Molecular techniques in phytoplankton research: from allozyme electrophoresis to genomics

Molecular techniques have become a valuable tool in phytoplankton studies over the past decades. The appropriate choice of a technique from an increasing array of methods can be rather complex, because different techniques are suitable for different questions or problems in ecology and evolution. Each technique has its particular strengths and weaknesses and is based upon different (theoretical) assumptions. Our aim is to give a better insight in the (correct) use of various molecular techniques in phytoplankton research, with special emphasis on the fields of strain identification, differentiation of populations and the establishment of phylogenetic relationships. The basic steps in the development of molecular techniques like allozyme electrophoresis, RFLP, DGGE, SSCP, RAPD, AFLP and microsatellites, and the application of these techniques in phytoplankton research, are discussed. Furthermore, recent developments in molecular biology, that have so far only found limited application in phytoplankton studies, such as single-cell PCR, PCR assays combined with molecular probes (Heteroduplex Mobility Assays or DNA arrays), Real-time PCR, complete genome sequencing, multi-gene expression studies using microarrays, and Single Nucleotide Polymorphism (SNPs), are discussed. We emphasise the relevance of fundamental and applied molecular studies on phytoplankton for a wider community of ecologists and evolutionary biologists.     

Diversity and Detection of Nitrate Assimilation Genes in Marine Bacteria

A PCR approach was used to construct a database of nasA genes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.     

Development of soil microbiology methods: from respirometry to molecular approaches

This review deals with techniques and methods used in the study of the function and development of microorganisms occurring in soil with emphasis on the contributions of Czech Academician Ivan Málek and his coworkers or fellows (Jiří Macura, František Kunc) to the development of basic techniques used in soil microbiology. Early studies, including batch cultivation and respirometric techniques, as well as later developments of percolation and continuous-flow methods of cultivation of soil microorganisms are discussed. Recent developments in the application of analytical chemistry (HPLC or GC) and of molecular biological techniques to ecological questions that have revolutionized concepts in soil microbiology and microbial ecology are also briefly mentioned, including denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), phospholipid fatty acid analysis (PLFA) and others. The shift of soil microbiology from the study of individual microorganisms to entire microbial communities, including nonculturable species, is briefly discussed.     

rRNA技术及其在分子微生物生态上的应用

传统的基于微生物培养与纯种分离的技术所具有的局限性,以及分子生物学及其有关技术的长足进展,使微生物生态学的研究进入了分子的阶段.其中rRNA技术的建立、发展及其成功应用,为分子微生物生态和微生物系统分类学的研究掀开了崭新的一页.对rRNA分子技术的研究进展、以之为基础的主要方法及其在环境微生物研究中的应用,以及应用过程中所存在的一些潜在问题及其解决办法等作了详细综述.     

Expanding the microbial monitoring toolkit: Evaluation of traditional and molecular monitoring methods

Evaluation of microbial populations in oilfield systems is critical to understand the risk of microbiologically influenced corrosion, reservoir and surface souring (hydrogen sulfide production), and biofouling. Although traditional culture based methods have dominated oilfield microbial monitoring for years, use of molecular tools is becoming more common for both field and laboratory evaluations. The implementation of these additional tools is in response to some of the disadvantages of culture-based methods such as long incubation times and underestimation of actual microbial populations. The current work provides a direct comparison of culture-based methods (serial dilution for sulfate reducing, acid producing, and general heterotrophic bacteria) with molecular methods including adenosine triphosphate (ATP) and adenosine monophosphate (AMP) quantification and quantitative polymerase chain reaction (qPCR). The results demonstrate that these technologies provide nearly identical results in untreated samples with known culturable organisms, but show some differences when used in the context of a planktonic kill study. The pros and cons of each technology were addressed with respect to their use in field monitoring, laboratory monitoring, and microbial kill studies. The authors found that none of the technologies described in this work provide an all-inclusive answer, but together they provide significant insight into the microbial population in an oilfield system. In short, the authors demonstrate that it is advantageous for oilfield stakeholders to expand their microbial monitoring toolkit with these new technologies to ensure sustainable, cost-effective operation.     

