RESUMO
Large-scale open microalgae cultivation has tremendous potential to make a significant contribution to replacing petroleum-based fuels with biofuels. Open algal cultures are unavoidably inhabited with a diversity of microbes that live on, influence, and shape the fate of these ecosystems. However, there is little understanding of the resilience and stability of the microbial communities in engineered semicontinuous algal systems. To evaluate the dynamics and resilience of the microbial communities in microalgae biofuel cultures, we conducted a longitudinal study on open systems to compare the temporal profiles of the microbiota from two multigenerational algal cohorts, which include one seeded with the microbiota from an in-house culture and the other exogenously seeded with a natural-occurring consortia of bacterial species harvested from the Pacific Ocean. From these month-long, semicontinuous open microalga Nannochloropsis salina cultures, we sequenced a time-series of 46 samples, yielding 8804 operational taxonomic units derived from 9,160,076 high-quality partial 16S rRNA sequences. We provide quantitative evidence that clearly illustrates the development of microbial community is associated with microbiota ancestry. In addition, N. salina growth phases were linked with distinct changes in microbial phylotypes. Alteromonadeles dominated the community in the N. salina exponential phase whereas Alphaproteobacteria and Flavobacteriia were more prevalent in the stationary phase. We also demonstrate that the N. salina-associated microbial community in open cultures is diverse, resilient, and dynamic in response to environmental perturbations. This knowledge has general implications for developing and testing design principles of cultivated algal systems.
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Bactérias/classificação , Microalgas/microbiologia , Microbiota , Bactérias/genética , Bactérias/isolamento & purificação , Biocombustíveis , Biomassa , DNA Bacteriano/genética , Biblioteca Gênica , Oceano Pacífico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Estramenópilas/microbiologia , Microbiologia da ÁguaRESUMO
To fully understand the interactions of a pathogen with its host, it is necessary to analyze the RNA transcripts of both the host and pathogen throughout the course of an infection. Although this can be accomplished relatively easily on the host side, the analysis of pathogen transcripts is complicated by the overwhelming amount of host RNA isolated from an infected sample. Even with the read depth provided by second-generation sequencing, it is extremely difficult to get enough pathogen reads for an effective gene-level analysis. In this study, we describe a novel capture-based technique and device that considerably enriches for pathogen transcripts from infected samples. This versatile method can, in principle, enrich for any pathogen in any infected sample. To test the technique's efficacy, we performed time course tissue culture infections using Rift Valley fever virus and Francisella tularensis. At each time point, RNA sequencing (RNA-Seq) was performed and the results of the treated samples were compared with untreated controls. The capture of pathogen transcripts, in all cases, led to more than an order of magnitude enrichment of pathogen reads, greatly increasing the number of genes hit, the coverage of those genes, and the depth at which each transcript was sequenced.
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Francisella tularensis/genética , Francisella tularensis/fisiologia , Interações Hospedeiro-Patógeno , Vírus da Febre do Vale do Rift/genética , Vírus da Febre do Vale do Rift/fisiologia , Análise de Sequência de RNA/métodos , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Macrófagos/microbiologia , Macrófagos/virologia , Hibridização de Ácido Nucleico , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Viral/genéticaRESUMO
Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.
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Escherichia coli/genética , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Reação em Cadeia da Polimerase/métodos , Transcrição Reversa , Análise de Sequência de RNA/métodos , Sequência de Bases , Linhagem Celular Tumoral , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala/métodos , HumanosRESUMO
Loss of algal production from the crashes of algal mass cultivation systems represents a significant barrier to the economic production of microalgal-based biofuels. Current strategies for crash prevention can be too costly to apply broadly as prophylaxis. Bacteria are ubiquitous in microalgal mass production cultures, however few studies investigate their role and possible significance in this particular environment. Previously, we demonstrated the success of selected protective bacterial communities to save Microchloropsis salina cultures from grazing by the rotifer Brachionus plicatilis. In the current study, these protective bacterial communities were further characterized by fractionation into rotifer-associated, algal-associated, and free-floating bacterial fractions. Small subunit ribosomal RNA amplicon sequencing was used to identify the bacterial genera present in each of the fractions. Here, we show that Marinobacter, Ruegeria, and Boseongicola in algae and rotifer fractions from rotifer-infected cultures likely play key roles in protecting algae from rotifers. Several other identified taxa likely play lesser roles in protective capability. The identification of bacterial community members demonstrating protective qualities will allow for the rational design of microbial communities grown in stable co-cultures with algal production strains in mass cultivation systems. Such a system would reduce the frequency of culture crashes and represent an essentially zero-cost form of algal crop protection.
