Strain specific Starmerella bacillaris and Saccharomyces cerevisiae interactions in mixed fermentations.

AIMS: Yeast interactions have a key role in the definition of the chemical profile of the wines. For this reason, winemakers are increasingly interested in mixed fermentations, employing Saccharomyces cerevisiae and non-Saccharomyces strains. However, the outcome of mixed fermentations is often contradictory because there is a great variability among strains within species. Previously, it was demonstrated that the loss of culturability of Starmerella bacillaris in mixed fermentations with S. cerevisiae was due to the physical contact between cells. Therefore, to further explore previous observations, the interaction mechanisms among different strains of Starm. bacillaris and S. cerevisiae during mixed fermentations were investigated. METHODS AND RESULTS: Fermentations were conducted under conditions that allow physical contact between cells (flasks) but also using a double-compartment fermentation system in which cells of both species were kept separate. The role of competition for nutrients and antimicrobial compounds production on yeast-yeast interaction mechanisms was also investigated. Three Starm. bacillaris and three S. cerevisiae strains were used to investigate if interaction mechanisms are modulated in a strain-specific way. Both species populations were affected by physical contact, particularly Starm. bacillaris that lost its culturability during fermentation. In addition, loss of culturability of Starm. bacillaris strains was observed earlier in flasks than in the double-compartment system. The phenomena observed occurred in a strain couple-dependent way. Starm. bacillaris disappearance seemed to be independent of nutrient depletion or the presence of inhibitory compounds (which were not measured in this study). CONCLUSION: Overall, the results of the present study reveal that cell-to-cell contact plays a role in the early death of non-Saccharomyces but the extent to which it is observed depends greatly on the Starm. bacillaris/S. cerevisiae strains tested.

Evaluation of the production and extraction of polyhydroxybutyrate from volatile fatty acids by means of mixed cultures and B. cepacia.

The global consumption of plastics generates accelerated environmental pollution in landfills and marine ecosystems. Biopolymers are the materials with the greatest potential to replace synthetic polymers in the market due to their good biodegradability, however, there are still several disadvantages, mainly related to their production cost. Considering the above, the generation of biodegradable and biocompatible bioplastics stands out as an alternative solution, some of which are made from renewable raw materials, including polyhydroxyalkanoates PHAs. Although much research has been done on bacteria with the capacity for intracellular accumulation of PHAs, among others, it is also possible to produce PHAs using mixed microbial cultures instead of a single microorganism, using natural microbial consortia that have the capacity to store high amounts of PHAs. In this contribution, three methods for the extraction and purification of PHAs produced by fermentation using volatile fatty acids as a carbon source at different concentrations were evaluated, using the pure strain Burkholderia cepacia 2G-57 and the mixed cultures of the activated sludge from the El Salitre WWTP, in order to select the best method from the point of view of environmental sustainability as this will contribute to the scalability of the process. The mixed cultures were identified by sequencing of the 16S gene. A yield of 89% was obtained from the extraction and purification of PHA using acetic acid as a solvent, which according to its properties is "greener" than chloroform. The polymer obtained was identified as polyhydroxybutylated PHB.

Bioplastic polyhydroxyalkanoate conversion in waste activated sludge.

Polyhydroxyalkanoates (PHA) have been proposed as a promising solution for plastic pollution due to their biodegradability and diverse applications. To promote PHA as a competitive commercial product, an attractive alternative is to produce and recover PHA in the use of mixed cultures such as waste activated sludge from wastewater treatment plants. PHA can accumulate in sludge with a potential range of 40%-65% g PHA/g VSS. However, wider challenges with PHA production efficiency, stability, and economic viability still persist for PHA application. This work provides an overview of the current understanding and status of PHA bioconversion in waste sludge with particular attention given to metabolic pathways, operation modes, factors affecting the process, and applications. Challenges and future prospectives for PHA bioconversion in sludge are discussed.

Impact of phosphorus limitation on medium-chain-length polyhydroxyalkanoate production by activated sludge.

