Heavy Metal Tolerance of Microorganisms Isolated from Coastal Marine Sediments and Their Lead Removal Potential.

In this study, 338 microorganisms, comprising 271 bacteria and 67 fungi, were isolated from sediment samples collected from underexplored Pacific and Caribbean regions of Colombia. Screening trials were conducted on selected strains (n = 276) to assess their tolerance to cadmium (Cd2+), lead (Pb2+), and zinc (Zn2+), leading to the identification of six bacteria capable of withstanding 750 mg·L-1 of each heavy metal ion. Three promising microorganisms, identified as Enterobacter sp. INV PRT213, Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216 were selected for lead removal experiments using LB broth medium supplemented with 400 mg·L-1 Pb2+. Among these, Pseudomonas sp. INV PRT215 exhibited significant potential, removing 49% of initial Pb2+ after 240 min of exposure (16.7 g wet biomass·L-1, pH 5, 30 °C). Infrared spectra of Pb-exposed biomass showed changes in functional groups, including carbonyl groups of amides, carboxylate, phosphate, hydroxyl, and amine groups, compared to the not-exposed control. These changes suggested interactions between the metal and functional groups in the biomass. The findings of this study highlight the potential of microorganisms derived from coastal marine environments as promising candidates for future applications in bioremediation of polluted environments contaminated with heavy metals.

Advances in microbial pretreatment for biorefining of perennial grasses.

Perennial grasses are potentially abundant sources of biomass for biorefineries, which can produce high yields with low input requirements, and many added environmental benefits. However, perennial grasses are highly recalcitrant to biodegradation and may require pretreatment before undergoing many biorefining pathways. Microbial pretreatment uses the ability of microorganisms or their enzymes to deconstruct plant biomass and enhance its biodegradability. This process can enhance the enzymatic digestibility of perennial grasses, enabling saccharification with cellulolytic enzymes to produce fermentable sugars and derived fermentation products. Similarly, microbial pretreatment can increase the methanation rate when the grasses are used to produce biogas through anaerobic digestion. Microorganisms can also increase the digestibility of the grasses to improve their quality as animal feed, enhance the properties of grass pellets, and improve biomass thermochemical conversion. Metabolites produced by fungi or bacteria during microbial pretreatment, such as ligninolytic and cellulolytic enzymes, can be further recovered as added-value products. Additionally, the action of the microorganisms can release chemicals with commercialization potential, such as hydroxycinnamic acids and oligosaccharides, from the grasses. This review explores the recent advances and remaining challenges in using microbial pretreatment for perennial grasses with the goal of obtaining added-value products through biorefining. It emphasizes recent trends in microbial pretreatment such as the use of microorganisms as part of microbial consortia or in unsterilized systems, the use and development of microorganisms and consortia capable of performing more than one biorefining step, and the use of cell-free systems based on microbial enzymes. KEY POINTS: • Microorganisms or enzymes can reduce the recalcitrance of grasses for biorefining • Microbial pretreatment effectiveness depends on the grass-microbe interaction • Microbial pretreatment can generate value added co-products to enhance feasibility.

The state of the art of marine natural products in Colombia / El estado del arte de los productos naturales marinos en Colombia / O estado da arte dos produtos naturais marinhos na Colômbia

Abstract Marine Natural Products (MNPs) isolated from samples collected in Colombia have been an object of study since the early 1980's; however, this information is neither integrated nor compiled. This systematic review describes the articles published in scientific journals up to December 2019. 173 papers met the inclusion criteria of focusing on MNPs obtained from specimens collected from Colombian seas; all original papers written in English, Portuguese or Spanish. The selected papers were mostly authored by researchers from Colombian groups, with low interaction amongst themselves. 99.4% of the papers studied samples collected from the Caribbean Sea; 183 species were studied, mainly sponges and octocorals. In this study, 1,690 compounds (238 new ones) were reviewed, mainly diterpenes and sterol derivatives. Of the selected papers, 76.8% measured various biological activities, including antibiotic (34%) and anticancer (30%). These papers were published in 51 journals (74.6% were international). In conclusion, scientific work on natural marine products of Colombian origin has incremented over time. The most relevant opportunities to address and fill existing gaps comprise: exploring Pacific Ocean organisms and several of the misrepresented taxa; promoting strong interactions amongst the MNPs research groups, and accordingly with other areas of knowledge; and having the productive sector participate in MNPs research.

