in leaching experiments. About half of the dissolved Zn and Cd from groundwater samples can be accounted for by leaching, suggesting desorption was a major control for Zn and Cd. Chemical equilibrium calculations indicated that several reactions, including CdCO3 precipitation, coprecipitation of Zn with CaCO3, and sorption to ferric oxide, may retain the metals in the subsurface. Aqueous Zn concentrations were greatest in aerobic microcosms, lowest in sulfate-reducing microcosms, and intermediate in iron-reducing microcosms. Zinc would thus be expected to be most mobile in DePue soils during the early stages of flooding, when the soils are saturated but before conditions become anaerobic. Illinois Waste Management and Research Center published or submitted for publication is peer reviewed
Nitrogen and heavy metals derived from sewage sludge may potentially affect N 2 fixation and soybean [ Glycine max (L.) Merr.] growth. Field studies were conducted at two locations in 1983 and 1984 to examine the residual effects of sewage sludge on N 2 fixation in soybean. The Fairland plots (Typic Hapludults) were amended with two rates of anaerobically digested sludge in 1975. The Beltsville plots (Typic Paleudults) were established in 1976 with limed‐anaerobically digested, limed‐raw, limed‐compost, or heat‐treated sludge, and in 1978 with Nu‐Earth (Chicago, IL) anaerobically digested sludge. Nonnodulating and nodulating isolines of ‘Clark’ soybean were grown on the sludge‐treated plots, which consisted of various sludge rates and pH regimes. Dry matter and total N accumulation were determined in the shoots at the R4 stage of maturity. Dry matter and N accumulation by nonnodulating soybean generally increased due to greater availability of soil N on the sludge plots. In nodulating soybean, dry matter production and N 2 fixation were enhanced by most sludges with the greatest increase occurring under moisture stress. In 1983, a dry year, dry matter of nodulating soybean and N 2 fixation (estimated by the difference method) were increased by all sludges at Beltsville. During this year, dry matter and amount of N fixed increased from 16.5 g plant ⁻¹ and 285 mg N plant ⁻¹ (control) to 31.1 g plant ⁻¹ and 706 mg N plant ⁻¹ at the 672 Mg ha ⁻¹ limed‐compost sludge rate, respectively. In 1984, a normal rainfall year, dry matter and N 2 fixation was moderately higher on sludge plots compared to control levels. Dry matter and N 2 fixation showed a quadratic response due to overliming by high applications of limed‐digested and limed‐raw sludges. Evidence for toxicity of sludge metals on N 2 fixation was not found at Beltsville sludge plots. However, a toxic effect may be suggested by high metal sludge at Fairland sludge plots. In addition to a decrease in the amount of N 2 fixed, nonnodulating plant dry matter increased with sludge, whereas nodulating plant dry matter decreased. Results suggest that while sludge amended soil may enhance soybean growth, such beneficial results may be limited to sludges low in heavy metals.
Evaluating the biodiversity of microbial communities remains an elusive task because of taxonomic and methodological difficulties. An alternative approach is to examine components of biodiversity for which there exists a reasonable chance of detecting patterns that are biologically meaningful. One such alternative is functional diversity. We propose a procedure based on the Biolog identification system to quickly, effectively, and inexpensively assess aspects of the functional diversity of microbial communities. The numbers and types of substrates utilized by bacterial communities, as well as the levels of activities on various substrates and patterns of temporal development, constitute an information-rich data set from which to assess functional diversity. Data from six plant communities (black grama grassland. Sporobolus grassland, creosotebush bajada, herbaceous bajada, mesquite-playa fringe, and playa grassland) located along an elevational and moisture gradient at the Jornada Long-Term Ecological Research site in the northern Chihuahuan Desert, are analyzed to illustrate the procedure and its relevance to biodiversity. Our analyses demonstrate that the Biolog system can detect considerable variation in the ability of microbial communities to metabolize different carbon compounds. Variation in substrate use was compartmentalized differently along the moisture gradient. Differences in functional diversity were dependent upon the class of carbon sources (guild-specific results). A multifaceted approach to biodiversity that comprises both functional and taxonomic perspectives represents fertile ground for future research endeavors.
