http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_10 Environmental contamination has been viewed as an ecological malaise for which bioremediation can be prescribed as a “perfect medicine.” The solution to the problems with bioremediation lies in analyzing to what extent the microbes’ physiological machinery contributes to the degradation process and which biomolecules and their mechanisms are responsible for regulatory factors within the degradation system, such as protein, metabolite, and enzymatic chemical transformation. In the post-genomic era, recent advances in proteomics have allowed us to elucidate many complex biological mechanisms. Two-dimensional gel electrophoresis (2DE) in conjunction with mass spectrometry (MS) can be utilized to identify the biomolecules and their molecular mechanisms in bioremediation. A set of highly abundant global proteins over a pI range 4–7 was separated and compared by size fractionation (25–100 kDa) on 2DE. We identified a set of catabolic proteins, enzymes, and heat shock molecular chaperones associated with the regulatory network that was found to be overexpressed under phenol-stressed conditions. This chapter also offers optimized ideal directions for 2DE, followed by easy-to-follow directions for a protein identification strategy using MALDI-TOF and targeting novel proteins/enzymes for a universal set of experiments. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_11 Wastewater treatment systems tend to be engineered to select for a few functional microbial groups that may be organized in various spatial structures such as activated sludge flocs, biofilm or granules and represented by single coherent phylogenic groups such as ammonia-oxidizing bacteria (AOB) and polyphosphate-accumulating organisms (PAO). In order to monitor and control engineered microbial structure in wastewater treatment systems, it is necessary to understand the relationships between the microbial community structure and the process performance. This review focuses on bacterial communities in wastewater treatment processes, the quantity of microorganisms and structure of microbial consortia in wastewater treatment bioreactors. The review shows that the application of molecular techniques in studies of engineered environmental systems has increased our insight into the vast diversity and interaction of microorganisms present in wastewater treatment systems. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_12 Xenobiotic degradation during biological wastewater treatment can be established or enhanced by bioaugmentation − the addition of biological agents carrying biodegradation genes required to perform the task. Whereas the addition of microbial cells carrying chromosomally encoded catabolic genes can be impaired by limited survival of the added microorganisms, the addition of donor organisms carrying a transmissible catabolic plasmid is a promising alternative. This plasmid can spread within the indigenous microbial community of the system, circumventing the need for extended survival of the introduced bacterial strain. Here we discuss how the catabolic plasmid pNB2 can be evaluated towards its potential to facilitate the degradation of a xenobiotic compound, 3-chloroaniline, and demonstrate the applicability of this plasmid to accomplish 3-chloroaniline degradation in a bioreactor setting after in situ transfer to suitable recipient strains. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_13 Despite the widespread availability of state-of-the-art biological techniques, remediation practitioners have been slow to adopt these technologies to assist in designing or indeed monitoring remediation strategies. In part, this is because practitioners are driven by cost and fail to see the benefit of emerging technologies, and in part because most companies have only a small portfolio of procedures available to them. Here, we review the component parts required to design a decision support tool, appraise one that the authors have developed and critically evaluate its application to case studies. If bioremediation is to become adopted, then it is likely to have to operate in parallel with other remediation methods. Furthermore, remediation strategies must couple effective technology with a transparency of information such that all parties (practitioners, developers and stakeholders) understand how decisions were reached. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_14 Monitored natural attenuation (MNA) is an in situ remediation technology that relies on naturally occurring and demonstrable processes in soil and groundwater which reduce the mass and concentration of the contaminants. Natural attenuation (NA) involves both aerobic and anaerobic degradation of the contaminants due to the fact that oxygen is used up near the core of the contaminant plume. The aerobic and anaerobic microbial processes can be assessed by microbial activity measurements and molecular biology methods in combination with chemical analyses. The sampling and knowledge on the site conditions are of major importance for the linkage of the results obtained to the conditions in situ. Rates obtained from activity measurements can, with certain limitations, be used in modeling of the fate of contaminants whereas most molecular methods mainly give qualitative information on the microbial community and gene abundances. However, molecular biology methods are fast and describe the in situ communities and avoid the biases inherent to activity assays requiring laboratory incubations. