Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient “flue gas to algae” system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.
19 Related JoVE Articles!
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production
Institutions: Leibniz Institute for Agricultural Engineering.
Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 °C) and thermophilic (55 °C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 °C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 LCH4
(VS: volatile solids) and 121 L CH4
at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kgdry_biochar
was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.
Environmental Sciences, Issue 88, Biomethane, Hydrothermal Carbonization (HTC), Calorific Value, Lignocellulosic Biomass, UASS, Anaerobic Digestion
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2
, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3
. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5
. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6
. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture
Institutions: Western Washington University, Washington State University Northwestern Research and Extension Center, Texas Tech University.
Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ
degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g.
fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.
Microbiology, Issue 75, Plant Biology, Environmental Sciences, Agricultural Sciences, Soil Science, Molecular Biology, Cellular Biology, Genetics, Mycology, Fungi, Bacteria, Microorganisms, Biodegradable plastic, biodegradable mulch, compostable plastic, compostable mulch, plastic degradation, composting, breakdown, soil, 18S ribosomal DNA, isolation, culture
Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry
Institutions: Max Planck Institute for Molecular Plant Physiology, Royal Holloway, University of London, VIB, UGhent, ETH Zurich, EMPA, Royal Institute of Technology (KTH), European Research and Project Office GmbH, ABBA Gaia S.L., Pflanzenöltechnologie, Capax Environmental Services, Green Fuels, Neutral Consulting Ltd, University of Melbourne.
The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca
) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum
. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.
Environmental Sciences, Issue 87, botany, plants, Biorefining, Poplar, Tobacco tree, Arabidopsis, suberin, lignin, cell walls, biomass, long-chain hydrocarbons, isoprenoids, Nicotiana glauca, systems biology
Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes
Institutions: North Carolina State University, North Carolina State University.
The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.
Environmental Sciences, Issue 82, biodegradation (bacterial degradation), pulp mill waste, black liquor, kraft process, lignocellulose extraction, microorganisms, fermentation products, GC-MS
Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana
Institutions: Institute for Environmental Protection and Research, Regional Agency for Environmental Protection in Emilia-Romagna.
Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia
to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia
) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia
). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2
. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3
. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4
. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.
Chemistry, Issue 62, Artemia franciscana, bioassays, chemical substances, crustaceans, marine environment
Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization
Institutions: UFZ - Helmholtz-Centre for Environmental Research.
The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax
) and the coulombic efficiency (CE
) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e
. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e
. turnover conditions, and in the absence of the substrate, i.e.
nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep
), peak current density (jp
), formal potential (Ef
) and peak separation (ΔEp
). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.
Environmental Sciences, Issue 82, Electrochemistry, Microbial fuel cell, microbial bioelectrochemical system, cyclic voltammetry, electroactive bacteria, microbial bioelectrochemistry, bioelectrocatalysis
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence
Institutions: University of Alberta, University of Calgary.
Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides
, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.
Chemistry, Issue 87, engineering (general), microbiology, bioengineering (general), Eukaryota Algae, Nile Red, Fluorescence, Oil Content, Oil Extraction, Oil Quantification, Neutral Lipids, Optical Microscope, biomass
Analysis of Fatty Acid Content and Composition in Microalgae
Institutions: Wageningen University and Research Center, Wageningen University and Research Center, Wageningen University and Research Center.
A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.
Environmental Sciences, Issue 80, chemical analysis techniques, Microalgae, fatty acid, triacylglycerol, lipid, gas chromatography, cell disruption
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
Institutions: Delft University of Technology, Delft University of Technology.
This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli
and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9
addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli
to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2
) of the alkane hydroxylase system from Gordonia
were transformed into E. coli
. For the conversion of long-chain alkanes (C15-C36), theladA
gene from Geobacillus thermodenitrificans
was implemented. For the required further steps of the degradation process, ADH
and ALDH (
originating from G. thermodenitrificans
) were introduced10,11
. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli
K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii
OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g.
under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n
-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli
to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.
Bioengineering, Issue 68, Microbiology, Biochemistry, Chemistry, Chemical Engineering, Oil remediation, alkane metabolism, alkane hydroxylase system, resting cell assay, prefoldin, Escherichia coli, synthetic biology, homologous interaction mapping, mathematical model, BioBrick, iGEM
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology
Institutions: San Diego State University, University of California San Diego.
Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.
Environmental Sciences, Issue 93, dissolved organic carbon, particulate organic matter, nutrients, DAPI, SYBR, microbial metagenomics, viral metagenomics, marine environment
Seawater Sampling and Collection
Institutions: University of British Columbia - UBC.
This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first. Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.
Molecular Biology, Issue 28, microbial biomass, nucleic acids, nutrients, trace gas, ammonia, sulfide, seawater, fjord, hypoxic, Saanich Inlet
Assay for Adhesion and Agar Invasion in S. cerevisiae
Institutions: Princeton University.
Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.
Microbiology, Issue 1, Yeast, Adhesion, Invasion
Small Volume (1-3L) Filtration of Coastal Seawater Samples
Institutions: University of British Columbia - UBC.
The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 μm filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.
Molecular Biology, Issue 28, microbiology, seawater, filtration, biomass concentration
Investigating the Microbial Community in the Termite Hindgut - Interview
Institutions: California Institute of Technology - Caltech.
Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.
Microbiology, issue 4, microbial community, diversity