Resumen Los productos naturales marinos (PNM) aislados de muestras recolectadas en Colombia han sido estudiados desde principios de los años 1980, mas esta información no está integrada, ni recopilada. Esta revisión sistemática describe los artículos publicados hasta diciembre de 2019. 173 artículos cumplieron los criterios de inclusión de enfoque en PNM obtenidos de especímenes recolectados en mares colombianos; trabajos originales escritos en inglés, portugués o español. La mayoría de los artículos fueron escritos por investigadores de grupos colombianos, con poca interacción entre ellos. El 99,4% de los artículos estudiaban muestras recolectadas del mar Caribe. Se estudiaron 183 especies, especialmente esponjas y octocorales. Se identificaron 1690 compuestos (238 nuevos), principalmente diterpenos y derivados de esteroles. En el 76,8% de los artículos se midió alguna actividad biológica, principalmente antibiótica (34%) y anticancerígena (30%). Los artículos se publicaron en 51 revistas (74,6% internacionales). En conclusión, la investigación sobre los PNM de origen colombiano ha crecido con el tiempo. Algunas oportunidades para abordar las lagunas encontradas comprenden: explorar los organismos del océano Pacífico y los taxa poco estudiados; promover interacciones entre los grupos de investigación de los PNM y de otras áreas del conocimiento; e involucrar al sector productivo en la investigación de los PMN.

Resumo Os Produtos Naturais Marinhos (PNMs) isolados de amostras coletadas na Colômbia têm sido objeto de estudo desde a década de 1980; porém, esta informação não está integrada nem compilada. Esta revisão sistemática descreve os artigos publicados em revistas científicas até dezembro de 2019. 173 artigos atenderam aos critérios de inclusão de foco em PNMs obtidos de espécimes coletados em mares colombianos; artigos originais escritos em inglês, português ou espanhol. A maioria dos autores dos artigos eram pesquisadores de grupos colombianos, com baixa interação entre eles. 99,4% dos artigos estudavam amostras coletadas no Mar do Caribe. Foram estudadas 183 espécies, especialmente esponjas e octocorais. Nesta revisão, identificaram-se 1690 compostos (238 novos), principalmente diterpenos e derivados de esterol. 76,8% dos artigos mediram algumas atividades biológicas, incluindo antibiótica (34%) e anticancerígena (30%). Os artigos analisados foram publicados em 51 periódicos (74,6% internacionais). Em conclusão, o trabalho científico sobre PNM de origem colombiana cresceu ao longo do tempo. As oportunidades mais relevantes para preencher as lacunas existentes incluem: explorar organismos do Oceano Pacífico e os taxa pouco estudados; promover interação entre os grupos de pesquisa de PNMs e com grupos de outras áreas do conhecimento; e envolver o setor produtivo na pesquisa de PNMs.

Diversity of cultivable bacteria from deep-sea sediments of the Colombian Caribbean and their potential in bioremediation.

The diversity of deep-sea cultivable bacteria was studied in seven sediment samples of the Colombian Caribbean. Three hundred and fifty two marine bacteria were isolated according to its distinct morphological character on the solid media, then DNA sequences of the 16S rRNA were amplified to identify the isolated strains. The identified bacterial were arranged in three phylogenetic groups, Firmicutes, Proteobacteria, and Actinobacteria, with 34 different OTUs defined at ≥ 97% of similarity and 70 OTUs at ≥ 98.65%, being the 51% Firmicutes, 34% Proteobacteria and 15% Actinobacteria. Bacillus and Fictibacillus were the dominant genera in Firmicutes, Halomonas and Pseudomonas in Proteobacteria and Streptomyces and Micromonospora in Actinobacteria. In addition, the strains were tested for biosurfactants and lipolytic enzymes production, with 120 biosurfactant producing strains (mainly Firmicutes) and, 56 lipolytic enzymes producing strains (Proteobacteria). This report contributes to the understanding of the diversity of the marine deep-sea cultivable bacteria from the Colombian Caribbean, and their potential application as bioremediation agents.