For a sustainable economy, biodegradable biopolymers polyhydroxyalkanoates (PHA) are desirable substitutes to petroleum-based plastics that contaminate our environment. Medium-chain-length (MCL) PHA bioplastics are particularly interesting due to their thermoplastic properties. To hamper the high cost associated to PHA production, the use of bacterial mixed cultures cultivated in open systems and using cheap resources is a promising strategy. Here, we studied the operating conditions favouring direct MCL accumulation by activated sludge, using oleic acid as a model substrate and phosphorus limitation in fed-batch bioreactors. Our results confirm the presence of PHA-accumulating organisms (PHAAO) in activated sludge able to accumulate MCL from oleic acid. A positive correlation between phosphorus (P) limitation and PHA accumulation was demonstrated, allowing up to 26% PHA/total biomass accumulation, and highlighted its negative impact on the MCL/PHA fraction in the polymer. Diversity analysis through 16S rRNA amplicon sequencing revealed a differential selection of PHAAO according to the P-limitation level. A differential behaviour for the orders Pseudomonadales and Burkholderiales at increasing P-limitation levels was revealed, with a higher abundance of the latter at high levels of P-limitation. The PHA accumulation observed in activated sludge open new perspectives for MCL-PHA production system based on P-limitation strategy applied to mixed microbial communities. KEY POINTS: • Direct accumulation of MCL-PHA in activated sludge was demonstrated. • MCL-PHA content is negatively correlated with P-limitation. • Burkholderiales members discriminate the highest P-limitation levels.

Catch me if you can: capturing microbial community transformation by extracellular DNA using Hi-C sequencing.

The transformation of environmental microorganisms by extracellular DNA is an overlooked mechanism of horizontal gene transfer and evolution. It initiates the acquisition of exogenous genes and propagates antimicrobial resistance alongside vertical and conjugative transfers. We combined mixed-culture biotechnology and Hi-C sequencing to elucidate the transformation of wastewater microorganisms with a synthetic plasmid encoding GFP and kanamycin resistance genes, in the mixed culture of chemostats exposed to kanamycin at concentrations representing wastewater, gut and polluted environments (0.01-2.5-50-100 mg L-1). We found that the phylogenetically distant Gram-negative Runella (102 Hi-C links), Bosea (35), Gemmobacter (33) and Zoogloea (24) spp., and Gram-positive Microbacterium sp. (90) were transformed by the foreign plasmid, under high antibiotic exposure (50 mg L-1). In addition, the antibiotic pressure shifted the origin of aminoglycoside resistance genes from genomic DNA to mobile genetic elements on plasmids accumulating in microorganisms. These results reveal the power of Hi-C sequencing to catch and surveil the transfer of xenogenetic elements inside microbiomes.

Mixed Cultures of Saccharomyces kudravzevii and S. cerevisiae Modify the Fermentation Process and Improve the Aroma Profile of Semi-Sweet White Wines.

The purpose of the study was to evaluate the impact of the Saccharomyces cerevisiae and S. kudriavzevii mixed culture on the fermentation, chemical and aromatic composition of semi-sweet white wines. The variables tested in the experiment were the initial ratio of yeast in mixed cultures and the time of inoculation of the S. kudriavzevii co-culture. The addition of S. kudriavzevii to the inoculum did not significantly change the chemical composition of the wines obtained. No reduction in ethanol yield was found in mixed culture fermented wines; however, in some variants of the experiment, the ethanol content was higher. The mixed cultures of S. cerevisiae and S. kudriavzevii increased the level of volatile compounds in white grape wines. Wines fermented with the co-culture of S. kudriavzevii were characterized by a more diversified ester profile. The mixed cultures of S. cerevisiae and S. kudriavzevii raised the levels of terpenes in white wines. The most promising results were obtained for mixed culture variants, in which S. kudriavzevii was sequentially inoculated on the sixth day of fermentation.

Interaction and Effects of Bacteria Addition on Dichlorodiphenyltrichloroethane Biodegradation by Daedalea dickinsii.

