Gerrit Jan (Ger) van den Engh - US grants

This project focuses on the intricate combination of biological and physicochemical processes by which dissolved organic matter (DOM) is formed and cycled in the ocean. The interlinked biological and physical processes by which bioactive elements are cycled in the ocean represent one of the most complicated and critical systems on earth. Although living organisms are the ultimate source and sink of the organic matter in which these elements primarily cycle, the major currency for both their active transfer and long?term storage in the ocean is as small, nonliving organic molecules. This "molecular prerogative" results because the biomacromolecules composing organisms and tissues must be broken down to inert subunits to pass bacterial cell walls prior to complete intracellular respiration. In the ocean, much of this molecular dismantling is accomplished by bacterial exoenzymes operating on organic substrates. Nutrient elements carried through this "bacterial loop" become available for conversion back to living particulate form either through photosynthesis or via transfer of bacterial production up food webs through protists and zooplankton. Such biologically mediated cycling between dissolved and particulate organic forms is critical on a larger scale to the transfer and fate of nutrients because only particles can sink to selectively transport bioactive elements from the lighted surface ocean into deep storage below the thermocline (i.e., the biological pump). Recent evidence for spontaneous assembly of colloidal marine molecules into microscopic polymer gels has fundamentally changed the way that oceanographers think about processes linking the microbial loop and biological pump to the rest of the biosphere and the geosphere. The key observation was that colloidal?size organic molecules found in surface seawater can coalesce spontaneously. The resulting particles (microgels) are sufficiently large to sink and might undergo other processes such as catastrophically collapsing to dense forms after passing sharp thresholds of temperature, pressure or pH. Our group has also recently found that these microgels are degraded approximately an order of magnitude faster by marine bacteria than the dispersed molecules from which they are formed, apparently by providing an effective means for abiotically concentrating substrate for efficient exoenzymatic dismantling. This project is an interdisciplinary focus on the role of these microgel particles in carbon cycling processes.

This award supports the development of a DNA-array scanner that detects fluorescence signals by means of a novel wave guide collector. The wave guide will collect light trapped inside the array substrate by total internal reflection (TIR). The proposed scanner combines the best properties of various alternative scanner designs, while avoiding some significant disadvantages. The use of orthogonal illumination will result in low background levels and improved signal-to-noise in comparison to existing commercial instruments. The light collection optics allow addition of a temperature kinetics module which will permit use of hybridization conditions that greatly increase binding specificity.

The use of DNA array hybridization has been widely adopted by biologists and biomedical researchers as a technique for detection of mutations in DNA and for monitoring of gene expression. Commercially available instruments for scanning arrays are relatively complex devices based on the same optics used in confocal microscopes, are expensive to manufacture and are unsuited to the real-time measurement of hybridization kinetics. The TIR design is simple, compact, requires little alignment and can be manufactured at low cost. Scanners with TIR collection will make possible a new generation of versatile laboratory instruments for hybridization array quantification with improved accuracy and speed.

The PI's request funding to develop a commercial detector for identifying and counting marine plankton that will be integrated into an instrument for extended use at sea. The micro-plankton detector will be based on the standard detector that Cytopeia uses in its flow cytometers and cell sorters. This detector will be supplemented with position sensitive detectors (PSDs) that sense the position of particles that flow through its observation area. Using well-established principles of flow cytometry and confocal microscopy, the detector will be able to discriminate between particles that flow through the system's optical axis and those particles that pass out of focus. The system will process fluorescence signals from in-focus particles only, and will ignore out-of-focus events that cannot be measured accurately. This will result in will a reliable in situ instrument for routine cytometric characterization of phytoplankton populations.

ABSTRACT

OCE-0452563

The chemical identity and dynamics of dissolved biopolymeric material is a standing question in oceanography. However, due to a prior dearth of techniques to study dissolved biopolymers, we are only beginning to understand the source, composition and reactivity at the molecular level. In this research, scientists from the Institute of Systems Biology will develop innovative tools (biosensors) for biopolymer research using high throughput proteomics and antibody display technology. Rather than solely examining single amino acids after hydrolysis, which erases the chemical-history of the proteins and the link between biology and ocean chemistry, the application of proteomics to characterize the secretory proteome of two model phytoplankton species will advance our understanding of the composition and reactivity of these compounds at the molecular level. Because secretory proteomes are thought to be conservative, knowledge of the proteome for these two ecologically important species should aid in the characterization of other phytoplankton secretory proteomes. High affinity biosensors (monoclonal antibodies from phage) that recognize and bind to specific molecular moieties will allow the identification and quantification of chemical species at low concentrations in complex heterogeneous samples. Ultimately this technology will allow in situ tracking of phytoplankton proteins, their biosynthesis, transformation, and degradation in unconcentrated seawater.

In terms of broader impacts, combining the direct monitoring of proteins released by phytoplankton and cyanobacteria and tracking of phytoplankton and cyanobacterial proteins with monoclonal probes should allow the linking of biological production and consumption to ocean chemistry through understanding the dynamics and turnover of proteins released by phytoplankton and cyanobacteria into the overall dissolved organic matter pool. Furthermore, this will lead to improved knowledge of their role in global carbon cycling and will greatly advance our knowledge of marine geochemistry and marine ecology. Undergraduate students will be exposed to these new techniques as part of this study. The PIs will play active roles in local and regional school district science educational initiatives.