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_15 The use of bioassays for soil characterization is receiving significant attention as a complementary tool to chemical analysis. Bioassays consist of direct toxicity assays of environmental samples that are transferred to the laboratory and analyzed for toxicity against selected organisms. Such soil samples contain the combination of the different pollutants present in situ and enable factors such as the bioavailability of contaminants or the interactions (synergic and antagonic) between them to be simultaneously studied. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_16 Community-level physiological profiling (CLPP) is a technique which offers an easily applied protocol yielding information regarding mixed microbial community function and functional adaptations over space and time. Different communities can be compared and classified based on sole carbon source utilization patterns (CSUPs) gathered using BIOLOG™ microplates. One of the most challenging aspects associated with the CLPP method is in the data analysis. This chapter describes the relatively simple CLPP laboratory protocol and provides a detailed description of different data analysis techniques. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_1 The quantification of organic contaminant bioaccessibility in soils and sediments is essential for the risk assessment and remediation of contaminated land. Within this framework, practitioners require standardised protocols. Cyclodextrins are a group of macrocyclic compounds that can form inclusion complexes with organic xenobiotics. This occurrence can be exploited to measure the labile/rapidly desorbable compound fraction, which correlates with microbial degradation. We present a rapid and easily reproducible HPCD shake extraction technique that has been experimentally demonstrated to directly predict microbial availability and degradation in soil. This method can provide practitioners with both an indication of bioremediation end-points and may be valuable in the risk assessment of contaminated land. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_2 This chapter considers the use of a variety of approaches to assess either the bioavailability or the bioaccessibility of metals in soil. The bioavailability of metals from soils is considered with respect to a series of single-extraction methods, including the use of ethylenediaminetetraacetic acid (EDTA), acetic acid, diethylenetriaminepentaacetic acid (DTPA), ammonium nitrate, calcium chloride and sodium nitrate. Then, a procedure for the recovery of metals using a three-stage sequential extraction protocol is described. Two alternate approaches for assessing the environmental health risk to humans by undertaking in vitro gastrointestinal extraction (also known as the physiologically based extraction test, PBET) are considered. Finally, two acid digestion protocols that allow the pseudo-total metal content of samples to be assessed are provided. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_3 Each step of a molecular environmental microbiology study is prone to errors, though the qualitative and quantitative biases of PCR amplification could result in the most serious biases. One has to be aware of this fact, and well-characterized PCR biases have to be avoided by using target-optimized PCR protocols. The most important tasks are primer and thermal profile optimization. We have shown that primer mismatches, even in the case of universal primers, can cause almost complete missing of common taxa from clone libraries, for example. Similarly high annealing temperatures can drastically distort community composition of the sample in the PCR product. Strategies of primer selection and PCR thermal profile design are discussed in detail. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_4 Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic–biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_5 Bioremediation has been identified as a beneficial and effective strategy for the removal of recalcitrant environmental contaminants. Bioaugmentation of polluted environments with exogenous degrading microorganisms constitutes a major strategy of bioremediation. However, the ecological role of these strains and their impact on the endogenous microbial community of the micro-ecosystems where they are released should be known. Fingerprinting PCR-based methods, like denaturating gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP), could be used in studies exploring the ecology of pollutant-degrading microorganisms and their effects on the structure of the soil microbial community. This chapter provides a brief outline of the technical details involved in the application of DGGE and TRFLP fingerprinting in soil microbial ecology, with particular reference to bioremediation studies. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_6 Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in composting systems. This analysis is based on the restriction endonuclease digestion of fluorescently end-labeled PCR products. The digested product is mixed with a DNA size standard, itself labeled with a distinct fluorescent dye, and the fragments are then separated by capillary or gel electrophoresis using an automated sequencer. Upon analysis, only the terminal end-labeled restriction fragments are detected. An electropherogram is produced, which shows a profile of compost microbial community as a series of peaks of varying height. This technique has also been effectively used in the exploration of complex microbial environments and in the study of bacterial, archaeal, and eukaryal populations in natural habitats. