Contrasting patterns of carbon cycling and dissolved organic matter processing in two phytoplankton–bacteria communities

Microbial consumption of phytoplankton-derived organic carbon in the pelagic food web is an important component of the global C cycle. We studied C cycling in two phytoplankton–bacteria systems (non-axenic cultures of a dinoflagellate Apocalathium malmogiense and a cryptophyte Rhodomonas marina) in two complementary experiments. In the first experiment we grew phytoplankton and bacteria in nutrient-replete conditions and followed C processing at early exponential growth phase and twice later when the community had grown denser. Cell-specific primary production and total community respiration were up to 4 and 7 times higher, respectively, in the A. malmogiense treatments. Based on the optical signals, accumulating dissolved organic C (DOC) was degraded more in the R. marina treatments, and the rate of bacterial production to primary production was higher. Thus, the flow of C from phytoplankton to bacteria was relatively higher in R. marina treatments than in A. malmogiense treatments, which was further supported by faster 14C transfer from phytoplankton to bacterial biomass. In the second experiment we investigated consumption of the phytoplankton-derived DOC by bacteria. DOC consumption and transformation, bacterial production, and bacterial respiration were all higher in R. marina treatments. In both experiments A. malmogiense supported a bacterial community predominated by bacteria specialized in the utilization of less labile DOC (class Bacteroidia), whereas R. marina supported a community predominated by copiotrophic Alpha- and Gammaproteobacteria. Our findings suggest that large dinoflagellates cycle relatively more C between phytoplankton biomass and the inorganic C pool, whereas small cryptophytes direct relatively more C to the microbial loop.

Assessing the Viability of Commercial Media for the Mass Culture of Chaetoceros muelleri

The microalgae Chaetoceros muelleri is considered a highly nutritious feed for the cultured larvae of the tropical sea cucumber Holothuria scabra. Due to the cost of analytical grade culture media used in the production of C. muelleri, there is a need to evaluate cheap alternative commercial media to decrease the cost of producing quality live microalgal food. In this study, two different indoor batch culture systems (1 L glass bottles and 10 L plastic carboys) were used to evaluate the effectiveness of two conventional (modified F/2 and Walne’s) and one commercial (Epizyme AGP complete) microalgal culture media. Results of the 1 L glass bottle experiment showed that the peak cell density of C. muelleri in AGP (1,241 ± 116 x 104 cells ml-1) was not significantly different from the modified F/2 (1,584 ± 41 x 104 cells ml-1) and Walne’s medium (1,319 ± 162 x 104 cells ml-1) (Kruskal-Wallis test, p=0.78). Likewise, in the plastic carboy experiment, the maximum cell density of C. muelleri in Walne’s medium (750 ± 144 x 104 cells ml-1) and F/2 medium (653 ± 79 x 104 cells ml-1) were higher, but not significantly different from AGP (496 ± 184 x 104 cells ml-1) (Kruskal-Wallis test, p=0.43). The highest growth rate in the glass bottle cultures was the modified F/2 (0.38 div day-1), while AGP was the lowest (0.34 div. day-1). On the other hand, in carboy culture, AGP was higher (0.17 div.day-1) compared to modified F/2 (0.15 div. day-1) and Walne’s medium (0.13 div. day-1). The exponential growth phase was similar in the glass bottles, while in the carboy, the exponential phase was reached at a shorter time in the AGP treatment than those in the modified F/2 and Walne’s media. The findings showed that AGP medium is an adequate alternative to replace the conventional media (modified F/2 and Walne’s) during the secondary stock culture for C. muelleri. The viability of using cheaper and more readily available commercial AGP media for the indoor culture production of C. muelleri can contribute to cost-effective scaling-up of the hatchery production of quality H. scabra larvae and early juveniles.