In vitro biological activity of extracts from marine bacteria cultures against Toxoplasma gondii and Mycobacterium tuberculosis.

AIMS: To evaluate the biological activity of extracts from cultures of marine bacteria against Toxoplasma gondii and Mycobacterium tuberculosis. METHODS AND RESULTS: Ethyl acetate extracts obtained from seven marine bacteria were tested against T. gondii GFP-RH and M. tuberculosis H37Rv. The cytotoxicity on HFF-1 cells was measured by a microplate resazurin fluorescent approach, and the haemolytic activity was determined photometrically. The extracts from Bacillus sp. (INV FIR35 and INV FIR48) affected the tachyzoite viability. The extracts from Bacillus, Pseudoalteromonas, Streptomyces and Micromonospora exhibited effects on infection and proliferation processes of parasite. Bacillus sp. INV FIR48 extract showed an minimum inhibitory concentration value of 50 µg ml-1 against M. tuberculosis H37Rv. All the extracts exhibited relatively low toxicity to HFF-1 cells and the primary culture of erythrocytes, except Bacillus sp. INV FIR35, which decreased cell viability under 20%. Liquid chromatography coupled to mass spectrometry analysis of the most active bacterial extract Bacillus sp. INV FIR48 showed the presence of peptide metabolites related to surfactin. CONCLUSIONS: The extract from culture of deep-sea Bacillus sp. INV FIR48 showed anti-T. gondii and anti-tuberculosis (TB) biological activity with low cytotoxicity. In addition, peptide metabolites were detected in the extract. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoplasmosis and TB are among the most prevalent diseases worldwide, and the current treatment drugs exhibit side effects. This study confirm that marine bacteria are on hand sources of anti-infective natural products.

Dereplication of antimicrobial biosurfactants from marine bacteria using molecular networking.

Biosurfactants are amphiphilic surface-active molecules of microbial origin principally produced by hydrocarbon-degrading bacteria; in addition to the bioremediation properties, they can also present antimicrobial activity. The present study highlights the chemical characterization and the antimicrobial activities of biosurfactants produced by deep-sea marine bacteria from the genera Halomonas, Bacillus, Streptomyces, and Pseudomonas. The biosurfactants were extracted and chemically characterized through Chromatography TLC, FT-IR, LC/ESI-MS/MS, and a metabolic analysis was done through molecular networking. Six biosurfactants were identified by dereplication tools from GNPS and some surfactin isoforms were identified by molecular networking. The half-maximal inhibitory concentration (IC50) of biosurfactant from Halomonas sp. INV PRT125 (7.27 mg L-1) and Halomonas sp. INV PRT124 (8.92 mg L-1) were most effective against the pathogenic yeast Candida albicans ATCC 10231. For Methicillin-resistant Staphylococcus aureus ATCC 43300, the biosurfactant from Bacillus sp. INV FIR48 was the most effective with IC50 values of 25.65 mg L-1 and 21.54 mg L-1 for C. albicans, without hemolytic effect (< 1%), and non-ecotoxic effect in brine shrimp larvae (Artemia franciscana), with values under 150 mg L-1, being a biosurfactant promising for further study. The extreme environments as deep-sea can be an important source for the isolation of new biosurfactants-producing microorganisms with environmental and pharmaceutical use.

Survival of eastern oysters Crassostrea virginica from three lines following experimental challenge with bacterial pathogens.