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_7 Fluorescence in situ hybridization (FISH) can be combined with a number of staining techniques to reveal the relationships between the microorganisms and their function in complex microbial systems with a single-cell resolution. In this chapter, we have focused on staining methods for intracellular storage compounds (polyhydroxyalkanoates, polyphosphate) and a measure for cell viability, reduction of the tetrazolium-based redox stain CTC. These protocols are optimized for the study of microorganisms in waste-water treatment (activated sludge and biofilms), but they may also be used with minor modifications in many other ecosystems. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_8 Ecophysiological analysis and functions of single cells in complex microbial systems can be examined by simple combinations of Fluorescence in situ hybridization (FISH) for identification with various staining techniques targeting functional phenotypes. In this chapter, we describe methods and protocols optimized for the study of extracellular enzymes, surface hydrophobicity and specific surface structures. Although primarily applied to the study of microbes in wastewater treatment (activated sludge and biofilms), the methods may also be used with minor modifications in several other ecosystems. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-439-5_9 DNA-stable isotope probing, a method to identify active microorganisms without the prerequisite of cultivation, has been widely applied in the study of microorganisms involved in the degradation of environmental pollutants. Recent advances and technique considerations in applying DNA-SIP in bioremediation are highlighted. A detailed protocol of a DNA-SIP experiment is provided. 2009-10-23T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-214-8_10 A variety of chemicals are produced upon pretreatment of lignocellulosic biomass. Aliphatic acids, aromatic acids, aldehydes, and phenolic compounds are of particular interest due to their presumed inhibitory influence on downstream enzymatic or microbial steps in biomass-to-ethanol conversion. Herein, we describe a series of analytical protocols that collectively enable quantitative monitoring of 40 potential fermentation inhibitors in biomass pretreatment samples. Solid samples are accommodated by first employing pressurized fluid extraction to generate an aqueous “wash stream.” Sample preparation for liquids involves an initial precipitation-filtration step, followed by liquid–liquid extraction and reconstitution of extracts in water. Samples are analyzed using high–performance liquid chromatography (HPLC) in combination with ultraviolet (UV) absorbance and tandem mass spectrometry (MS/MS) detection. A standard addition approach is utilized for quantitation to alleviate complications arising from co-extracted sample matrix. 2009-10-05T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-214-8_11 In its broadest definition, biomass can be described as all material that was or is a part of a living organism. For renewable energy applications, however, the definition of biomass is usually limited to include only materials that are plant-derived such as agricultural residues (e.g., wheat straw, corn stover) by-products of industrial processes (e.g., sawdust, sugar cane bagasse, pulp residues, distillers grains), or dedicated energy crops (e.g., switchgrass, sorghum, Miscanthus, short-rotation woody crops). This chapter describes analytical methods developed to measure plant components with an emphasis on the measurement of components that are important for biomass conversion. The methods described here can be viewed as a portfolio of analytical methods, with consistent assumptions and compatible sample preparation steps, selected for simplicity, robust application, and the ability to obtain a summative mass closure on most samples that accurately identifies greater than 95% of the mass of a plant biomass sample. The portfolio of methods has been successfully applied to a wide variety of biomass feedstock as well as liquid and solid fractions of both thermochemical pretreatment and enzymatic saccharification (1). 2009-10-05T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-214-8_12 We describe a high-throughput method for estimating cell-wall chemistry traits using analytical pyrolysis. The instrument used to perform the high-throughput cell-wall chemistry analysis consists of a commercially available pyrolysis unit and autosampler coupled to a custom-built molecular beam mass spectrometer. The system is capable of analyzing approximately 42 biomass samples per hour. Lignin content and syringyl to guaiacol (S/G) ratios can be estimated directly from the spectra and differences in cell wall chemistry in large groups of samples can easily be identified using multivariate statistical data analysis methods. The utility of the system is demonstrated on a set of 800 greenhouse-grown poplar trees grown under two contrasting nitrogen treatments. High-throughput analytical pyrolysis was able to determine that the lignin content varied between 13 and 28% and the S/G ratio ranged from 0.5 to 1.5. There was more cell-wall chemistry variation in the plants grown under high nitrogen conditions than trees grown under nitrogen-deficiency conditions. Analytical pyrolysis allows the user to rapidly screen large numbers of samples at low cost, using very little sample material while producing reliable and reproducible results. 