Ontogenetic allometry of the bluemouth, <Emphasis Type="Italic">Helicolenus dactylopterus dactylopterus</Emphasis> (Teleostei: Scorpaenidae), in the Northeast Atlantic and Mediterranean based on geometric morphometrics

Since the emergence of the ‘microbial loop’ concept, heterotrophic flagellates have received particular attention as grazers in aquatic ecosystems. These microbes have historically been regarded incorrectly as a homogeneous group of bacterivorous protists in aquatic systems. More recently, environmental rDNA surveys of small heterotrophic flagellates in the pelagic zone of freshwater ecosystems have provided new insights. (i) The dominant phyla found by molecular studies differed significantly from those known from morphological studies with the light microscope, (ii) the retrieved phylotypes generally belong to well-established eukaryotic clades, but there is a very large diversity within these clades and (iii) a substantial part of the retrieved sequences cannot be assigned to bacterivorous but can be assigned instead to parasitic and saprophytic organisms, such as zoosporic true fungi (chytrids), fungus-like organisms (stramenopiles), or virulent alveolate parasites (Perkinsozoa and Amoebophrya sp.). All these microorganisms are able to produce small zoospores to assure dispersal in water during their life-cycles. Based on the existing literature on true fungi and fungus-like organisms, and on the more recently published eukaryotic rDNA environmental studies and morphological observations, we conclude that previously overlooked microbial diversity and related ecological potentials require intensive investigation (i) for an improved understanding of the roles of heterotrophic flagellates in pelagic ecosystems and (ii) to properly integrate the concept of ‘the microbial loop’ into modern pelagic microbial ecology.     

实时荧光定量PCR及其在微生物生态学中的应用

定量描述微生物群落的组成,在微生物生态学的许多研究领域都是非常重要的。然而由于可培养技术的局限性,定量描述微生物群落成为比较困难的事情。最近包括PCR技术在内的分子生物学技术为人们提供了有力的工具,使对微生物群落的分布、丰度等有了进一步的了解。实时荧光定量PCR技术作为核酸定量检测技术,自从发明以来在微生物生态学研究中逐渐得到了广泛的应用。从微生物生态学角度,综述了实时荧光定量PCR技术的原理、发展、优缺点及其在微生物生态学研究中的应用与研究进展,并探讨了实时荧光定量PCR技术的发展和应用前景。     

Microbial Colonization and Decomposition of Carex Litter in an Arctic Lake

The decomposition and microbial colonization of Carex leaf litter were examined in an arctic lake in Alaska during the summer of 1978. Dried leaf segments in screen bags were placed at various locations and depths for 13 and 26 days. Weight loss varied from 24.15 to 33.56% and from 27.69 to 65.01% after 13 and 26 days, respectively. Abiotic controls lost approximately 19.5% with no subsequent change. Weight loss significantly correlated with microbial colonization as measured by alkaline phosphatase activity (r = 0.780), cellulase activity (r = 0.613), heterotrophic CO₂ fixation (r = 0.835), and acetate incorporation into microbial lipids (r = 0.618). Alkaline phosphatase activity correlated with cellulase activity (r = 0.889), and heterotrophic CO₂ fixation correlated with acetate incorporation into lipids (r = 0.712). Weight loss after 26 days inversely correlated with the logarithm of the depth of incubation regardless of whether incubation occurred on the sediment surface or in the water column. These findings suggest that a rapid initial period of microbial colonization is driven by nutrients derived from the litter and that the rate of these processes is controlled by a factor(s) inversely related to the logarithm of depth, such as light intensity, primary production, or turbulence.     

Measurement of rRNA Variations in Natural Communities of Microorganisms on the Southeastern U.S. Continental Shelf

The development of a clear understanding of the physiology of marine prokaryotes is complicated by the difficulties inherent in resolving the activity of various components of natural microbial communities. Application of appropriate molecular biological techniques offers a means of overcoming some of these problems. In this regard, we have used direct probing of bulk RNA purified from selective size fractions to examine variations in the rRNA content of heterotrophic communities and Synechococcus populations on the southeastern U.S. continental shelf. Heterotrophic communities in natural seawater cultures amended with selected substrates were examined. Synechococcus populations were isolated from the water column by differential filtration. The total cellular rRNA content of the target populations was assayed by probing RNA purified from these samples with an oligonucleotide complementing a universally conserved region in the eubacterial 16S rRNA (heterotrophs) or with a 1.5-kbp fragment encoding the Synechococcus sp. strain WH 7803 16S rRNA (cyanobacteria). The analyses revealed that heterotrophic bacteria responded to the addition of glucose and trace nutrients after a 6-h lag period. However, no response was detected after amino acids were added. The cellular rRNA content increased 48-fold before dropping to a value 20 times that detected before nutrients were added. Variations in the rRNA content from Synechococcus spp. followed a distinct diel pattern imposed by the phasing of cell division within the irradiance cycle. The results indicate that careful application of these appropriate molecular biological techniques can be of great use in discerning basic physiological characteristics of selected natural populations and the mechanisms which regulate growth at the subcellular level.     