Shellfish production is often affected by bacterial pathogens that cause high losses in hatcheries and nurseries. We evaluated the relative survival of larvae and juveniles of 3 Crassostrea virginica oyster lines: (1) GHP, a Rhode Island line; (2) NEHY, a line resistant to dermo and multinucleated sphere X diseases; and (3) FLOWERS, a line resistant to Roseovarius oyster disease, experimental challenge with Vibrio spp. isolates RE22 and RE101, causative agents of bacillary necrosis in Pacific oyster larvae, and the type strain of Roseovarius crassostreae, causative agent of Roseovarius oyster disease. All of the isolates were able to induce significant mortalities in oyster larvae and juveniles. Susceptibility to bacterial challenge in larvae was significantly higher at 25 degrees C than at 20 degrees C. Susceptibility decreased with oyster age; mean survival time ranged from 24 h in oyster larvae to more than 6 wk in juveniles. Significant differences in susceptibility to bacterial challenge were observed between oyster lines; NEHY was the most resistant line overall. Extracellular products (ECPs) from Vibrio sp. RE22 and R. crassostreae, as well as viable bacteria, were toxic to hemocytes from the 3 oyster lines, suggesting that ECPs are involved in pathogenesis and that external and mucosal barriers to infection are major contributors to resistance to bacterial challenge. These protocols will be useful in the elucidation of mechanisms of bacterial pathogenesis and resistance to infection in oysters.

Numerical quantification of Perkinsus marinus in the American oyster Crassostrea virginica (Gmelin, 1791) (Mollusca: Bivalvia) by modern stereology.

Species of Perkinsus are responsible for high mortalities of bivalve molluscs world-wide. Techniques to accurately estimate parasites in tissues are required to improve understanding of perkinsosis. This study quantifies the number and tissue distribution of Perkinsus marinus in Crassostrea virginica by modern stereology and immunohistochemistry. Mean total number of trophozoites were (mean +/- SE) 11.80 +/- 3.91 million and 11.55 +/- 3.88 million for the optical disector and optical fractionator methods, respectively. The mean empirical error between both stereological approaches was 3.8 +/- 1.0%. Trophozoites were detected intracellularly in the following tissues: intestine (30.1%), Leydig tissue (21.3%), hemocytes (14.9%), digestive gland (11.4%), gills (6.1%), connective tissues (5.7%), gonads (4.1%), palps (2.2%), muscle (1.9%), mantle connective (0.8%), pericardium (0.7%), mantle epithelium (0.1%), and heart (0.1%). The remaining 0.6% were found extracellularly. Percentages of trophozoite stages were (mean +/- SE): large, log-phase trophonts, i.e., signet rings, 97.0 +/- 1.2%; meronts, 2.0 +/- 0.9%; clusters of small, log-phase trophonts, i.e., merozoites, 1.0 +/- 0.5%. Levels of infection in hemocytes and Leydig tissue were representative of total parasite intensity. These techniques are a powerful tool to follow parasite distribution and invasion, and to further explore mechanisms of Perkinsus spp. pathogenesis in bivalves.

Role of nitric oxide in the defenses of Crassostrea virginica to experimental infection with the protozoan parasite Perkinsus marinus.

We investigated the role of nitric oxide (NO) in the responses of the Eastern oyster, Crassostrea virginica, to the protozoan parasite Perkinsus marinus, causative agent of Dermo disease. P. marinus induced a slight but significant increase in NO production by oyster hemocytes in vitro, comparable to the increase induced by the immune stimulants phorbol myristrate acetate (PMA) and lipopolysaccharide (LPS). P. marinus also activated the NO response in oysters in vivo, as shown by induction of a protein reacting with a universal NO synthase (NOS) antibody in hemocytes and the presence of high levels of nitrite in plasma. Treatment of experimentally infected oysters with the NOS inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) resulted in a transient decrease in NO levels in oyster plasma and a significant increase in the number of parasites at early time points after infection. The NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) caused a significant inhibition in the proliferation of P. marinus cultured cells after 24 h of incubation. These results indicate that NO has a role in decreasing parasite loads at early time points after infection.