Profiles of potential utilization of 95 separate C sources by microbial communities can be readily generated from direct incubation of environmental samples in BIOLOG microplates. Color formation from a redox indicator dye is used to quantify the degree of C source utilization. I aimed to examine different analytical approaches for classifying microbial communities based on these profiles. Specifically, the relative effects of average rate of color development versus the pattern of relative C source utilization on the classification of rhizosphere samples from different crop types were evaluated. The average rate of color development was correlated to the density of total (acridine-orange) bacterial cells (R2 = 0.52) and active (5-cyano-2,3-ditolyl tetrazolium chloride) bacterial cells (R2 = 0.70) inoculated into the plate. Analysis of plates with different rates of color development after a specific incubation period resulted in samples with variation in the overall extent of color development (expressed as average well color development, or AWCD). Classification of these samples using principal component analysis was significantly influenced by the variation in AWCD, resulting in the classification of samples based on the density of inoculum rather than the pattern of C source utilization. The effect of variation in AWCD was eliminated by normalizing data prior to ordination, or by using an alternative ordination technique, detrended correspondence analysis. Variation in AWCD can be limited through multiple-plate readings and subsequent selection of plates with a common reference point in AWCD. The specific AWCD used for analysis does not appear important for classification purposes; consistent discrimination of rhizosphere samples from different crop types was apparent for analysis across a wide range of AWCD (0.25–1.00 abs. units). The specific differences in C source utilization between rhizosphere sample types did depend on the set point used for analysis due to the differences in the rate of color formation among wells. Results suggest that single-plate readings can be used to classify samples, but only if potential differences in AWCD are accounted for in the data analysis. Repeated plate readings will provide a more complete understanding of differences in C source utilization among samples.
Four acid soils from Hawaii and three calcareous soils from Pakistan were used to study the effect of suspension pH on Zn solubility. The soils, at different pH values, were equilibrated with added Zn for 6 days at 25[degrees]C. After equilibration, Zn concentrations in the supernatants were determined by atomic absorption spectrophotometry. The effect of suspension pH was quantitatively evaluated by plotting the negative log Zn in solution (pZn) against pH. A linear relationship was observed up to pH 7.0 for acid soils. As the system passed neutrality, a general increase was noted in the amounts of Zn in solutions for the soils high in organic matter. The increase in Zn solubility over the alkaline range was assumed to be due to dispersion of organic matter, which either released complexed Zn or provided chelating agents for added Zn and reduced adsorption or precipitation. For the calcareous soils, the relationship between Zn solubility and pH was nonlinear. It was assumed that at higher pH, Zn in solutions precipitated as Zn(OH)2, ZnCO3, or Ca-zincate. There was no indication of colloidal peptization or formation of soluble zincate ions with NaOH.
Amounts of microbial biomass were measured in soils from two different U.K. field experiments, one on a sandy loam (15% clay) at Luddington (Wick series) and the other on a silty loam soil (21% clay) at Lee Valley (Hamble series), where sewage sludges, mainly enriched with single metals, were applied 22 yr ago. No single metal (Zn, Cu, Ni and Cd) at or below current EC permitted total soil metal concentrations, or limits, decreased the amounts of soil microbial biomass. However, Cu at about two and a half times permitted metal limits decreased the amounts of biomass by about 40% at both sites and caused an increased accumulation of organic C and total N of about 30% in the sandy loam and about 13% in the silty loam soil. Zinc, at about the same concentration, decreased the biomass by about 40% in the sundy loam and 30% in the silty loam soil while soil organic matter accumulation increased by only 9–14%. Cadmium, at about twice current EC limits did not affect the amount of biomass or soil organic matter in the silty loam soil. Similarly, neither were affected by Ni at 2–3 times current metal limits. The amount of microbial biomass C as a percentage of total soil organic C was much lower (< 1.0%) in soils contaminated with Zn and Cu at about two-and-a-half times current permitted limits than in soils containing less metal. This also suggested that the metals were causing decreased microbial biomass at these metal concentrations.