Assessment of Changes in Microbial Community Structure during Operation of an Ammonia Biofilter with Molecular Tools

Biofiltration has been used for two decades to remove odors and various volatile organic and inorganic compounds in contaminated off-gas streams. Although biofiltration is widely practiced, there have been few studies of the bacteria responsible for the removal of air contaminants in biofilters. In this study, molecular techniques were used to identify bacteria in a laboratory-scale ammonia biofilter. Both 16S rRNA and ammonia monooxygenase (amoA) genes were used to characterize the heterotrophic and ammonia-oxidizing bacteria collected from the biofilter during a 102-day experiment. The overall diversity of the heterotrophic microbial population appeared to decrease by 38% at the end of the experiment. The community structure of the heterotrophic population also shifted from predominantly members of two subdivisions of the Proteobacteria (the beta and gamma subdivisions) to members of one subdivision (the gamma subdivision). An overall decrease in the diversity of ammonia monooxygenase genes was not observed. However, a shift from groups dominated by organisms containing Nitrosomonas-like and Nitrosospira-like amoA genes to groups dominated by organisms containing only Nitrosospira-like amoA genes was observed. In addition, a new amoA gene was discovered. This new gene is the first freshwater amoA gene that is closely affiliated with Nitrosococcus oceanus and the particulate methane monooxygenase gene from the methane oxidizers belonging to the gamma subdivision of the Proteobacteria.     

Fate of Heterotrophic Microbes in Pelagic Habitats: Focus on Populations

Major biogeochemical processes in the water columns of lakes and oceans are related to the activities of heterotrophic microbes, e.g., the mineralization of organic carbon from photosynthesis and allochthonous influx or its transport to the higher trophic levels. During the last 15 years, cultivation-independent molecular techniques have substantially contributed to our understanding of the diversity of the microbial communities in different aquatic systems. In parallel, the complexity of aquatic habitats at a microscale has inspired research on the ecophysiological properties of uncultured microorganisms that thrive in a continuum of dissolved to particulate organic matter. One possibility to link these two aspects is to adopt a “Gleasonian” perspective, i.e., to study aquatic microbial assemblages in situ at the population level rather than looking at microbial community structure, diversity, or function as a whole. This review compiles current knowledge about the role and fate of different populations of heterotrophic picoplankton in marine and inland waters. Specifically, we focus on a growing suite of techniques that link the analysis of bacterial identity with growth, morphology, and various physiological activities at the level of single cells. An overview is given of the potential and limitations of methodological approaches, and factors that might control the population sizes of different microbes in pelagic habitats are discussed.     

Toolboxes for cyanobacteria: Recent advances and future direction

Photosynthetic cyanobacteria are important primary producers and model organisms for studying photosynthesis and elements cycling on earth. Due to the ability to absorb sunlight and utilize carbon dioxide, cyanobacteria have also been proposed as renewable chassis for carbon-neutral “microbial cell factories”. Recent progresses on cyanobacterial synthetic biology have led to the successful production of more than two dozen of fuels and fine chemicals directly from CO₂, demonstrating their potential for scale-up application in the future. However, compared with popular heterotrophic chassis like Escherichia coli and Saccharomyces cerevisiae, where abundant genetic tools are available for manipulations at levels from single gene, pathway to whole genome, limited genetic tools are accessible to cyanobacteria. Consequently, this significant technical hurdle restricts both the basic biological researches and further development and application of these renewable systems. Though still lagging the heterotrophic chassis, the vital roles of genetic tools in tuning of gene expression, carbon flux re-direction as well as genome-wide manipulations have been increasingly recognized in cyanobacteria. In recent years, significant progresses on developing and introducing new and efficient genetic tools have been made for cyanobacteria, including promoters, riboswitches, ribosome binding site engineering, clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) systems, small RNA regulatory tools and genome-scale modeling strategies. In this review, we critically summarize recent advances on development and applications as well as technical limitations and future directions of the genetic tools in cyanobacteria. In addition, toolboxes feasible for using in large-scale cultivation are also briefly discussed.     

Agreement between amoA Gene-Specific Quantitative PCR and Fluorescence In Situ Hybridization in the Measurement of Ammonia-Oxidizing Bacteria in Activated Sludge

Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase (amoA) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA gene-specific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers.     

