Biology

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Impact of salinity on morphology, growth, and pigment profiles of Scenedesmus obliquus HTB1 under ambient air and elevated CO2 (10%) conditions
(2024) Jiao, Fanglue; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Certain microalgal species tolerate high CO2 concentrations and proliferate faster with elevated CO2 than with ambient air. This feature makes them attractive for carbon sequestration, a tool for mitigating climate change due to increasing atmospheric CO2. Scenedesmus species are among these microalgae. Scenedesmus obliquus strain HTB1 is a microalgal strain isolated from the Baltimore Inner Harbor (brackish water) and has shown a faster growth with 10% CO2 compared to air. However, how HTB1 grows under different salinity and if the salt response is affected by elevated CO2 remains elusive. Two experiments were set up to address these questions. The first experiment tested the impact of salinity gradient (0, 17.5, 20, 22.5, 25, 27.5, and 30 ppt) on HTB1 under ambient air. With increasing salinity, HTB1 cells became smaller, and the cultures changed color from green to brown, yellowish brown, and then to pale white. The pigment analysis showed that HTB1 reduced several pigments (i.e. zeaxanthin, lutein, chlorophyll b) in response to salt stress. However, HTB1 produced higher concentrations of canthaxanthin under the salt stress. The growth of HTB1 decreased with increasing salinity and was inhibited when the salinity was greater than 22.5 ppt. In the second experiment, we compared the impact of salinity (0, 10, and 20 ppt) on HTB1 under air and 10% CO2, respectively. HTB1 cultures showed little color change with increasing salinity under 10% CO2. In contrast, the change of culture color from dark green to brown was observed with increasing salinity when HTB1 was grown with air. Interestingly, the growth of HTB1 was less inhibited with salt under 10% CO2 than with air, suggesting that elevated CO2 mitigates the salt stress of HTB1. Lutein and canthaxanthin increased with increasing salinity when HTB1 was grown with 10% CO2. Our results indicate that increased salinity affects the growth of Scenedesmus obliquus HTB1 more with air than with 10% CO2. This study provides insight into the impact of salt stress on algal morphology, growth, and pigment composition.
Methane Biogeochemistry and Microbial Communities in Natural and Restored Freshwater Depressional Wetlands
(2024) Hamovit, Nora David; Yarwood, Stephanie A; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Wetlands are the largest natural source of methane (CH4), a potent greenhouse gas. Wetland CH4 emissions are dependent on rates of microbial CH4 production (methanogenesis) and consumption (methanotrophy). These processes vary spatially and temporally with environmental conditions, edaphic characteristics, and microbial community structure, making it difficult to predict wetland CH4 emissions. This high variability can be further pronounced in restored wetlands that have undergone environmental and edaphic disturbances. The following work aims to understand this variability by assessing patterns of methanogenesis and methanotrophy, and their associated microbial communities, across natural and restored freshwater depressional wetlands on the Delmarva Peninsula (USA). Sites addressed in this work were restored from agricultural land between 1986 and 2004 through multiple programs funded by the United States Department of Agriculture (USDA). In the first set of experiments, we identified a high abundance of active acetoclastic methanogens in intact core incubations from a restored wetland suggesting a higher potential for methanogenesis in situ compared to the natural wetland assessed. The co-occurrence of active methanogens and Fe-reducing bacteria in these restored wetland cores contradicted the hypothesis that loss of competition may allow methanogens to be the primary users of acetate. Following assessments across vegetative-hydrologic zones in a series of restored wetlands of varying ages, and their natural counterparts, highlighted vegetation type and extent as a driver of methanogen community abundance, composition, and activity. In turn, restored wetlands showed elevated potentials rates of methanogenesis compared to natural sites. Potential rates of methanotrophy (aerobic and anaerobic), however, were also elevated in restored wetlands, which could constrain CH4 emissions in situ. Variability of environmental conditions (ie. hydrology and vegetation) and edaphic measures (ie. soil organic matter (SOM)) across all sites sampled are reflected in distinct microbial community composition and CH4 biogeochemistry. Clear patterns of SOC accumulation and CH4 biogeochemistry with restoration age were not observed for these wetlands, and variability in environmental conditions and edaphic measures across the sites (restored and natural), emphasize the need for continued monitoring and maintenance of the wetlands. Our results suggest efforts to manage herbaceous vegetation extent and maintain regular seasonal hydrology in future restorations may help prevent high potentials for CH4 production, and thus emissions.
