Ryan Paerl

The metabolism of marine heterotrophic bacterioplankton significantly impacts global elemental cycling, productivity, and water quality; thus, it is critical to understand the factors controlling bacterioplankton metabolism. Recent evidence shows that most marine bacterioplankton rely on exogenous vitamin B1 (thiamin; B1 herein) or congeners (B1 precursors, phosphorylated B1) to survive, and addition of either can stimulate bacterial production in estuarine Baltic Sea surface waters. This suggests that it is favorable for most marine bacterioplankton to rely on exogenous, biologically produced B1/congeners rather than produce them individually, making B1 dynamics an environmentally relevant test case to study metabolite exchanges within the planktonic microbiome. Here, it is proposed to investigate the impact of B1/congener on bacterioplankton in ecosystems where vitamins have garnered little consideration as influential nutrients, in the second largest estuary in the lower USA and in the non-upwelling influenced coastal ocean. Proposed nutrient amendment experiments test if B1/congeners modulate bacterioplankton growth, composition, and gene transcription (reflecting putative function). Complementary lab-based experiments with wildtype and genetically engineered B1-auxotrophic bacterioplankton are also proposed to test if use of exogenous B1/congener significantly improves (growth) fitness ������������������ directly addressing why reliance upon exogenous B1/congener is so prevalent among wild bacterioplankton. Overall the proposed work reaches beyond recent genome-based extrapolations to address how exogenous B1/precursor availability shapes bacterioplankton metabolism, community structure/intractions, and fitness.

The environmental health of the Sacramento-San Joaquin River�������� Bay Delta (SR/SJR-BD), CA is threatened by the recent expansion of toxigenic cyanobacterial harmful algal blooms (CyanoHABs), particularly Microcystis, linked to increased occurrences of the hepatotoxin microcystin. Several studies have investigated the impacts of CyanoHABs and microcystin on water quality, but recent findings suggest that they also threaten air quality via 1. primary spray aerosol emissions carrying cells and cyanotoxins and 2. secondary organic aerosol (SOA) formation via algal volatile organic compound (VOC) emissions. Given CA��������s historical air quality concerns, many communities in the SR/SJR-BD region are already at a heightened risk of respiratory illness associated with environmental exposures. Therefore, to assess the public health risks posed by the inhalation of cyanobacterial compounds, it is crucial that we generate more information on the physicochemical and ecological controls which favor their aerosolization. Our proposed study aims to investigate the transport of primary spray aerosol containing cyanotoxins, and the contribution of CyanoHABs to SOA formation in the airshed of the SR/SJR-BD. To do so, we will 1) utilize mixed aerosol collection methods in the field coupled with targeted microbial analyses of cyanobacterial cells, DNA, and microcystin. Aerosol samples will be collected via high-volume impactors and efficient soft-impingers. Water samples and meteorological data will be collected in parallel to model environmental factors which influence the primary aerosolization of CyanoHAB compounds. To evaluate the production of SOA from biogenic cyanobacterial emissions, as well as nutrient related growth and toxin production factors in SR/SJR-BD cyanobacterial communities, we will 2) perform a series of in situ, microcosm, nutrient addition experiments. SOA precursor gases and mass concentrations will be measured directly from the headspace of the microcosm chambers. Nutrient modulation of phytoplankton (chiefly CyanoHAB) growth and microcystin production will be assessed alongside SOA formation measurements. Findings from will serve to inform both human exposure guidelines to CyanoHAB aerosols and CyanoHAB mitigation policy. The ultimate goal of this research is to diminish the adverse impacts of CyanoHABs on water and air quality in the SR/SJR-BD region.