2009-10-05T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-214-8_13 Lignin, comprised primarily of three randomly polymerized phenylpropenyl monomers, is, arguably, the second most common organic molecule on earth. In current biorefinery applications, lignin is burned, usually in concentrated pulping or hydrolysis liquor, as a source of process steam and both internal and exported electricity. The aromatic content of lignin makes it a potentially attractive feedstock for high-value aromatic chemicals, polymers, and carbon products (graphite, activated carbon, and carbon fiber). Revenue from production of lignin-based chemicals could play a major role in biorefinery profitability if cost-effective methods for lignin separation and purification can be developed. This chapter presents descriptions of methods for assessing and purifying biorefinery lignins so that they can be evaluated for use as feedstock for production of chemical products. Areas covered are: (1) initial evaluations of as-received lignin samples (visual, microscopic, separable organics); (2) analysis of common contaminants (bulk and filterable ash and particulate contaminants in liquid and dry lignin samples); (3) preparation of lignins for experimental use as chemical feedstock (prefiltration, filtration using bench-scale chemical apparatus and larger scale bag filters, one-step lignin precipitation, two-step carbohydrate and lignin precipitation, desalting of dry powdered or precipitated lignin, and lyophilization). These methods have been used successfully at the bench scale to produce the 1–50 kg amounts of wood and grass lignins typically required for bench-scale assessment as chemical feedstocks. 2009-10-05T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-214-8_14 Cellulose is a heterogeneous polysaccharide, and its enzymatic hydrolysis requires endoglucanase, exoglucanase (cellobiohydrolase), and β-glucosidase to work together. We summarize the most commonly used assays for individual enzymes and cellulase mixture. 2009-10-05T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-214-8_15 Cellulase enzyme is a key cost component in the production of fuels and chemicals from lignocellulosic biomass. Cellulolytic ability of the enzyme preparation is often measured by activity assays using model substrates such as filter paper. Using lignocellulosic biomass as the substrate to assess enzyme performance has the potential of being more process relevant. We describe two procedures that use washed pretreated cellulosic material to measure the efficacy of cellulase enzymes. First, a saccharification assay that measures glucose yield as a function of the amount of cellulase used in the process. And second, the simultaneous saccharification and fermentation (SSF) assay measures cellulase performance by the amount of ethanol produced from enzymatic hydrolysis of the cellulosic material. You can use both assays to screen cellulases under a variety of substrate types, loadings, and process conditions. 2009-10-05T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-214-8_16 Oxygen is either limiting or absent in many ecosystems. Anaerobic bacteria are often key players in such environments and these organisms have important roles in geo-elemental cycling, agriculture, and medicine. The metabolic versatility of anaerobes is exploited in a variety of industrial processes including fermented food production, biochemical synthesis, and bioremediation. There has been recent considerable interest in developing and enhancing technologies that employ anaerobes as biocatalysts. The study of anaerobic bacteria requires specialized techniques, and specific methods are described for the culture and manipulation of these microbes. 2009-10-05T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-214-8_17 Ethanol production by fermentation of lignocellulosic biomass-derived sugars involves a fairly ancient art and an ever-evolving science. Production of ethanol from lignocellulosic biomass is not avant-garde, and wood ethanol plants have been in existence since at least 1915. Most current ethanol production relies on starch- and sugar-based crops as the substrate; however, limitations of these materials and competing value for human and animal feeds is renewing interest in lignocellulose conversion. Herein, we describe methods for both simultaneous saccharification and fermentation (SSF) and a similar but separate process for partial saccharification and cofermentation (PSCF) of lignocellulosic biomass for ethanol production using yeasts or pentose-fermenting engineered bacteria. These methods are applicable for small-scale preliminary evaluations of ethanol production from a variety of biomass sources. 2009-10-05T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-214-8_18 Biodiesel is produced by reacting vegetable oils or animal fats with alcohol in the presence of an alkaline catalyst. The resulting methyl esters, which are the biodiesel fuel, are separated from the by-product glycerin, and then washed with water and dehydrated to produce fuel that must meet standardized specifications. Degraded oils containing high levels of free fatty acids can also be converted to biodiesel, but pretreatment with acid-catalyzed esterification is required. The resulting fuel is suitable for use as a neat fuel in diesel engines or blended with conventional diesel fuel. 2009-10-05T04:00:00Z