Impact of excluding nursing home COVID-19 cases when assessing the relationship between county-level social distancing behavior and COVID-19 cases across the US during the early phase of the pandemic, February 2020-May 2020

Objectives To conduct a cross-sectional nationwide study examining how exclusion of nursing home COVID-19 cases influences the association between county level social distancing behavior and COVID-19 cases throughout the US during the early phase of the pandemic (February 2020-May 2020). Methods Using county-level COVID-19 data and social distancing metrics from tracked mobile devices, we investigated the impact social distancing had on a county’s total COVID-19 cases (cases/100,000 people) between when the first COVID-19 case was confirmed in a county and May 31st, 2020 when most statewide social distancing measures were lifted, representing the pandemic’s exponential growth phase. We created a mixed-effects negative binomial model to assess how implementation of social distancing measures when they were most stringent (March 2020-May 2020) influenced total COVID-19 cases while controlling for social distancing and COVID-19 related covariates in two scenarios: (1) when COVID-19 nursing home cases are not excluded from total COVID-19 cases and (2) when these cases are excluded. Model findings were compared to those from February 2020, a baseline when social distancing measures were not in place. Marginal effects at the means were generated to further isolate the influence of social distancing on COVID-19 from other factors and determine total COVID-19 cases during March 2020-May 2020 for the two scenarios. Results Regardless of whether nursing home COVID-19 cases were excluded from total COVID-19 cases, a 1% increase in average % of mobile devices leaving home was significantly associated with a 5% increase in a county’s total COVID-19 cases between March 2020-May 2020 and about a 2.5% decrease in February 2020. When the influence of social distancing was separated from other factors, the estimated total COVID-19 cases/100,000 people was comparable throughout the range of social distancing values (25%-45% of mobile phone devices leaving home between March 2020-May 2020) when nursing home COVID-19 cases were not excluded (25% of mobile phones leaving home: 163.84 cases/100,000 people (95% CI: 121.81, 205.86), 45% of mobile phones leaving home: 432.79 cases/100,000 people (95% CI: 256.91, 608.66)) and when they were excluded (25% of mobile phones leaving home: 149.58 cases/100,000 people (95% CI: 111.90, 187.26), 45% of mobile phones leaving home: 405.38 cases/100,000 people (95% CI: 243.14, 567.62)). Conclusions Exclusion of nursing home COVID-19 cases from total COVID-19 case counts has little impact when estimating the relationship between county-level social distancing and preventing COVID-19 cases with additional research needed to see whether this finding is also observed for COVID-19 growth rates and mortality.

Gene expression analysis of Alcaligenes faecalis during induction of heterotrophic nitrification

Alcaligenes faecalis is a heterotrophic nitrifying bacterium that oxidizes ammonia and generates nitrite and nitrate. When A. faecalis was cultivated in a medium containing pyruvate and ammonia as the sole carbon and nitrogen sources, respectively, high concentrations of nitrite accumulated in the medium whose carbon/nitrogen (C/N) ratio was lower than 10 during the exponential growth phase, while the accumulation was not observed in the medium whose C/N ratio was higher than 15. Comparative transcriptome analysis was performed using nitrifying and non-nitrifying cells of A. faecalis cultivated in media whose C/N ratios were 5 and 20, respectively, to evaluate the fluctuations of gene expression during induction of heterotrophic nitrification. Expression levels of genes involved in primary metabolism did not change significantly in the cells at the exponential growth phase under both conditions. We observed a significant increase in the expression levels of four gene clusters: pod cluster containing the gene encoding pyruvic oxime dioxygenase (POD), podh cluster containing the gene encoding a POD homolog (PODh), suf cluster involved in an iron-sulfur cluster biogenesis, and dnf cluster involved in a novel hydroxylamine oxidation pathway in the nitrifying cells. Our results provide valuable insight into the biochemical mechanism of heterotrophic nitrification.

Metabolic Flux Analysis of VERO Cells under Various Culture Conditions

Although the culture of VERO cells in bioreactors is an important industrial bioprocess for the production of viruses and vaccines, surprisingly few reports on the analysis of the flux distribution in the cell metabolism have been published. In this study, an attempt is made to fill this gap by providing an analysis of relatively simple metabolic networks, which are constructed to describe the cell behavior in different culture conditions, e.g., the exponential growth phase (availability of glucose and glutamine), cell growth without glutamine, and cell growth without glucose and glutamine. The metabolic networks are kept as simple as possible in order to avoid underdeterminacy linked to the lack of extracellular measurements, and a unique flux distribution is computed in each case based on a mild assumption that the macromolecular composition of the cell is known. The result of this computation provides some insight into the metabolic changes triggered by the culture conditions, which could support the design of feedback control strategies in fed batch or perfusion bioreactors where the lactate concentration is measured online and regulated by controlling the delivery rates of glucose and, possibly, of some essential amino acids.