Picophytoplankton, small phytoplankton <3 ��������m in diameter, are important primary producers in large coastal North Carolina ecosystems. In the Neuse River Estuary (NRE), a major tributary of the Pamlico Albemarle Sound System (PASS), picophytoplankton are significant contributors to primary productivity (PP) and phytoplankton biomass (PB) (on avg. ~40% and >70% during summer periods) (Gaulke et al. 2010; Paerl et al. 2020) and desirable prey for nano- and micro-zooplankton (Gaulke et al. 2010; Wetz et al. 2011). Recent flow cytometry data shows that phycocyanin-rich (green pigmented) Synechococcus-like cells (PC-SYN) are the dominant picophytoplankton group for much of the year in terms of biomass, especially during warmer summer and fall months where they reach upwards of 1 million cells mL-1 or more. While it is now clear that PC-SYN are key primary producers in coastal NC waters, what are their physiological capabilities and potential to impact ecosystem health? We need to expand our knowledge of PC-SYN in coastal NC waters as they may influence ecosystem health and function beyond carbon fixation contribution in surface waters. 1) Picocyanobacteria can be resistant to grazing and viral lysis (Zwirglmaier et al. 2009; Apple et al. 2011; Zoborowsky and Lindell 2019). If this is true in the NRE and PASS, it would reduce their true contribution to nutrient and energy flow to upper trophic levels of food webs and instead increase their importance in carbon export (Richardson and Jackson 2007). 2) PC-SYN and marine picocyanobacteria can produce secondary metabolites that inhibit co-occurring phytoplankton (Paz-Yepes et al. 2013) and impact behavior of co-occurring fish and filter feeders (Wall et al. 2012; Hamilton et al. 2014). 3) Picocyanobacteria vary in their ability to use inorganic and organic nutrients (Moore et al. 2002) as well as to tolerate changes in water chemistry and light conditions (Stomp et al. 2007; Shmidt et al. 2020). Increased precipitation is predicted in the future for eastern NC (Kunkel et al. 2020) and with it reduced PC-SYN numbers are expected (Paerl et al. 2020). However, a better understanding of PC-SYN resilience ������������������ esp. abilities to use organic nitrogen/phosphorous, tolerate metals and harvest low light ������������������ would enable better predictions of their ability to support food webs in the future. Genomics, i.e. sequencing of genomes and comparisons of genome sequences, offers the ability to identify the broad metabolic potential of PC-SYN cells without extensive laboratory-based experiments with isolates. In this project, sequencing of picocyanobacterial isolate genomes and metagenomes from sorted naturally occurring cells is proposed to gain insight into the capabilities of this dominant primary producers in local NC estuaries.

Fluorescence activated cell sorting (FACS) is a technique that involves sorting away select cells (or objects) from complex mixtures based on their intrinsic or acquired fluorescence. FACS is a transformative technology that allows the study of the unculturable microbes (which are numerically dominant in nature) and accomplish tasks that are highly laborious or impossible complete in other ways; the technology has led to significant discoveries in many microbial research fields (e.g. ecology, genetics, physiology, symbiosis/interactions, bioengineering, and bio-prospecting), and nearly single-handedly forged new fields of research, e.g. single cell genomics and transcriptomics, which involves the study of DNA and mRNA from individual cells. More than 25 North Carolina State University (NCSU) faculty, belonging to 4 colleges, have needs for FACS in their research or teaching programs; however, NCSU lacks a FACS instrument optimized for the analysis of non-mammalian microbial cells (e.g. bacteria, archaea and fungi), and to our knowledge, no ���������������microbe optimized������������������ instrument is available at research universities within the Research Triangle of North Carolina (e.g. UNC-CH, Duke University). Here, funds are requested to acquire a Becton Dickinson FACSMelody flow cytometer, a versatile (3 excitation laser, 9-color detection) and ���������������turn key������������������ system, which fundamentally enables microbiological research that is highly laborious or impossible to accomplish without it. The FACSMelody is powerful yet simple to use and generates easy to grasp visual (flow cytometric) data ������������������ making it a good potential training and educational tool for undergraduate/graduate courses and workshops. A FACSMelody system is ideal for getting FACS technology rapidly and easily into the hands of faculty in need. Overall, a FACS system for non-mammalian microbial research is needed for NCSU to be innovative, internationally competitive at attracting new faculty and highly talented students, and foster creative future proposals.

Health impacts are usually quantified in terms of exposure to mass concentrations of particulate matter with aerodynamic diameter ��������������� 2.5 ��������m (PM_{2.5}) and are well documented. Sub-10 nm particles contribute negligible mass to atmospheric PM_{2.5}. Yet they may contribute strongly to the overall toxic dose delivered through the aerosol. The toxicity of sub-10 nm particles is higher when compared to particles of larger size with the same composition. Sub-10 nm particles are readily taken up by cells, can cross the skin barrier, air-blood barrier, and blood-brain barrier and in turn reach sensitive organs. However, the paucity of information about their abundance, morphology, and toxicity has hampered efforts to attribute their potential health effects with disease. We have recently identified substantial direct emissions of sub-10 nm particles in an urban environment and found that there are multiple temporally stable and spatially confined point sources within the city. It is unclear how the toxicity of environmental sub-10 nm particles differs from engineered nanoparticles, and how the toxicity might change upon release into the atmosphere. Current in in-vitro and in vivo exposure studies deliver milligrams of material. In contrast, ambient concentrations of sub-10 nm particles are on the order of ng m^-3, making collection of mg amounts unrealistic. This proposal addresses this challenge. The specific aim is to test new methods that can be used to evaluate the influence of sub-10 nm particles on living cells at ambient mass concentrations.