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Algal biofuels are a growing interest worldwide due to their potential in terms of sustainable greenhouse gas displacement and energy production. This article describes a comparative survey of biodiesel production and conversion yields of biodiesel via alkaline transesterification of acylglycerols extracted from the microalgae Thalassiosira pseudonana and Phaeodactylum tricornutum, grown under silicate or nitrate limitation, and that of model vegetable oils: soybean, and rapeseed oil. Acylglycerols were extracted with n-hexane and the total yield per biomass was determined by gravimetric assay. Under our conditions, the total acylglycerol yield from the microalgae studied was 13-18% of total dry weight. The biodiesel samples were analyzed using gas chromatography-flame ionization detector to determine quantitative information of residual glycerol, mono-, di-, and tri-acylglycerol concentrations in the biodiesel. All of the algal-based biodiesel demonstrated less mono-, di-, and tri-acylglycerol concentrations than the vegetable-based biodiesel under identical transesterification conditions. The fatty acid compositions of all the feedstock oils and their resultant biodiesel were also analyzed and reported. Based on the fatty acid methyl ester compositions of our samples we qualitatively assessed the suitability of the algal-derived biodiesel in terms of cetane number (CN), cold-flow properties, and oxidative stability.
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Biocombustíveis , Diatomáceas/metabolismo , Glicerídeos/análise , Glicerídeos/isolamento & purificação , Óleos de Plantas/química , Óleo de Soja/química , Cromatografia Gasosa , Diatomáceas/crescimento & desenvolvimento , Ácidos Graxos Monoinsaturados , Nitrogênio/metabolismo , Óleo de Brassica napus , Silicatos/metabolismoRESUMO
Economically successful microalgal mass cultivation is dependent on overcoming several barriers that contribute to the cost of production. The severity of these barriers is dependent on the market value of the final product. These barriers prevent the commercially viable production of algal biofuels but are also faced by any producers of any algal product. General barriers include the cost of water and limits on recycling, costs and recycling of nutrients, CO2 utilization, energy costs associated with harvesting and biomass loss due to biocontamination and pond crashes. In this paper, recent advances in overcoming these barriers are discussed.
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Microalgas , Biocombustíveis , Biomassa , Reciclagem , Águas ResiduáriasRESUMO
While algae demonstrate potential as a sustainable fuel source, low productivities limit the economic realization of algal biofuels. High-throughput strain engineering, omics-informed genome-scale modeling, and microbiome engineering are key technologies for enabling algal biofuels. High-throughput strain engineering efforts generate improved traits, including high biomass productivity and lipid content, in diverse algal species. Genome-scale models, constructed with the aid of omics data, provide insight into metabolic limitations and guide rational algal strain engineering efforts. As outdoor cultivation systems introduce exogenous organisms, microbiome engineering seeks to eliminate harmful organisms and introduce beneficial species. Optimizing algal biomass production and lipid content using these technologies may overcome the productivity barrier for the commercialization of algal biofuels.
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Biocombustíveis , Microalgas , Microalgas/genética , Microalgas/metabolismo , Biomassa , Plantas , LipídeosRESUMO
Outdoor cultivation of microalgae has promising potential for renewable bioenergy, but there is a knowledge gap on the structure and function of the algal microbiome that coinhabits these ecosystems. Here, we describe the assembly mechanisms, taxonomic structure, and metabolic potential of bacteria associated with Microchloropsis salina cultivated outdoors. Open mesocosms were inoculated with algal cultures that were either free of bacteria or coincubated with one of two different strains of alga-associated bacteria and were sampled across five time points taken over multiple harvesting rounds of a 40-day experiment. Using quantitative analyses of metagenome-assembled genomes (MAGs), we tracked bacterial community compositional abundance and taxon-specific functional capacity involved in algal-bacterial interactions. One of the inoculated bacteria (Alteromonas sp.) persisted and dispersed across mesocosms, whereas the other inoculated strain (Phaeobacter gallaeciensis) disappeared by day 17 while a taxonomically similar but functionally distinct Phaeobacter strain became established. The inoculated strains were less abundant than 6 numerically dominant newly recruited taxa with functional capacities for mutualistic or saprophytic lifestyles, suggesting a generalist approach to persistence. This includes a highly abundant unclassified Rhodobacteraceae species that fluctuated between 25% and 77% of the total community. Overall, we did not find evidence for priority effects exerted by the distinct inoculum conditions; all mesocosms converged with similar microbial community compositions by the end of the experiment. Instead, we infer that the 15 total populations were retained due to host selection, as they showed high metabolic potential for algal-bacterial interactions such as recycling alga-produced carbon and nitrogen and production of vitamins and secondary metabolites associated with algal growth and senescence, including B vitamins, tropodithietic acid, and roseobacticides. IMPORTANCE Bacteria proliferate in nutrient-rich aquatic environments, including engineered algal biofuel systems, where they remineralize photosynthates, exchange secondary metabolites with algae, and can influence system output of biomass or oil. Despite this, knowledge on the microbial ecology of algal cultivation systems is lacking, and the subject is worthy of investigation. Here, we used metagenomics to characterize the metabolic capacities of the predominant bacteria associated with the biofuel-relevant microalga Microchloropsis salina and to predict testable metabolic interactions between algae and manipulated communities of bacteria. We identified a previously undescribed and uncultivated organism that dominated the community. Collectively, the microbial community may interact with the alga in cultivation via exchange of secondary metabolites which could affect algal success, which we demonstrate as a possible outcome from controlled experiments with metabolically analogous isolates. These findings address the scalability of lab-based algal-bacterial interactions through to cultivation systems and more broadly provide a framework for empirical testing of genome-based metabolic predictions.