MICROBIAL COMMUNITIES IN COASTAL ECOSYSTEMS
(2024) Kim, Carol; Malkin, Sairah Y; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Firstly, I examined microbial community succession along a chronosequence of constructed salt marshes using the Poplar Island restoration project site as a case study. By comparing 16S rRNA gene amplicon sequences across 6 constructed low marshes spanning a chronosequence of 1-16 years at Poplar Island (Chesapeake Bay) and a nearby natural reference marsh, I found strong evidence that the development of soil microbial communities is on a trajectory towards natural marsh conditions following marsh restoration with successional rates within timescales expected for soil development. Results from this study showed the value of microbial communities to serve as effective bioindicators for monitoring the recovery of microbially mediated biogeochemical processes in restored or newly constructed salt marshes, as well as potentially for assessing the marsh inundation period and by extension marsh health and resiliency. Secondly, I conducted a manipulation experiment to explore microbial communities associated with cable bacteria using RNA stable isotope probing (RNA-SIP). I traced the uptake of isotopically labeled bicarbonate and acetate in sediments with baseline and with stimulated cable bacteria activity, to test the hypothesis that cable bacteria activity can stimulate chemoautotrophic bacteria in anaerobic sediments. I used 16S rRNA sequencing to identify the active “incorporators” of bicarbonate (as a tracer of chemoautotrophy) and acetate (as a tracer of heterotrophy). I found that estuarine cable bacteria activity stimulated the chemoautotrophic activity of Gammaproteobacteria (Nitrosomonas, Thioalkalispira-Sulfurivermis) and Campylobacterota (Sulfurovum, Sulfurimonas) at anaerobic depths. This result is not explainable with conventional understanding of chemoautotrophic activity. Rather, this study contributes to the emerging concept that cable bacteria activity stimulates metabolic activities at suboxic sediment depths, potentially by serving as an electron sink for other microbes. Furthermore, I found that heterotrophic activity, measured as 13C-acetate assimilation into RNA, was stimulated amongst known chemoautotrophic sulfur oxidizers at depth, highlighting that metabolic flexibility, and specifically mixotrophy, may be widespread in complex natural sediment environments. Lastly, I characterized the composition and metabolic potential of microbial communities in estuarine sediment enriched with cable bacteria. By using metagenomic and 16S rRNA sequencing, I constructed 23 medium- to high-quality metagenome-assembled genomes (MAGs) that span across 9 phyla. I retrieved MAGs exhibiting mixotrophy and a range of capabilities for extracellular electron transport. This study revealed a diverse range of metabolically flexible communities of microbes that contribute to the biogeochemical cycling of carbon, nitrogen, and sulfur.
EVOLUTION OF THE CRISPR IMMUNE SYSTEM FROM ECOLOGICAL TO MOLECULAR SCALES
(2024) Xiao, Wei; Johnson, Philip LF; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Bacteria and archaea inhabit environments that constantly face viral infections and other external genetic threats. They have evolved an arsenal of defense strategies to protect themselves. My research delves into the CRISPR immune system, the only known adaptive immune system of prokaryotes. My work explores three different dimensions of the CRISPR immune system, ranging from ecological to molecular scales.From an evolutionary perspective, CRISPR is widely distributed across the prokaryotic tree, underscoring its immune effectiveness. However, the CRISPR distribution is uneven and some lineages are devoid of CRISPR. Here, I identify two ecological drivers of the CRISPR immune system. By analyzing both 16S rRNA data and metagenomic data, I find the CRISPR system is favored in less abundant prokaryotes in the saltwater environment and higher diverse prokaryote communities in the human oral environment. On the molecular level, the CRISPR system selects and cleaves its “favorite” DNA segments (also known as “spacers”) from invading viral genomes to form immune memories. I explore how the spacer sequence composition affects its acquisition rate by the CRISPR system. I develop a convolutional neural network model to predict the spacer acquisition rate based on the spacer sequence composition in natural microbial communities. The model interpretation reveals that the PAM-proximal end of the spacer is more important in predicting the spacer abundance, which is consistent with previous findings from controlled experimental studies. Combining these scales, CRISPR repeat sequences coevolve with the rest of the genome. Thus, I explore the potential of utilizing CRISPR repeat sequences for taxonomy profiling. I find a strong relationship between unique repeat sequences and taxonomy in both the RefSeq database and a human metagenomic dataset. Then I show high accuracy when utilizing repeat sequences in taxonomy annotation of human metagenomic contigs. This novel method not only aids in annotating CRISPR arrays but also introduces a novel tool for metagenomic sequence annotation.
