Instruments and MethodsFixation filter, device for the rapid in situ preservation of particulate samples
Introduction
Because the majority of microorganisms in the aquatic environment are not yet cultured, many microbiologists and microbial ecologists have adopted culture-independent methods for investigating microorganism diversity and function. One powerful method, metatranscriptomics, involves the procurement and sequencing of messenger RNA (mRNA) from the environment for understanding the linkage between diversity and activity (Frias-Lopez et al., 2008, Stewart et al., 2011), for examining microbial metabolic responses to changing environmental conditions (Pachiadaki et al., 2014) and estimation of rates of select microbial processes (e.g., Helbling et al., 2012). At a minimum, the approach provides an overview of highly expressed genes in a sample, and provides information on the metabolic pathways utilized by microbiota and specific gene products expressed at the time of sample preservation. The method has been successfully used in studies of gene expression in varied marine and fresh water habitats. Marine examples include deep subsurface sediments (Orsi et al., 2013), the North Pacific Subtropical gyre (Frias-Lopez et al., 2008, Poretsky et al., 2009, Shi et al., 2010), eastern tropical South Pacific oxygen minimum zone (Ulloa et al. 2012), coastal waters (Hollibaugh et al., 2011, Gifford et al., 2011), hydrothermal vent plumes (Li et al., 2014), deep sea normal saline and hypersaline environments (Edgcomb et al.,, Pachiadaki et al., 2014) and microcosm experiments on mixed layer samples from the NE Pacific Ocean (Marchetti et al., 2012). Additionally, more focused RNA-based investigations of expression of particular genes of interest using Reverse Transcription PCR (RT-PCR) or Reverse Transcription quantitative PCR (RT-qPCR) are becoming routine in oceanography.
Oceanographers examining marine microbiota have historically relied on ship-based hydrocasting operations for collection of water samples from various depths in the ocean via Niskin rosette samplers, followed by shipboard processing. When large and variable lapses in time occur between sample capture and preservation, or when samples are exposed to significant physico-chemical changes (e.g., changes in pressure, light, temperature, redox state, etc.) during transport to the surface, the Niskin-based approach is likely to introduce artifacts (Feike et al., 2012, Edgcomb et al.,). These concerns have long been recognized as particularly significant even when working with upper water column phototrophs (sudden light changes, e.g., Williams and Robertson, 1989; also see discussion in Gomelsky and Hoff (2011)), collecting samples from the deep sea (e.g., Bartlett, 2002) and/or from low-oxygen or anoxic zones (intrusion of oxygen or other gasses dissolved in Niskin bottle walls and elastomers, personal experience; Doherty et al., 2003). The degree of impact that sampling strategy has on transcription and protein synthesis, is likely to vary for different microbial taxa, depending on delicacy of cells and the degree to which they metabolically respond to such changes. Given that average lifetimes of prokaryotic transcripts can be on the order of several minutes (e.g., Wang et al., 2002, Andersson et al., 2006, Steglich et al., 2010), microorganism gene expression profiles can potentially be significantly altered between the time of collection and preservation using traditional approaches.
Delays between collection and preservation of DNA and ribosomal RNA (often used as a phylogenetic identifier of viable organisms) may introduce fewer artifacts because of their significantly longer half-lives (Hallsworth et al., 2007, preservation in hypersaline waters; see also preservation of ribosomal RNA in deep subsurface marine sediments, Orsi et al., 2013; medical literature, e.g., Miller, 1973). However, if sample handling induces cell lysis, delayed preservation can still lead to biases. This issue is particularly significant for sampling many relatively fragile microbial eukaryotes, and is potentially compounded when sampling at greater depths (Edgcomb et al., 2011).
The importance of in situ preservation was recently illustrated by Feike et al. (2012), who reported differences in microbial gene expression in suboxic samples from the Baltic Sea at 70–120 m depth, when preserved in situ vs. samples recovered to the deck prior to preservation. Edgcomb et al. (in press) also observed significant differences in gene expression profiles from deep (2200 m) Mediterranean Sea samples preserved in situ vs. those collected via Niskin casts and filtered/preserved in the ship׳s laboratory. Some categories of gene transcripts were more highly expressed in the Niskin samples, while many others were more highly expressed in the samples preserved in situ. For example, in Niskin water samples transcripts annotated to genes associated with stress responses (post-translational modification, protein turnover, and chaperone functions) were much more abundant (as a percentage of total annotated reads for each sample). In samples that were preserved in situ, expression levels of genes associated with functions such as energy production and conversion, replication, recombination and repair, inorganic ion transport and metabolism, and amino acid transport and metabolism were higher for most taxonomic groups than in Niskin samples, suggesting that cellular processes were less disturbed in samples that had not experienced decompression and additional handling (Edgcomb et al., in press).
As the scientific community transitions toward studies focusing on gathering more accurate assessment of in situ microbial activities and responses of the microbial community to environmental change, particularly in the deep sea, new technologies are required to support these aims. For example, in cabled or moored observatory work, the capability of the time series collection and preservation of filtered water samples in a manner that minimizes sample handling artifacts is essential for accurately representing ecosystem processes and response to environmental change.
Accurate data on microbial processes are fundamentally important to understanding microbially-driven ocean processes and responses of microbiota (and the major biogeochemical cycles that they mediate) to global climate change (e.g., Honjo et al., 2014).