The residue of organochlorine pesticides (OCPs) has been a major pollution problem in our environment. Dichlorodiphenyltrichloroethane (DDT) is one of the most common persistent OCPs that continue to pose a serious risk to human health and the environment. Some treatment methods have been developed to reduce and minimize the adverse impacts of the use of DDT, including biodegradation with brown-rot fungi (BRF). However, DDT degradation using BRF has still low degradation rate and needs a long incubation time. Therefore, the ability of BRF need to be enhanced to degrade DDT. Interaction and effect of bacteria addition on biodegradation of DDT by brown-rot fungus Daedalea dickinsii were investigated. The interaction assay between D. dickinsii with bacteria addition showed that the addition of bacterium Pseudomonas aeruginosa did not provide resistance to the growth of D. dickinsii. Meanwhile, bacterium Bacillus subtilis addition has an inhibitory effect on the growth of D. dickinsii. The addition of 10 ml (1 ml = 1.05 × 109 CFU/ml bacteria cell) of P. aeruginosa and B. subtilis was able to improve DDT biodegradation by D. dickinsii from 53.61% to 96.70% and 67.60%, respectively. The highest biodegradation capability of DDT was obtained through addition of 10 ml of P. aeruginosa into the D. dickinsii culture in which the mixed cultures produce final metabolites of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD) and 1-chloro-2,2-bis(4-chlorophenyl)ethylene (DDMU). This study indicated that the addition of P. aeruginosa can be used for optimization of DDT biodegradation by D. dickinsii.

The Use of Yeast Mixed Cultures for Deacidification and Improvement of the Composition of Cold Climate Grape Wines.

Interest in the use of non-Saccharomyces yeast in mixed cultures is increasing due to the perceived improvement in the quality and complexity of the resulting wines. The aim of the study was to determine the ability of monocultures and mixed yeast cultures for deacidification and improvement of the composition of cold climate grape wines. Fermentation of grape musts with increased total acidity was carried out with the use of monocultures of Saccharomyces cerevisiae MH020215 (Sc), Zygosaccharomyces bailii 749 (Zb) and Metschnikowia pulcherrima MG970690 (Mp), and their mixed cultures, inoculated simultaneously and sequentially. Oenological parameters, organic acids and volatile compounds profiles of obtained wines were characterized. The fermentation kinetics and analytical profiles of the obtained wines showed that the use of mixed yeast cultures contributed to the reduction of volatile acidity and acetic acid content in the wines, as well as obtaining a favorable aromatic profile of the wines. The dominant higher alcohols in all wines were 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Significantly higher amounts of the first two compounds were found in wines obtained with M. pulcherrima MG070690, both in monoculture and in mixed cultures. The monocultures of M. pulcherrima MG070690 (Mp) compared with Z. bailli 749 (Zb) synthesized higher levels of esters in wines, including ethyl acetate, ethyl propionate, isobutyl acetate, ethyl pyroracemate and isoamyl acetate.

Metabolic modeling for predicting VFA production from protein-rich substrates by mixed-culture fermentation.

Proteinaceous organic wastes are suitable substrates to produce high added-value products in anaerobic mixed-culture fermentations. In these processes, the stoichiometry of the biotransformation depends highly on operational conditions such as pH or feeding characteristics and there are still no tools that allow the process to be directed toward those products of interest. Indeed, the lack of product selectivity strongly limits the potential industrial development of these bioprocesses. In this work, we developed a mathematical metabolic model for the production of volatile fatty acids from protein-rich wastes. In particular, the effect of pH on the product yields is analyzed and, for the first time, the observed changes are mechanistically explained. The model reproduces experimental results at both neutral and acidic pH and it is also capable of predicting the tendencies in product yields observed with a pH drop. It also offers mechanistic insights into the interaction among the different amino acids (AAs) of a particular protein and how an AA might yield different products depending on the relative abundance of other AAs. Particular emphasis is placed on the utility of this mathematical model as a process design tool and different examples are given on how to use the model for this purpose.

Yeasts for low input winemaking: Microbial terroir and flavor differentiation.

Vitis vinifera flowers and grape fruits are one of the most interesting ecosystem niches for native yeasts development. There are more than a 100 yeast species and millions of strains that participate and contribute to design the microbial terroir. The wine terroir concept is understood when grape and wine micro-regions were delimited by different quality characteristics after humans had been growing vines for more than 10,000 years. Environmental conditions, such as climate, soil composition, water management, winds and air quality, altitude, fauna and flora and microbes, are considered part of the "terroir" and contribute to a unique wine style. If "low input winemaking" strategies are applied, the terroir effect will be expected to be more authentic in terms of quality differentiation. Interestingly, the role of the microbial flora associated with vines was very little study until recently when new genetic technologies for massive species identification were developed. These biotechnologies allowed following their environmental changes and their effect in shaping the microbial profiles of different wine regions. In this chapter we explain the interesting positive effects on flavor diversity and wine quality obtained by using "friendly" native yeasts that allowed the microbial terroir flora to participate and contribute during fermentation.