AN INVESTIGATION ON THE MOLECULAR BASIS FOR DIMER FORMATION OF A BACTERIOPHAGE ENDOLYSIN POSSESSING ANTIMICROBIAL ACTIVITY AGAINST STREPTOCOCCUS PNEUMONIAE
(2023) Alreja, Adit Bipin; Nelson, Daniel C; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The global rise of antibiotic resistance casts a shadow on treating infectious disease. An alternative to the use of antibiotics is bacteriophage-derived peptidoglycan hydrolases called endolysins. Endolysins, produced at the end of a bacteriophage replication cycle, cause bacterial cell lysis and virion release. When applied exogenously as recombinant proteins, they are also capable of cleaving the Gram-positive bacterial peptidoglycan. Various studies conducted in vitro and in vivo showcase the therapeutic potential of endolysins as the next generation of antimicrobials. Streptococcus pneumoniae is the most common cause of a variety of infections ranging from otitis media to invasive bloodstream infection (bacteremia) and meningitis (brain infection). While pneumococcal vaccination programs have proven to be effective, the high rates of antibiotic resistance reported for S. pneumoniae has led to the CDC classifying it as a “serious” threat. One of the most studied endolysins targeting S. pneumoniae is Cpl-1. This thesis represents an investigation into the molecular basis for dimer formation of the Cpl-1 endolysin which displays antibacterial activity against S. pneumoniae. In addition to disproving a long-accepted mechanism of dimerization of Cpl-1 in the presence of choline, we have conclusively identified the residue involved in the formation of the Cpl-1 dimer. Our findings led to the discovery of a novel C-terminal consensus sequence shared by all pneumococcal endolysins that informs their propensity to form dimers in the presence of choline. Next, through a bioinformatics approach we identified a new endolysin containing this C-terminal consensus sequence, produced it, named it SP-CHAP, and showed that it forms a dimer in the presence of choline, indicative of the widespread dimerization phenomenon associated with pneumococcal endolysins. Of interest, SP-CHAP is the first endolysin with antimicrobial activity against S. pneumoniae that possesses a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain. SP-CHAP was subsequently characterized for its biochemical and antimicrobial properties and benchmarked against Cpl-1. SP-CHAP is active in all physiologically relevant conditions (pH, temperature) against various S. pneumoniae strains and displays no activity towards oral/nasal commensal organisms. This enzyme also displays pneumococcal biofilm eradication ability to a greater extent than Cpl-1, as visualized by confocal microscopy. To further translate the antimicrobial potential of this enzyme, the antimicrobial efficacy of SP-CHAP was validated in a S. pneumoniae mouse nasopharyngeal colonization model. Our results demonstrate the therapeutic potential of SP-CHAP as an attractive endolysin to treat S. pneumoniae infections and warrant further translational development of this enzyme.