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Biocombustíveis , Microbiota , Biomassa , Metagenoma , SimbioseRESUMO
The ocean biota contains a vast reservoir of genomic diversity. Here we present the sequence and preliminary characterization of a protein that is a cadmium-containing carbonic anhydrase from the marine diatom Thalassiosira weissflogii. The existence of a cadmium enzyme in marine phytoplankton may indicate that there is a unique selection pressure for metalloenzymes in the marine environment, and our discovery provides a long-awaited explanation for the nutrient-like behaviour of cadmium in the oceans.
Assuntos
Cádmio/metabolismo , Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Diatomáceas/enzimologia , Fitoplâncton/enzimologia , Animais , Sítios de Ligação , Cádmio/farmacologia , Anidrases Carbônicas/genética , Diatomáceas/efeitos dos fármacos , Diatomáceas/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Biologia Marinha , Dados de Sequência Molecular , Fitoplâncton/efeitos dos fármacos , Fitoplâncton/genética , Água/químicaRESUMO
Open microalgal ponds used in industrial biomass production are susceptible to a number of biotic and abiotic environmental stressors (e.g., grazers, pathogens, pH, temperature, etc.) resulting in pond crashes with high economic costs. Identification of signature chemicals to aid in rapid, non-invasive, and accurate identification of the stressors would facilitate targeted and effective treatment to save the algal crop from a catastrophic crash. Specifically, we were interested in identifying volatile organic compounds (VOCs) that can be used to as an early diagnostic for algal crop damage. Cultures of Microchloropsis gaditana were subjected to two forms of algal crop damage: (1) active grazing by the marine rotifer, Brachionus plicatilis, or (2) repeated freeze-thaw cycles. VOCs emitted above the headspace of these algal cultures were collected using fieldable solid phase microextraction (SPME) fibers. An untargeted analysis and identification of VOCs was conducted using gas chromatography-mass spectrometry (GC-MS). Diagnostic VOCs unique to each algal crop damage mechanism were identified. Active rotifer grazing of M. gaditana was characterized by the appearance of carotenoid degradation products, including ß-cyclocitral and various alkenes. Freeze-thaw algae produced a different set of VOCs, including palmitoleic acid. Both rotifer grazing and freeze-thawed algae produced ß-ionone as a VOC, possibly suggesting a common stress-induced cellular mechanism. Importantly, these identified VOCs were all absent from healthy algal cultures of M. gaditana. Early detection of biotic or abiotic environmental stressors will facilitate early diagnosis and application of targeted treatments to prevent algal pond crashes. Thus, our work further supports the use of VOCs for monitoring the health of algal ponds to ultimately enhance algal crop yields for production of biofuel.
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Here, we report the annotated genome sequence for a heterokont alga from the class Xanthophyceae. This high-biomass-producing strain, Tribonema minus UTEX B 3156, was isolated from a wastewater treatment plant in California. It is stable in outdoor raceway ponds and is a promising industrial feedstock for biofuels and bioproducts.