In recent years several investigators have developed micro-laboratories and samplers that have brought sample manipulation and analysis, normally reserved for the laboratory, out into the field for extended periods of time, typically on moorings or integrated within the instrumentation network of ocean observatories. The Autonomous Microbial Genosensor (AMG, Fries et al., 2007), Environmental Sample Processor (ESP, Scholin et al., 2006, 2009; Roman et al., 2007; smaller version in development) and various versions of the Submersible Incubation Device (SID, Taylor and Doherty, 1990, Taylor et al., 1993, Taylor and Howes, 1994; Microbial Sampling-SID, MS-SID, Edgcomb et al.,, Pachiadaki et al., 2014) provide a glimpse into the developments for fielding molecular analytical and microbial activity techniques in a remote ocean setting.
Various in situ water samplers are in operation, examples of which include the Remote Access Sampler (RAS, Honda and Watanabe, 2007) for time series collection/chemical preservation of whole water samples, usually for various chemical analyses, the Phytoplankton Sampler (PPS, http://www.mclanelabs.com/master_page/product-type/samplers/phytoplankton-sampler) for time series collection of phytoplankton and other particulate samples on filters, and the Autonomous Microbial Sampler (AMS, Taylor et al., 2006) a manned submersible or Remote Operated Vehicle (ROV) operated device possessing protected inlets which completely eliminates the potential for nucleic acid and microbial “crosstalk” between samples. Autonomous Underwater Vehicle (AUV)-based whole water samplers were recently described by Dodd, et al. (2006), Ryan et al. (2010) and the SUspended Particulate Rosette (SUPR, Breier et al., 2009) is a manned submersible or ROV-deployed device for collection of hydrothermal vent plume particulate samples. These samplers, however, lack a means for sample preservation and generally require that samples either be processed relatively quickly or be unaffected by possible decomposition processes.
In situ preservation of samples, particularly concentrated particulate samples imposes an additional technical challenge. One recent approach involved modifying the Niskin bottle concept by injection of preservative into the enclosed sample (Automatic Flow Injection Sampler, AFIS, Feike et al., 2012) for subsequent metatranscriptomic analysis. The various SID concepts early on possessed means for chemically terminating biological activity and preserving filtered particulate samples, but the approach was primarily applicable to tracer incubation studies. The relatively complex ESP possesses an archiving function for in situ collection and storage of chemically preserved filtered samples and was the technology behind a metatranscriptomic study assessing the suitability of RNAlater for longer term preservation of microbes under environmental conditions (Ottesen et al., 2011). In situ preservation of filtered samples is a capability of a second generation particulate sampler based on the SUPR concept (SUPR-V2 and smaller version SUPR-V3; http://www.whoi.edu/science/AOPE/dsl/jbreier/SUPR.html) and a free-fall SUPR analog (CLIO, http://www.whoi.edu/science/AOPE/dsl/jbreier/Clio.html) are in advanced development; both require a pumping system for in situ delivery of an appropriate preservative (e.g., RNAlater) into the filter housing to preserve the particulate samples.
We report here the development of a new stand-alone technology for the in situ filtration and preservation of particulate samples in a manner compatible with microbial metagenomic and metatranscriptomic analyses. The device, the Fixation Filter Unit 3 (FF3), requires no additional electromechanical support for preservative delivery and therefore can be adapted to a variety of existing mooring-, hydrocasting-, submersible-based sampling platforms (e.g., SID, MS-SID, PPS, RAS, AMS, etc.), allowing retrofit enhancement of existing in situ sampling capability.
Section snippets
Description of the filter unit
The FF3 (Fig. 1A, Taylor et al., 2013) is a stand-alone 10.2 cm×5.5 cm diameter (4″×2.2″ diameter) in-line 47 mm filter system for in situ collection and chemical preservation of filtered particulate samples. They carry a preservative of the user׳s choice, (RNAlater® was used as an example in this study), permit filtration of water samples through 47 mm diameter filters of the user׳s choice and upon completion of filtration, chemically preserve the retained particulate sample within 10׳s of
Mechanism of operation
The assembled device is deployed into the environment as diagrammatically illustrated in Fig. 2A with the entire dead volume spaces (3.52 mL) within the FF3 containing the preservative (shown in magenta). Upon initiation of filtration, sample flows via the Sample Inlet into the interior, through a filter medium of choice and out, the particulate fraction being impinged on the filter surface (Fig. 2B and green inset) as occurs in a normal in-line filter. Sample flows are shown in blue and by the
Conclusions
A new technology (FF3) has been developed in response to the increasing role metatranscriptomics and other analyses involving capture and preservation of labile biomolecules is playing in understanding the linkages between organisms and their environment. The FF3 filtration device allows the collection and chemical preservation of particulate microbial samples directly in situ. This eliminates the attendant stresses resulting from a host of physico-chemical changes that can occur in the
Author contributions
CT and KD co-developed the concept behind the FF3 and earlier prototypes, and were in communication throughout the development. CT, KD, IE and TS participated in the detailed engineering design of the FF3 and earlier prototypes. CT and VE conducted FF3 experiments, analyzed results and jointly participated in the writing of the manuscript. VE and MP conducted the transcriptomic study and SM conducted early fixation filter studies. SH generally supported the fixation filter development and
Acknowledgments
We would like to thank the captain and crew of the R/V Urania for their hard work in obtaining the samples for this work. Cruise participation was partially supported by Deutsche Forschungsgemeinschaft (DFG) Grant STO414/10-1 to T. Stoeck. This research was funded by NSF OCE-1061774 to VE and CT, NSF DBI-0424599 (C-MORE) to CT and McLane Research Laboratories, Falmouth, MA to VE and CT.
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