Direct Effects of Temperature on Growth of Different Tropical Phytoplankton Species.

Temperature increase may influence competition among phytoplankton species, potentially intensifying cyanobacteria blooms that can be favored by direct and indirect effects of temperature. In this study, we aimed to clarify how cyanobacteria can be favored by the direct effects of increased temperature compared to diatoms and chlorophytes. Strains of the most representative species of a eutrophic coastal lagoon (Microcystis aeruginosa, Planktothrix agardhii, Desmodesmus communis, and Cyclotella meneghiniana) were used to test the hypothesis that cyanobacteria would be favored by the direct effect of temperature increase. First, we evaluated the effect of temperature increase on growth in monocultures (batch and chemostats) at 25 and 30 °C and after in mixed cultures (chemostats). In batch monocultures, the cyanobacteria showed higher growth rates in 30 °C than in 25 °C. However, in continuous culture experiments (chemostats), growth rates of M. aeruginosa and P. agardhii were not affected by temperature, but the strains showed higher biovolume in steady-state with the temperature increase. In continuous mixed cultures, M. aeruginosa was always dominant and C. meneghiniana was excluded, regardless of temperature tested. D. communis was able to coexist with lower biomass. This study shows that rising temperatures can be detrimental to diatoms, even for a tropical strain. Although some studies indicate that the dominance of cyanobacteria in warmer climates may be due to the indirect effect of warming that will promote physical conditions in the environment more favorable to cyanobacteria, the outcomes of mixed cultures demonstrate that the direct effect of temperature can also favor the dominance of cyanobacteria.

RNA-seq based transcriptional analysis of Saccharomyces cerevisiae and Lachancea thermotolerans in mixed-culture fermentations under anaerobic conditions.

BACKGROUND: In wine fermentation starter cultures, the blending of non-Saccharomyces yeast with Saccharomyces cerevisiae to improve the complexity of wine has become common practice, but data regarding the impact of co-cultivation on yeast physiology and on genetic and metabolic regulation remain limited. Here we describe a transcriptomic analysis of mixed fermentations of Saccharomyces cerevisiae and Lachancea thermotolerans. The fermentations were carried out in carefully controlled environmental conditions in a bioreactor to reduce transcriptomic responses that would be due to factors other than the presence of the second species. RESULTS: The transcriptomic data revealed that both yeast species showed a clear response to the presence of the other. Affected genes primarily belonged to two groups: genes whose expression can be linked to the competition for certain trace elements such as copper and iron, as well as genes required for cell wall structure and integrity. Furthermore, the data revealed divergent transcriptional responses with regard to carbon metabolism in response to anoxic conditions. CONCLUSIONS: The results suggest that the mixed fermentation created a more competitive and stressful environment for the two species than single strain fermentations independently from total biomass, i.e. competition between cells of the same species is less stressful, or may present a different set of challenges, than interspecies competition. The changes in cell wall and adhesion properties encoding genes suggest that the adjustment of physical contact between cells may play a direct role in the response to the presence of competing species.

Assessing the efficiency in assisted depuration of coffee processing wastewater from mixed wild microbial selected inoculum.