Biodesulfurization of Dibenzothiophene and Its Alkylated Derivatives in a Two-Phase Bubble Column Bioreactor by Resting Cells of Rhodococcus erythropolis IGTS8

Biodesulfurization (BDS) is considered a complementary technology to the traditional hydrodesulfurization treatment for the removal of recalcitrant sulfur compounds from petroleum products. BDS was investigated in a bubble column bioreactor using two-phase media. The effects of various process parameters, such as biocatalyst age and concentration, organic fraction percentage (OFP), and type of sulfur compound—namely, dibenzothiophene (DBT), 4-methyldibenzothiophene (4-MDBT), 4,6-dimethyldibenzothiophene (4,6-DMDBT), and 4,6-diethyldibenzothiophene (4,6-DEDBT)—were evaluated, using resting cells of Rhodococcus erythropolis IGTS8. Cells derived from the beginning of the exponential growth phase of the bacterium exhibited the highest biodesulfurization efficiency and rate. The biocatalyst performed better in an OFP of 50% v/v. The extent of DBT desulfurization was dependent on cell concentration, with the desulfurization rate reaching its maximum at intermediate cell concentrations. A new semi-empirical model for the biphasic BDS was developed, based on the overall Michaelis-Menten kinetics and taking into consideration the deactivation of the biocatalyst over time, as well as the underlying mass transfer phenomena. The model fitted experimental data on DBT consumption and 2-hydroxibyphenyl (2-HBP) accumulation in the organic phase for various initial DBT concentrations and different organosulfur compounds. For constant OFP and biocatalyst concentration, the most important parameter that affects BDS efficiency seems to be biocatalyst deactivation, while the phenomenon is controlled by the affinities of biodesulfurizing enzymes for the different organosulfur compounds. Thus, desulfurization efficiency decreased with increasing initial DBT concentration, and in inverse proportion to increases in the carbon number of alkyl substituent groups.

Growth and Nutrient Uptake Characteristics of Heterosigma akashiwo (Raphidophyceae) under Nitrogen and Phosphorus Concentrations in the East China Sea

Heterosigma akashiwo is classified as a harmful algal bloom (HAB) species that frequently occurs in eutrophic coastal waters and results in the contamination and mortality of fish and shellfish. The growth of H. akashiwo in four phosphate and nitrate concentration scenarios, representing the observed nutrient concentration ranges in the East China Sea (ECS), was evaluated to further understand the effect of nutrient concentrations on H. akashiwo blooms. The specific growth rate in the exponential growth phase (µ′) and the maximum cell density were lower (17–21% and 41%, respectively) under low phosphorus concentration scenarios, compared to the rates observed under high phosphorus concentration scenarios. The cellular nitrogen-to-phosphorus ratios of H. akashiwo were influenced by the initially supplied N:P ratio and the allocation strategy employed. Phosphorus concentration had a greater influence on the total growth of H. akashiwo than nitrate did, within the natural nutrient conditions of the ECS. These results could serve as a reference for coastal water management and marine ecological management and may be useful for further studies on the simulation and prediction of H. akashiwo blooms, particularly in the ECS.