Climate Change and Vibrio species: Investigation of Environmental Parameters Associated with Occurrence and Transmission
(2023) Brumfield, Kyle David; Colwell, Rita R.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Climate change, associated with shifts in the geographical range of biological species, has become increasingly important in emergence and re-emergence of disease. Vibrio spp., native to aquatic ecosystems, are commonly associated with aquatic invertebrates, notably crustaceans and zooplankton. Some species of the genus Vibrio cause infection in humans, of which Vibrio cholerae, the etiological agent of pandemic cholera, is the most documented. Pathogenic non-cholera Vibrio spp., namely Vibrio parahaemolyticus and Vibrio vulnificus, cause gastroenteritis and also septicemia and extra-intestinal infections. They are responsible for a large number of public health emergencies in developed countries, including the United States. As sea temperatures rise and salinity profiles are altered, a pattern of poleward spreading of non-cholera Vibrio spp. has been observed globally, demonstrating significant geographic expansion of these bacterial populations, corroborated by an associated increase in the number of reported vibriosis cases. Since Vibrio spp., including pathogenic vibrios, play an important role in the degradation of polymeric substances, such as chitin, and in biogeochemical processes, they cannot be eradicated. Hence, routine monitoring and an early warning system are needed for public health preparedness. Since the 1960’s, ongoing research has focused on environmental factors linked with occurrence and distribution of clinically relevant Vibrio spp. and their role in disease transmission. We have reported that lack of, or damage to, water, sanitation, and hygiene (WASH) infrastructure, coupled with elevated air temperatures, and followed by above average rainfall promotes exposure of a population to contaminated water, hence increases the risk of an outbreak of cholera. Global predictive intelligence models applicable to diseases caused by non-cholera Vibrio spp. are in development. The research reported here describes results of intensive sampling to detect and characterize Vibrio spp. in the Chesapeake Bay, Maryland, and the Florida Gulf Coast, the latter an area significantly impacted by Hurricane Ian, September 2022, with a spike in confirmed vibriosis cases and deaths during weeks following the storm. Results of this study provide confirmation of environmental predictors for Vibrio spp. and document long-term increase and extended seasonality of Vibrio populations in the Chesapeake Bay. Using satellite remote sensing data, we demonstrate the impact of extreme heat, precipitation, and other key environmental and geophysical factors (e.g., temperature, salinity, and chlorophyll) on prevalence of pathogenic Vibrio spp. in aquatic systems. This study lays the groundwork for a predictive intelligence system for Vibrio spp. and other pathogens under varying climatic scenarios.
BIOPROSPECTING MARINE ACTINOMYCETES FOR NOVEL ANTI-TUBERCULOSIS DRUGS
(2022) Tizabi, Daniela Rose; Hill, Russell T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Mycobacterium tuberculosis (M. tb), the causative agent of the infectious lung disease tuberculosis (TB), is estimated to infect approximately 1.7 billion people worldwide. This pathogen was responsible for more than 1.5 million deaths in 2020, and is likely to remain a global threat for many years to come due to the rising incidence of antibiotic resistance, as well as dramatic setbacks in treatment due to the ongoing COVID-19 pandemic. There is an urgent demand for novel therapeutics to treat the disease through unique mechanisms of action. In the search for these drugs, a novel collection of 101 marine actinomycetes previously isolated from a Caribbean giant barrel sponge Xestospongia muta was investigated for their ability to inhibit M. tb growth. Thirteen novel strains of Micrococcus, Micromonospora, Brevibacterium, and Streptomyces were identified as consistently producing extracts that inhibit M. tb in a dose-dependent manner. After sequencing the genomes of these strains, a comparative analysis between three assembly algorithms (SPAdes, A5-miseq, Shovill) was performed to determine which program yielded the best assembly from Illumina MiSeq data for biosynthetic gene cluster (BGC) mining. Upon characterizing the biosynthetic potential of each strain, two isolates generating highly potent extracts – Micrococcus sp. strain R8502A1 and Micromonospora sp. strain R45601 – were selected for further analysis through a dual genomics and chemistry-enabled approach. No compounds with obvious anti-TB activity were detected in the genome of Micrococcus sp. strain R8502A1, suggesting production of an elusive and novel anti-TB compound through a cryptic pathway. A comprehensive examination of all BGC-associated domains was conducted to evaluate possible biosynthetic pathways linked to the anti-TB activity observed. The active component of the Micrococcus extract was further isolated with high performance liquid chromatography (HPLC) and is under investigation with liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). In contrast, a BGC with 94% similarity to the selective and potent but poorly soluble anti-TB compound diazaquinomycin H/J was identified in the genome of Micromonospora sp. strain R45601, suggesting production of a chemical analog. LC-MS detected four peaks of interest, two of which are associated with mass-to-charge (m/z) values that do not correlate with any previously identified diazaquinomycin analogs. This analysis has identified at least two potentially novel anti-TB compounds, supporting continued investigation into sponge-associated marine actinomycetes for novel therapeutics.
SOIL MICROBIAL COMMUNITIES IN URBAN STREAM RESTORATIONS AND FORESTED SITES IN FAIRFAX COUNTY, VA
(2022) Wood, Lindsay; Yarwood, Stephanie; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Urbanization is rapidly occurring worldwide and can increase hydrological flows into urban streams and alter forest structure and soil properties. Stream restoration projects are ongoing in Fairfax County, Virginia in order to reconnect the channel to the floodplain and increase nutrient removal via microbially mediated processes. Ecological assessment of urban forests is also ongoing to understand the ecosystem services that urban forests provide. Using Illumina sequencing and qPCR, the bacterial and fungal communities were analyzed between stream riparian zones and reference sites, and between different forest qualities. Fungal communities differed significantly after stream restoration and between forest quality types. qPCR was also used to quantify denitrifying genes between restoration types. Post restoration sites had higher abundances of nirS, while reference sites were higher in nirK. The high quality forest sites were most colonized by arbuscular mycorrhizal fungi and were highest in ectomycorrhizal fungal sequences.