RESUMO
Photosynthetic algae represent a large, diverse bioresource potential. Yellow-green algae of the genus Tribonema are candidates for production of biofuels and other bioproducts. We report on a filamentous isolate from an outdoor raceway polyculture growing on municipal reclaimed wastewater which we classified as T. minus. Over one year of cultivation in 3.5 m2 raceway ponds fed by reclaimed municipal wastewater, T. minus cultures were more productive than the native algal polycultures, with annual average productivities of 15.9 ± 0.3 and 13.4 ± 0.4 g/m2/day, respectively. The biochemical composition of T. minus biomass grown outdoors was constant year-round, with 28.3 ± 0.4% carbohydrates, 37.6 ± 0.7% proteins, and 6.1 ± 0.3% fatty acids (measured as methyl esters), with up to 4.0% of the valuable omega-3 eicosapentaenoic acid, on an ash-free dry-weight basis. In summary, T. minus was more productive, easier to harvest and produced higher quality biomass than the native polycultures.
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Microalgas , Estramenópilas , Biocombustíveis , Biomassa , LagoasRESUMO
Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if compensation for autofluorescence and background signals can be performed accurately. We demonstrate the importance of accurate autofluorescence modeling and the utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when Francisella novicida invade host macrophage cells. The resulting analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion, we present the spectral properties and cellular origin of macrophage autofluorescence.
Assuntos
Francisella tularensis/citologia , Processamento de Imagem Assistida por Computador/métodos , Macrófagos/microbiologia , Microscopia Confocal/métodos , Animais , Linhagem Celular , CamundongosRESUMO
Microalgae produce specific chemicals indicative of stress and/or death. The aim of this study was to perform non-destructive monitoring of algal culture systems, in the presence and absence of grazers, to identify potential biomarkers of incipient pond crashes. Here, we report ten volatile organic compounds (VOCs) that are robustly generated by the marine alga, Microchloropsis salina, in the presence and/or absence of the marine grazer, Brachionus plicatilis. We cultured M. salina with and without B. plicatilis and collected in situ volatile headspace samples using thermal desorption tubes over the course of several days. Data from four experiments were aggregated, deconvoluted, and chromatographically aligned to determine VOCs with tentative identifications made via mass spectral library matching. VOCs generated by algae in the presence of actively grazing rotifers were confirmed via pure analytical standards to be pentane, 3-pentanone, 3-methylhexane, and 2-methylfuran. Six other VOCs were less specifically associated with grazing but were still commonly observed between the four replicate experiments. Through this work, we identified four biomarkers of rotifer grazing that indicate algal stress/death. This will aid machine learning algorithms to chemically define and diagnose algal mass production cultures and save algae cultures from imminent crash to make biofuel an alternative energy possibility.
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New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five phages were engineered for nonlysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, killed P. aeruginosa PAO1 in liquid culture as well as in a waxworm (Galleria mellonella) model of infection.IMPORTANCE The antibiotic resistance crisis has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that computationally identified prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.
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Algae ponds used in industrial biomass production are susceptible to pathogen or grazer infestation, resulting in pond crashes with high economic costs. Current methods to monitor and mitigate unhealthy ponds are hindered by a lack of early indicators that precede culture crash. We used solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) to identify volatiles emitted from healthy and rotifer infested cultures of Microchloropsis salina. After 48 hours of algal growth, marine rotifers, Brachionus plicatilis, were added to the algae cultures and volatile organic compounds (VOC) were sampled from the headspace using SPME fibers. A GC-MS approach was used in an untargeted analysis of VOCs, followed by preliminary identification. The addition of B. plicatilis to healthy cultures of M. salina resulted in decreased algal cell numbers, relative to uninfected controls, and generated trans-ß-ionone and ß-cyclocitral, which were attributed to carotenoid degradation. The abundances of the carotenoid-derived VOCs increased with rotifer consumption of algae. Our results indicate that specific VOCs released by infected algae cultures may be early indicators for impending pond crashes, providing a useful tool to monitor algal biomass production and pond crash prevention.
Assuntos
Eutrofização , Lagoas/química , Compostos Orgânicos Voláteis/análise , Animais , Biomarcadores/análise , Ecologia , Biomarcadores Ambientais , Lagoas/microbiologia , Rotíferos , Compostos Orgânicos Voláteis/metabolismoRESUMO
Improving the economic feasibility is necessary for algae-based processes to achieve commercial scales for biofuels and bioproducts production. A closed-loop system for fusel alcohol production from microalgae biomass with integrated nutrient recycling was developed, which enables the reuse of nitrogen and phosphorus for downstream application and thus reduces the operational requirement for external major nutrients. Mixed fusel alcohols, primarily isobutanol and isopentanol were produced from Microchloropsis salina hydrolysates by an engineered E. coli co-culture. During the process, cellular nitrogen from microalgae biomass was converted into ammonium, whereas cellular phosphorus was liberated by an osmotic shock treatment. The formation of struvite from the liberated ammonium and phosphate, and the subsequent utilization of struvite to support M. salina cultivation was demonstrated. The closed loop system established here should help overcome one of the identified economic barriers to scale-up of microalgae production, and enhance the sustainability of microalgae-based chemical commodities production.