1249. Metabolomic Profile of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA) in Latin-American MRSA Isolates

Background Vancomycin (VAN) is a first-line therapeutic option in severe infections caused by MRSA in Latin-America (LA). Development of reduced susceptibility to VAN has been associated with multiple changes in genes encoding pathways for cell wall metabolism and envelope stress responses. Nevertheless, a detailed and coherent mechanistic model to explain the phenotype remains elusive. To gain further insights into the hVISA phenotype, we sought to explore the metabolomic profile of hVISA isolates from LA. Methods The undirected profile of intracellular S. aureus metabolites was analysed in four clinical isolates (two hVISA and two VSSA [Vancomycin susceptible S. aureus]) belonging to the Chilean/Cordobes clone-ST5 (the predominant hVISA lineage in LA), and two reference strains Mu3 and N315. The metabolites were obtained in mid-exponential growth phase in trypticase soy broth in five independent replicates. The metabolites were determined by reverse phase liquid chromatography and hydrophilic interaction. The metabolic profile was determined by variable importance in the projection score (VIP > 1). The differences between hVISA and VSSA were maximized by orthogonal partial least squares discriminant analysis (OPLS-DA) and the affected metabolic pathways were identified with MetaboAnalyst. Results Among the differences identified in the metabolic profiles of hVISA respect to VSSA, 69 metabolites were relevant. Of these, 47 were fatty acids (including glycerol), 7 amino acids and 6 nucleosides (Table 1). These changes mainly impact the biosynthesis of amino acids derived from pyruvate since tyrosine, valine and leucine, had a reduction of 34%, 57% and 41% in hVISA compared to VSSA, respectively, which suggests alterations of the acid cycle tricarboxylic (TCA). Additionally, a reduction in purine and pyrimidine metabolism in hVISA was identified with reduction of nucleosides and dinucleotides derived from the pentose phosphate pathway. Table 1. Metabolites with higher VIP scores in the comparison of the hVISA and VSSA profile Conclusion We were able to observe metabolic alterations in TCA, pentose phosphate pathway and purine intermediates in hVISA-ST5 isolates. Our results support that gluconeogenesis and biosynthesis of carbohydrates and nucleic acids are the main pathways involved in the reduced susceptibility to VAN as reported in VISA isolates. Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support) Lorena Diaz, PhD , Nothing to disclose

Superoxide Production by the Red Tide-Producing Chattonella marina Complex (Raphidophyceae) Correlates with Toxicity to Aquacultured Fishes

The marine raphidophyte Chattonella marina complex forms red tides, causing heavy mortalities of aquacultured fishes in temperate coastal waters worldwide. The mechanism for Chattonella fish mortality remains unresolved. Although several toxic chemicals have been proposed as responsible for fish mortality, the cause is still unclear. In this study, we performed toxicity bioassays with red sea bream and yellowtail. We also measured biological parameters potentially related to ichthyotoxicity, such as cell size, superoxide (O2•−) production, and compositions of fatty acids and sugars, in up to eight Chattonella strains to investigate possible correlations with toxicity. There were significant differences in moribundity rates of fish and in all biological parameters among strains. One strain displayed no ichthyotoxicity even at high cell densities. Strains were categorized into three groups based on cell length, but this classification did not significantly correlate with ichthyotoxicity. O2•− production differed by a factor of more than 13 between strains at the late exponential growth phase. O2•− production was significantly correlated with ichthyotoxicity. Differences in fatty acid and sugar contents were not related to ichthyotoxicity. Our study supports the hypothesis that superoxide can directly or indirectly play an important role in the Chattonella-related mortality of aquacultured fishes.

Early-life exposure to Ivermectin alters long-term growth and disease susceptibility

Ivermectin is a broad-spectrum antiparasitic medicine, which is often used as a treatment for parasites or as a prophylaxis. While studies have looked at the long-term effects of Ivermectin on helminths, studies have not considered the long-term impacts of this treatment on host health or disease susceptibility. Here, we tracked the effects of early life Ivermectin treatment in Cuban tree frogs (Osteopilus septentrionalis) on growth rates, mortality, metabolically expensive organ size, and susceptibility to Batrachochytrium dendrobatidis (Bd) infection. One year after exposure, there was no effect of Ivermectin exposure on frog mass (X21 = 0.904, p = 0.34), but when tracked through the exponential growth phase (~2.5 years) the Ivermectin exposed individuals had lower growth rates and were ultimately smaller (X21 = 7.78, p = 0.005; X21 = 5.36, p = 0.02, respectively). These results indicate that early life exposure is likely to have unintended impacts on organismal growth and potentially reproductive fitness. Additionally, we exposed frogs to Bd, a pathogenic fungus that has decimated amphibian populations globally, and found early life exposure to Ivermectin decreased disease susceptibility (disease load: X21 = 17.57, p = 0.0002) and prevalence (control: 55%; Ivermectin: 22%) over 2 years after exposure. More research is needed to understand the underlying mechanism behind this phenomenon. Given that Ivermectin exposure altered disease susceptibility, proper controls should be implemented when utilizing this drug as an antiparasitic treatment in research studies.