This work evaluated the efficiency of bacterial bio-augmentation to the biological treatment of coffee processing wastewater (CPWW) in a pilot wastewater treatment plant (WTP). Biochemical oxygen demand (BOD) and chemical oxygen demand (COD) values were the basis for the treatment efficiency. Serratia marcescens CCMA 1010 and CCMA 1013, Corynebacterium flavescens CCMA 1006 and Acetobacter indonesiensis CCMA 1002 were previously selected. The microbial cocktail was inoculated and persisted in CPWW during all treatments. The richness of wild species was a little altered over time and up to nine species were found in each sampled season. The microbiota composition presented variation of a total of 13 species, despite the inoculation of the microbial inoculum. The biodegradability index of effluent, close to 0.5, was favourable to biological treatment. The pollution parameters of CPWW were decreased in function of the variation of community composition and microbial activity. The greatest reduction of BOD (~ 33%) and COD (~ 25%) was observed between 72 h and 8 days of the biological treatment. The CPWW toxicity in Allium cepa seeds was lower by up to 60%, and the germination index (GI) exceeded 100% in the treated CPWW. The results of the CPWW biological treatment by bio-augmentation from native micro-organisms in the pilot-scale WTP indicated the greatest efficiency relating to the spontaneous biological treatment of CPWW. After this treatment, the discharge of effluent in the environment would not have toxic effects on the plants.

Susceptibility to Oral Antiseptics and Virulence Factors Ex Vivo Associated with Candida spp. Isolated from Dental Prostheses.

PURPOSE: To isolate Candida spp. from dental prosthesis users' saliva and to evaluate the isolates for the presence of several virulence factors. This research also aimed to investigate the antifungal activity of 3 commercial mouthwashes/oral antiseptic formulations containing 0.12% chlorhexidine, 0.07% cetylpyridinium, or 0.075% cetylpyridinium against planktonic and sessile (biofilm mode) yeast cells. MATERIALS AND METHODS: Forty-three Candida yeasts were isolated from 32 of 70 selected patients, and the virulence factors of C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis species were investigated by polymerase chain reaction (PCR) and proteinase in plates. Minimum inhibitory concentration (MIC), and in vitro biofilm assay evaluated the antifungal activity of antiseptics. RESULTS: C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis were detected in mono and mixed cultures. Only C. albicans displayed genes related to adhesion and proteinases (ALS2, ALS3, SAP1, and SAP3). The aspartate proteinase activity was found in 60.46% of isolates. The tested antiseptic formulations exhibited a MIC less than 1.25% toward yeasts in the planktonic mode. According to XTT ((2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay results, most Candida isolates and all mixed cultures formed biofilms within 24 hours. The evaluated antiseptic formulations were also active against biofilms. CONCLUSION: Most virulence factors investigated here (ALS2, ALS3, SAP1, and SAP3) occurred in the majority of the Candida spp. isolates, especially in C. albicans. The tested mouthwash formulations were effective against all the yeast isolates in both the planktonic and sessile growth modes. Developing alternative therapies that can avoid or control biofilm formation is necessary to prevent oral candidiasis and other Candida spp. infections.

Metabolic footprint analysis of metabolites that discriminate single and mixed yeast cultures at two key time-points during mixed culture alcoholic fermentations.

INTRODUCTION: There has been a growing interest towards creating defined mixed starter cultures for alcoholic fermentations. Previously, metabolite differences between single and mixed cultures have been explored at the endpoint of fermentations rather than during fermentations. OBJECTIVES: To create metabolic footprints of metabolites that discriminate single and mixed yeast cultures at two key time-points during mixed culture alcoholic fermentations. METHODS: 1H NMR- and GC-MS-based metabolomics was used to identify metabolites that discriminate single and mixed cultures of Lachancea thermotolerans (LT) and Saccharomyces cerevisiae (SC) during alcoholic fermentations. RESULTS: Twenty-two metabolites were found when comparing single LT and mixed cultures, including both non-volatiles (carbohydrate, amino acid and acids) and volatiles (higher alcohols, esters, ketones and aldehydes). Fifteen of these compounds were discriminatory only at the death phase initiation (T1) and fifteen were discriminatory only at the death phase termination (T2) of LT in mixed cultures. Eight metabolites were discriminatory at both T1 and T2. These results indicate that specific metabolic changes may be descriptive of different LT growth behaviors. Fifteen discriminatory metabolites were found when comparing single SC and mixed cultures. These metabolites were all volatiles, and twelve metabolites were discriminatory only at T2, indicating that LT-induced changes in volatiles occur during the death phase of LT in mixed cultures and not during their initial growth stage. CONCLUSIONS: This work provides a detailed insight into yeast metabolites that differ between single and mixed cultures, and these data may be used for understanding and eventually predicting yeast metabolic changes in wine fermentations.