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Álcoois/metabolismo , Biomassa , Microalgas/metabolismo , Nutrientes/metabolismo , Estramenópilas/metabolismo , Escherichia coli/metabolismo , Microalgas/crescimento & desenvolvimento , Nitrogênio/metabolismo , Fósforo/metabolismo , Reciclagem , Estramenópilas/crescimento & desenvolvimento , Estruvita/metabolismoRESUMO
We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. This micro fluorescence-activated cell sorter (microFACS) uses an infrared laser to laterally deflect cells into a collection channel. Green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. To rule out potential photoinduced cell damage during optical deflection, we investigated the response of mouse macrophage to brief exposures (<4 ms) of focused 1064-nm laser light (9.6 W at the sample). We found no significant difference in viability, cell proliferation, activation state, and functionality between infrared-exposed and unexposed cells. Activation state was measured by the phosphorylation of ERK and nuclear translocation of NF-kappaB, while functionality was assessed in a similar manner, but after a lipopolysaccharide challenge. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida. A total of 10,738 infected cells were sorted at a throughput of 11 cells/s with 93% purity and 39% recovery.
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Separação Celular/métodos , Diagnóstico por Imagem , Francisella tularensis/efeitos da radiação , Macrófagos/efeitos da radiação , Microfluídica/métodos , Pinças Ópticas , Animais , Núcleo Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Fluorescência , Corantes Fluorescentes , Francisella tularensis/imunologia , Francisella tularensis/metabolismo , Proteínas de Fluorescência Verde , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos da radiação , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , NF-kappa B/metabolismo , Fosforilação/efeitos da radiação , Transporte Proteico , Transdução de Sinais , Tularemia/imunologiaRESUMO
Evidence indicates that nurses inconsistently engage in evidence-based practice (EBP). This cross-sectional study of 402 nurses at a medical-surgical hospital identifies strategies for augmenting EBP. Nurses' EBP beliefs scores were higher than their EBP implementation scores. Those with baccalaureate/postgraduate degrees had higher EBP beliefs and implementation scores than those with associate degrees or diplomas. Bedside or direct care nurses were less likely to have baccalaureate/higher degrees and had lower EBP beliefs and implementation scores than did those nurses not serving in direct care roles.
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Enfermagem Baseada em Evidências/estatística & dados numéricos , Conhecimentos, Atitudes e Prática em Saúde , Recursos Humanos de Enfermagem Hospitalar/educação , Desenvolvimento de Pessoal , Adulto , Atitude do Pessoal de Saúde , Estudos Transversais , Educação em Enfermagem , Educação Continuada em Enfermagem , Feminino , Humanos , Internet , Masculino , Cultura Organizacional , Inquéritos e QuestionáriosRESUMO
Open microalgae cultures host a myriad of bacteria, creating a complex system of interacting species that influence algal growth and health. Many algal microbiota studies have been conducted to determine the relative importance of bacterial taxa to algal culture health and physiological states, but these studies have not characterized the interspecies relationships in the microbial communities. We subjected Nanochroloropsis salina cultures to multiple chemical treatments (antibiotics and quorum sensing compounds) and obtained dense time-series data on changes to the microbial community using 16S gene amplicon metagenomic sequencing (21,029,577 reads for 23 samples) to measure microbial taxa-taxa abundance correlations. Short-term treatment with antibiotics resulted in substantially larger shifts in the microbiota structure compared to changes observed following treatment with signaling compounds and glucose. We also calculated operational taxonomic unit (OTU) associations and generated OTU correlation networks to provide an overview of possible bacterial OTU interactions. This analysis identified five major cohesive modules of microbiota with similar co-abundance profiles across different chemical treatments. The Eigengenes of OTU modules were examined for correlation with different external treatment factors. This correlation-based analysis revealed that culture age (time) and treatment types have primary effects on forming network modules and shaping the community structure. Additional network analysis detected Alteromonadeles and Alphaproteobacteria as having the highest centrality, suggesting these species are "keystone" OTUs in the microbial community. Furthermore, we illustrated that the chemical tropodithietic acid, which is secreted by several species in the Alphaproteobacteria taxon, is able to drastically change the structure of the microbiota within 3 h. Taken together, these results provide valuable insights into the structure of the microbiota associated with N. salina cultures and how these structures change in response to chemical perturbations.