MICROBIAL BIOFILMS ON MICROPLASTICS: A LOOK INTO THE ESTUARINE PLASTISPHERE OF THE CHESAPEAKE BAY
(2021) Sosa , Ana Paula; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Microplastics are plastic particles that are smaller than 5 millimeters and are often found as pollution in our waterways. These polymer particles are globally distributed and are a direct result of human activity. Because of their rigidity and durability, microplastics are an ideal substrate for enhanced microbial growth and biofilm development. While microplastics have been studied in various contexts, only few studies have characterized the microbial communities on different types of plastic particles, but no study has been done in the estuarine water. In this study, we exposed three different types of plastics (polypropylene, polystyrene, and polylactic acid) to the water of Baltimore’s Inner Harbor, along with a non-plastic glass control. We used both in situ and in vitro incubations to understand the development of biofilm communities on microplastics. Microbial communities were analyzed based on the 16S rRNA gene sequences. We found that microbial composition on biofilm is distinct from that in the surrounding water, and different microplastic types have a minor impact on the composition of biofilm communities. The similarity between microbial communities on plastic and non-plastic particles suggests that surface supports rather than material types could be more critical for biofilm formation. Succession of microbial communities on the microplastics and interesting bacterial groups were described. Isolation and microscopic observations were also applied in this study. The presence of phototrophic organisms like filamentous cyanobacteria and Auxenochlorella on microplastic biofilms is interesting, and little is known about their contribution to carbon fixation in the ocean. Biofilms formed on microplastic surfaces could potentially affect the ecosystems via different mechanisms, including local nutrient cycling and the transportation of invasive or harmful species. As plastic production and mismanagement continues to be pervasive in our society, it is paramount that we include biofilm development into the framework of general ecology in order to truly understand the impact of plastic pollution and safeguard our ecosystems.
Viromics and biogeography of estuarine virioplankton
(2021) Sun, Mengqi; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Viruses are the most abundant biological entity in the ocean, and they can influence microbial mortality, evolution and biogeochemical cycles in marine ecosystems. Virioplankton communities in oceans have been studied extensively using viral metagenomics (viromics), but the estuarine viromes remain relatively unexplored. Estuaries are a complex and dynamic ecosystem. My dissertation is dedicated to understanding the composition and distribution of the virioplankton community in the Delaware Bay and Chesapeake Bay by investigating 16 viromes collected from these two bays. A total of 26,487 viral populations (contigs > 5kb) were identified in the two bays, establishing a high quality viromic dataset. The vast majority of the dominant viral populations are unclassified viruses. Viral sequences obtained from marine single cell genomes or long read single molecule sequencing comprised 13 of the top 20 most abundant viral populations, suggesting that we are still far from understanding the diversity of viruses in estuaries. Abundant viral populations (top 5,000) are significantly different between the Delaware Bay and Chesapeake Bay, indicating a strong niche adaptation of the viral community to each estuary. Surprisingly, no clear spatiotemporal patterns were observed for the viral community based on water temperature and salinity. The composition of known viruses (i.e. phages infecting Acinetobacter, Puniceispirillum, Pelagibacter, Synechococcus, Prochlorococcus, etc.) appeared to be relatively consistent across a wide range of salinity gradients and different seasons. Overall, the estuarine viral community is distinct from that in the ocean according to the composition of known viruses. N4-like viruses belong to a newly established viral family and have been isolated from diverse bacterial groups. Marine N4-like viruses were first found in the Chesapeake Bay, but little is known about their biogeographic pattern in the estuarine environment. N4-like viruses were confirmed to be rare in the estuary, and relatively more abundant in the samples from lower water temperature. Viruses which infect SAR11 bacteria (pelagiphage) are one of most abundant viral groups in the open ocean. We found that the abundance and community profile of pelagiphage in the estuaries is similar to that in the open ocean, and has no correlation with environmental factors.

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