Analysis of a microbial community oxidizing inorganic sulfide and mercaptans

Successful treatment of refinery spent-sulfidic caustic (which results from the addition of sodium hydroxide solutions to petroleum refinery waste streams) was achieved in a bioreactor containing an enrichment culture immobilized in organic polymer beads with embedded powdered activated carbon (Bio-Sep). The aerobic enrichment culture had previously been selected using a gas mixture of hydrogen sulfide and methyl mercaptan (MeSH) as the sole carbon and energy sources. The starting cultures for the enrichment consisted of several different Thiobacilli spp. (T. thioparus, T. denitrificans, T. thiooxidans, and T. neopolitanus), as well as activated sludge from a refinery aerobic wastewater treatment system and sludge from an industrial anaerobic digester. Microscopic examination (light and SEM) of the beads and of microbial growth on the walls of the bioreactor revealed a great diversity of microorganisms. Further characterization was undertaken starting with culturable aerobic heterotrophic microorganisms (sequencing of PCR-amplified DNA coding for 16S rRNA, Gram staining) and by PCR amplification of DNA coding for 16S rRNA extracted directly from the cell mass, followed by the separation of the PCR products by DGGE (denaturing gradient gel electrophoresis). Eight prominent bands from the DGGE gel were sequenced and found to be closest to sequences of uncultured Cytophagales (3 bands), Gram-positive cocci (Micrococcineae), alpha proteobacteria (3 bands), and an unidentified beta proteobacterium. Culturable microbes included several genera of fungi as well as various Gram-positive and Gram-negative heterotrophic bacteria not seen in techniques using direct DNA extraction.     

Changes in the pelagic microbial food web due to artificial eutrophication

The effect of nutrient enrichment on the structure and carbon flow in the pelagic microbial food web was studied in mesocosm experiments using seawater from the northern Baltic Sea. The experiments included food webs of at least four trophic levels; (1) phytoplankton–bacteria, (2) flagellates, (3) ciliates and (4) mesozooplankton. In the enriched treatments high autotrophic growth rates were observed, followed by increased heterotrophic production. The largest growth increase was due to heterotrophic bacteria, indicating that the heterotrophic microbial food web was promoted. This was further supported by increased growth of heterotrophic flagellates and ciliates in the high nutrient treatments. The phytoplankton peak in the middle of the experiments was mainly due to an autotrophic nanoflagellate, Pyramimonas sp. At the end of the experiment, the proportion of heterotrophic organisms was higher in the nutrient enriched than in the nutrient-poor treatment, indicating increased predation control of primary producers. The proportion of potentially mixotrophic plankton, prymnesiophyceans, chrysophyceans and dinophyceans, were significantly higher in the nutrient-poor treatment. Furthermore, the results indicated that the food web efficiency, defined as mesozooplankton production per basal production (primary production + bacterial production − sedimentation), decreased with increasing nutrient status, possibly due to increasing loss processes in the food web. This could be explained by promotion of the heterotrophic microbial food web, causing more trophic levels and respiration steps in the food web.     

Plankton dynamics in the marginal ice zone of the central Barents Sea during spring: carbon flow and structure of the grazer food chain

Plankton community structure was analysed during spring at four stations along a transect from the polar ice into open waters of the Barents Sea. The transect mimicks a time span of months in the biological succession during the Arctic summer. The significance of the microbial food web vs the more classical food web was evaluated using carbon budget models. The standing stocks of diatom-dominated phytoplankton and bacteria were generally high especially in connection to ice. The biomass of microzooplankton, dominated by heterotrophic dinoflagellates was significantly high, with specific growth rates following the in situ temperature. The mean ± SE specific growth rate was 0.40±0.12 d^?1 for ciliates and 0.24 ± 0.05 d^?1 for heterotrophic dinoflagellates, indicating no food limitation. The estimated total carbon requirement for microzooplankton was, at the ice-covered station, approximately 100% of the daily primary production, decreasing to 25% in the open water. Carbon-specific secondary production of the copepodsCalanus finmarchicus (Gunnerus),C. glacialis (Jaschnov),C. hyperboreus (Krøyer) andMetridia longa (Lubbock) were analysed by egg production.C. finmarchicus andM. longa were productive at all stations, including the ice-covered locations, with a maximum at 0.08 d^?1 and 0.035 d^?1, respectively. The other, more Arctic-related,Calanus spp. were virtually outspawned. The standing stock of copepods was only 10–20% of the total microbial grazer biomass. The community growth and grazing by copepods showed significantly less quantitative importance for the pelagic carbon flow than the microbial processes.     