Concentrations of the earthy/musty taste/odor compounds geosmin and MIB (2-methylisoborneol) annually exceed the threshold of human detection (~10 ng/L) in key drinking water reservoirs in Durham County, specifically Lake Michie. This causes a notable rise in customer complaints to Durham County Department of Water Management (DCDWM), especially in late Spring and Summer, and a perception that drinking water quality is poor. The microbial source(s) of these taste & odor (T&O) compounds is assumed; thus targets for effective monitoring and mitigation are not well-established. Recent phytoplankton monitoring, via flow cytometry (FCM) and microscopy, suggests picocyanobacteria (PicoC; cyanobacteria <3 ���������m diameter) have a role in elevated geosmin concentrations in Spring and early Summer������������������ an intriguing result as PicoC������������������s are often overlooked as geosmin sources. In summer ������������������filamentous cyanobacteria are more prevalent and may contribute to high geosmin and MIB levels during this time of the year. DCDWM uses copper-sulfate based algicide to reduce cyanobacterial biomass and T&O but the effectiveness of the treatment and whether reduced application could better aid T&O mitigation is unknown. It is hypothesized that: (H1) high net growth of PicoC is linked to geosmin peaks in Spring, while filamentous cyanobacteria are key producers in Summer; (H2) PicoC and filamentous cyanobacteria are the most prevalent geosmin producers in LM; (H3) reduced algicide addition will have a bacteriostatic effect ������������������ where cyanobacteria are not extensively lysed and concentrations of T&O compounds in the dissolved phase are reduced. H1 will be tested via improved time-series monitoring of phytoplankton (PicoC, filamentous cyanobacteria) and concentrations of T&O earlier in the phytoplankton growth season. H2 will tested using metagenomic sequencing of Lake Michie plankton DNA to directly identify populations possessing geosmin or MIB synthase genes. Last, algicide gradient addition experiments will be performed to test H3. Expected outcomes include: (1) establishment of phytoplankton targets for future monitoring and/or prediction of elevated T&O concentrations and (2) an evaluation of reduced algicide addition as an approach for water managers to lower dissolved T&O compound levels and save on costs. An educational outcome of the project will be T&O teaching modules that convey basics of what compounds are involved and who produces them to the general public. The module will be implemented as part of an ongoing ���������������mobile laboratory������������������ outreach program (called MAML) at Lake Johnson, Raleigh, and will include pre- and post-questionnaires to assess the effectiveness of these modules.

Freshwater mussel populations throughout North America have declined precipitously during the last three decades. Captive propagation and release of the captive reared stock has become an integral component of efforts to mitigate the decline and augment the reproductive capacity of remaining populations. Freshwater mussels are reared in captivity using two alternative approaches that focus on different approaches to facilitating the metamorphosis of juveniles. Either host-fish are used to support the metamorphosis of the larval stage or the larvae are reared in vitro in petri dishes and the metamorphosis is nutritionally supported with culture media. The initial survival of in vitro reared mussels is poor and their initial growth lags behind that of host-fish reared animals. Our limited understanding of the nutritional needs of freshwater mussels and how specific components of their diet contribute to their nutritional health impedes our ability to sustain them in captivity. In addition, declines noted in some free-ranging populations appear to be associated with poor nutrition. We propose studies to further inform our understanding of the role of different food-web resources in the diets of freshwater mussels. Specific objectives include: 1) Examining the role of pollen in the diet of freshwater mussels; 2) Assessing the role of detritus in freshwater mussel diets; 3) Quantifying the filtration ability of selected freshwater mussels species; 4) Preliminary studies of diet and freshwater mussel nutritional health; and 5) Refining procedures for characterizing the chemical composition of freshwater mussel shells.