Transcriptomic responses of mixed cultures of ascomycete fungi to lignocellulose using dual RNA-seq reveal inter-species antagonism and limited beneficial effects on CAZyme expression.

Gaining new knowledge through fungal monoculture responses to lignocellulose is a widely used approach that can lead to better cocktails for lignocellulose saccharification (the enzymatic release of sugars which are subsequently used to make biofuels). However, responses in lignocellulose mixed cultures are rarely studied in the same detail even though in nature fungi often degrade lignocellulose as mixed communities. Using a dual RNA-seq approach, we describe the first study of the transcriptional responses of wild-type strains of Aspergillus niger, Trichoderma reesei and Penicillium chrysogenum in two and three mixed species shake-flask cultures with wheat straw. Based on quantification of species-specific rRNA, a set of conditions was identified where mixed cultures could be sampled so as to obtain sufficient RNA-seq reads for analysis from each species. The number of differentially-expressed genes varied from a couple of thousand to fewer than one hundred. The proportion of carbohydrate active enzyme (CAZy) encoding transcripts was lower in the majority of the mixed cultures compared to the respective straw monocultures. A small subset of P. chrysogenum CAZy genes showed five to ten-fold significantly increased transcript abundance in a two-species mixed culture with T. reesei. However, a substantial number of T. reesei CAZy transcripts showed reduced abundance in mixed cultures. The highly induced genes in mixed cultures indicated that fungal antagonism was a major part of the mixed cultures. In line with this, secondary metabolite producing gene clusters showed increased transcript abundance in mixed cultures and also mixed cultures with T. reesei led to a decrease in the mycelial biomass of A. niger. Significantly higher monomeric sugar release from straw was only measured using a minority of the mixed culture filtrates and there was no overall improvement. This study demonstrates fungal interaction with changes in transcripts, enzyme activities and biomass in the mixed cultures and whilst there were minor beneficial effects for CAZy transcripts and activities, the competitive interaction between T. reesei and the other fungi was the most prominent feature of this study.

Use of a mixed culture strategy to isolate halophilic bacteria with antibacterial and cytotoxic activity from the Manaure solar saltern in Colombia.

BACKGROUND: Water evaporation in solar salterns creates salinity gradients that promote the adaptation of microbial species to different salinities. This competitive habitat challenges the metabolic capabilities of microorganisms and promotes alterations in their production of secondary metabolites. Thus, solar salterns are a potentially important source of new natural products. In Colombia, the most important and representative solar saltern is located in Manaure (La Guajira) in the north of Colombia. The aim of this study was to develop an alternative screening strategy to select halophilic bacteria as producers of bioactive compounds from mixed microbial cultures rather than individual environmental isolates. Brine and sediment samples from different ponds (across a salinity gradient) were inoculated in seven different culture media to grow bacteria and archaea, allowing for a total of 40 different mixed cultures. An organic extract from each mixed culture was obtained and tested against multidrug resistant pathogens, including Klebsiella pneumoniae, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus and Bacillus subtilis. In addition, the extracts were tested against two human cancer cell lines, cervical adenocarcinoma (SiHa) and lung carcinoma (A-549). RESULTS: Twenty-four of the forty extracts from mixed cultures obtained from brine and sediment samples from the Manaure solar saltern showed antibacterial activity against Bacillus subtilis. Two extracts, referred to as A1SM3-29 and A1SM3-36, were also active against a methicillin-resistant Staphylococcus aureus, with the latter extract also showing slight cytotoxic activity against the assayed human lung cancer cell line. From this mixed culture, nine isolates were cultivated, and their extracts were tested against the same pathogens, resulting in the identification of a Vibrio sp. strain (A1SM3-36-8) with antimicrobial activity that was similar to that observed for the mixed culture extract. The extract of this strain was subjected to a bioautography assay, and 3 different fractions exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. Based on the amount obtained for each fraction, F3 was selected to isolate and identify its metabolites. The major compound was identified by NMR and HRMS as 13-cis-docosenamide, an amide that has been previously reported to be an antimicrobial and cytotoxic compound. CONCLUSIONS: Our results shows the utility of our strategy in detecting bioactive molecules in initial mixed cultures by biological assays, resulting in the isolation and characterization of Vibrio sp. A1SM3-36-8, a halophilic strain with great antibacterial and cytotoxic potential.