The prokaryotic community of subglacial bottom sediments of Antarctic Lake Untersee: Detection by cultural and direct microscopic techniques

The heterotrophic mesophilic microbial component was studied in microbial communities of the samples of frozen regolith collected from the glacier near Lake Untersee collected in 2011 during the joint Russian-American expedition to central Dronning Maud Land (Eastern Antarctica). Cultural techniques revealed high bacterial numbers in the samples. For enumeration of viable cells, the most probable numbers (MPN) method proved more efficient than plating on agar media. Fluorescent in situ hybridization with the relevant oligonucleotide probes revealed members of the groups Eubacteria (Actinobacteria, Firmicutes) and Archaea. The application of the methods of cell resuscitation, such as the use of diluted media and prevention of oxidative stress, did not result in a significant increase in the numbers of viable cells retrieved from subglacial sediment samples. Our previous investigations demonstrated the necessity for special procedures for efficient reactivation of the cells from microbial communities of replace with buried soil and permafrost samples collected in the Arctic zone. The differing responses to the special resuscitation procedures may reflect the differences in the physiological and morphological state of bacterial cells in microbial communities subject to continuous or periodic low temperatures and dehydration.     

Seasonal Dynamics of Prokaryotic Abundance and Activities in Relation to Environmental Parameters in a Transitional Aquatic Ecosystem (Cape Peloro,Italy)

This study examines the effects of temporal changes on microbial parameters in a brackish aquatic ecosystem. To this aim, the abundances of prokaryotes and vibrios together with the rates of enzymatic hydrolysis of proteins by leucine aminopeptidase (LAP), polysaccharides by β-glucosidase (GLU) and organic phosphates by alkaline phosphatase (AP), heterotrophic prokaryotic production (HPP), respiration (R), were seasonally investigated, during a 2-year period in the coastal area of Cape Peloro (Messina, Italy), constituted by two brackish lakes (Faro and Ganzirri). In addition, physical and chemical parameters (temperature, salinity, nutrients) and particulate organic carbon and nitrogen (POC, PN) were measured. The influence of multiple factors on prokaryotic abundances and activities was analysed. The results showed that Cape Peloro area is characterised by high seasonal variability of the microbial parameters that is higher than the spatial one. Combined changes in particulate matter and temperature (T), could explain the variability in vibrios abundance, GLU and R activities in both lakes, indicating a direct stimulation of the warm season on the heterotrophic prokaryotic metabolism. Positive correlations between T (from 13.3 to 29.6 °C) and HPP, LAP, AP, POC, PN are also observed in Ganzirri Lake. Moreover, the trophic status index and most of the microbial parameters show significant seasonal differences. This study demonstrates that vibrios abundance and microbial activities are responsive to the spatial and seasonal changes of examined area. The combined effects of temperature and trophic conditions on the microbial parameters lead us to suggest their use as potential indicators of the prokaryotic response to climate changes in temperate brackish areas.     

Influence of an Oyster Reef on Development of the Microbial Heterotrophic Community of an Estuarine Biofilm

We characterized microbial biofilm communities developed over two very closely located but distinct benthic habitats in the Pensacola Bay estuary using two complementary cultivation-independent molecular techniques. Biofilms were grown for 7 days on glass slides held in racks 10 to 15 cm over an oyster reef and an adjacent muddy sand bottom. Total biomass and optical densities of dried biofilms showed dramatic differences for oyster reef versus non-oyster reef biofilms. This study assessed whether the observed spatial variation was reflected in the heterotrophic prokaryotic species composition. Genomic biofilm DNA from both locations was isolated and served as a template to amplify 16S rRNA genes with universal eubacterial primers. Fluorescently labeled PCR products were analyzed by terminal restriction fragment length polymorphism, creating a genetic fingerprint of the composition of the microbial communities. Unlabeled PCR products were cloned in order to construct a clone library of 16S rRNA genes. Amplified ribosomal DNA restriction analysis was used to screen and define ribotypes. Partial sequences from unique ribotypes were compared with existing database entries to identify species and to construct phylogenetic trees representative of community structures. A pronounced difference in species richness and evenness was observed at the two sites. The biofilm community structure from the oyster reef setting had greater evenness and species richness than the one from the muddy sand bottom. The vast majority of the bacteria in the oyster reef biofilm were related to members of the γ- and δ-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium -Bacteroides cluster, and the phyla Planctomyces and Holophaga-Acidobacterium. The same groups were also present in the biofilm harvested at the muddy sand bottom, with the difference that nearly half of the community consisted of representatives of the Planctomyces phylum. Total species richness was estimated to be 417 for the oyster reef and 60 for the muddy sand bottom, with 10.5% of the total unique species identified being shared between habitats. The results suggest dramatic differences in habitat-specific microbial diversity that have implications for overall microbial diversity within estuaries.