Contribution of Lysinibacillus sphaericus hemolysin and chitin-binding protein in entomopathogenic activity against insecticide resistant Aedes aegypti.

Given its toxicity against culicid larvae, Lysinibacillus sphaericus is used for the biological control of mosquitoes such as Culex sp. and Anopheles sp. The toxicity factors currently reported for L. sphaericus include the Binary toxin, Mtx toxins, and the S-layer. However, Aedes aegypti is refractory to the Binary toxin, the most toxic larvicidal protein of L. sphaericus. Until now, there are no evidences of the hemolytic and chitinolytic capacity of L. sphaericus. Herein, the expression of the hemolysin D (hlyD) and the chitin-binding protein genes of L. sphaericus III(3)7, OT4b.25, and 2362 was quantified. Gene expression was assessed 24 and 48 h after field-collected and Rockefeller A. aegypti larvae were fed with the bacteria. The hlyD gene showed the highest expression at 24 h whilst the expression of the chitin-binding protein gene increases at 48 h. The highest hlyD expression was seen in the III(3)7 strain and the highest chitin-binding protein gene expression was in the 2362 strain. The consortium of L. sphaericus III(3)7 and 2362 showed the highest expression of both genes being with field-collected and Rockefeller larvae. The results suggest that hemolysin D and the chitin-binding protein can be two novel toxic elements involved in the entomopathogenic activity of L. sphaericus. These proteins, along with the other L. sphaericus toxins, make this bacterium a suitable alternative to replace the chemical insecticides used for the control of A. aegypti populations.

Cytokine Production in Mixed Cultures of Mesenchymal Stromal Cells from Wharton's Jelly and Peripheral Blood Lymphocytes.

We compared the production of 19 humoral factors in mixed cultures of mesenchymal stromal cells from Wharton's jelly and allogenic peripheral blood lymphocytes. For evaluation of the specificity of immunosuppressive activity of mesenchymal stromal cells, comparative analysis of the production of these humoral factors in mixed cultures of lymphocytes and epithelial BxPC-3 cells was conducted. The production of soluble factors in both mono- and mixed cultures significantly correlated (p<0.05). The maximum production was found for proinflammatory chemokine IP-10 and IFN-γ and anti-inflammatory cytokine IL-10. The major difference of mesenchymal stromal cells from epithelial BxPC-3 cells was 7-fold higher production of IL-10, which can explain the immunosuppressive effect of mesenchymal stromal cells.

Sensitivity of Scenedesmus obliquus and Microcystis aeruginosa to atrazine: effects of acclimation and mixed cultures, and their removal ability.

Atrazine is an herbicide frequently detected in watercourses that can affect the phytoplankton community, thus impacting the whole food chain. This study aims, firstly, to measure the sensitivity of monocultures of the green alga Scenedemus obliquus and toxic and non-toxic strains of the cyanobacteria Microcystis aeruginosa before, during and after a 30-day acclimation period to 0.1 µM of atrazine. Secondly, the sensitivity of S. obliquus and M. aeruginosa to atrazine in mixed cultures was evaluated. Finally, the ability of these strains to remove atrazine from the media was measured. We demonstrated that both strains of M. aeruginosa had higher growth rate-based EC50 values than S. obliquus when exposed to atrazine, even though their photosynthesis-based EC50 values were lower. After being exposed to 0.1 µM of atrazine for 1 month, only the photosynthesis-based EC50 of S. obliquus increased significantly. In mixed cultures, the growth rate of the non-toxic strain of M. aeruginosa was higher than S. obliquus at high concentrations of atrazine, resulting in a ratio of M. aeruginosa to total cell count of 0.6. This lower sensitivity might be related to the higher growth rate of cyanobacteria at low light intensity. Finally, a negligible fraction of atrazine was removed from the culture media by S. obliquus or M. aeruginosa over 6 days. These results bring new insights on the acclimation of some phytoplankton species to atrazine and its effect on the competition between S. obliquus and M. aeruginosa in mixed cultures.