Vinasse odyssey: sugarcane vinasse remediation and laccase production by Trametes sp. immobilized in polyurethane foam.

Vinasse is a high pollutant liquid residue from bioethanol production. Due to its toxicity, most vinasse is used not disposed of in water bodies but employed for the fertigation of sugarcane crops, potentially leading to soil salinization or heavy metal deposition. The anaerobic digestion of vinasse for energy production is the main alternative to fertigation, but the process cannot eliminate colored compounds such as melanoidins, caramels, or phenolic compounds. The treatment of raw vinasse with white-rot fungi could remove colored and persistent toxic compounds, but is generally considered cost-ineffective. We report the treatment of vinasse by an autochthonous Trametes sp. strain immobilized in polyurethane foam and the concomitant production of high titers of laccase, a high value-added product that could improve the viability of the process. The reuse of the immobilized biomass and the discoloration of raw vinasse, the concentration of phenolic compounds, BOD and COD, and the phytotoxicity of the treated vinasse were measured to assess the viability of the process and the potential use of treated vinasse in fertigation or as a complementary treatment to anaerobic digestion. Under optimal conditions (vinasse 0.25X, 30 °C, 21 days incubation, 2% glucose added in the implantation stage), immobilized Trametes sp. causes a decrease of 75% in vinasse color and total phenolic compounds, reaching 1082 U L-1 of laccase. The fungi could be used to treat 0.50X vinasse (BOD 44,400 mg O2 L-1), causing a 26% decolorization and a 30% removal of phenolic compounds after 21 days of treatment with maximum laccase titers of 112 U L-1, while reducing COD and BOD from 103,290 to 42,500 mg O2 L-1 (59%) and from 44,440 to 21,230 mg O2 L-1 (52%), respectively. The re-utilization of immobilized biomass to treat 0.50X vinasse proved to be successful, leading to the production of 361 U L-1 of laccase with 77% decolorization, 61% degradation of phenolic compounds, and the reduction of COD and BOD by 75% and 80%, respectively. Trametes sp. also reduced vinasse phytotoxicity to Lactuca sativa seedlings. The obtained results show that the aerobic treatment of vinasse by immobilized Trametes sp. is an interesting technology that could be employed as a sole treatment for the bioremediation of vinasse, with the concomitant the production of laccase. Alternatively, the methodology could be used in combination with anaerobic digestion to achieve greater decolorization and reduction of phenolic compounds, melanoidins, and organic load.

Proteomic and enzymatic response under Cr(VI) overload in yeast isolated from textile-dye industry effluent.

Cyberlindnera jadinii M9 and Wickerhamomyces anomalus M10 isolated from textile-dye liquid effluents has shown capacity for chromium detoxification via Cr(VI) biological reduction. The aim of the study was to evaluate the effect of hexavalent chromium on synthesis of novel and/or specific proteins involved in chromium tolerance and reduction in response to chromium overload in two indigenous yeasts. A study was carried out following a proteomic approach with W. anomalus M10 and Cy. jadinii M9 strains. For this, proteins extracts belonging to total cell extracts, membranes and mitochondria were analyzed. When Cr(VI) was added to culture medium there was an over-synthesis of 39 proteins involved in different metabolic pathways. In both strains, chromium supplementation changed protein biosynthesis by upregulating proteins involved in stress response, methionine metabolism, energy production, protein degradation and novel oxide-reductase enzymes. Moreover, we observed that Cy. jadinii M9 and W. anomalus M10 displayed ability to activate superoxide dismutase, catalase and chromate reductase activity. Two enzymes from the total cell extracts, type II nitroreductase (Frm2) and flavoprotein wrbA (Ycp4), were identified as possibly responsible for inducing crude chromate-reductase activity in cytoplasm of W. anomalus M10 under chromium overload. In Cy.jadinii M9, mitochondrial Ferredoxine-NADP reductase (Yah1) and membrane FAD flavoprotein (Lpd1) were identified as probably involved in Cr(VI) reduction. To our knowledge, this is the first study proposing chromate reductase activity of these four enzymes in yeast and reporting a relationship between protein synthesis, enzymatic response and chromium biospeciation in Cy. jadinii and W. anomalus.

Biodecoloration of Reactive Black 5 by the methylotrophic yeast Candida boidinii MM 4035.

Azo dyes are extensively used in textile dyeing and other industries. Effluents of dying industries are specially colored and could cause severe damage to the environment. The anaerobic treatment of textile dying effluents is nowadays the preferred option, but it could generate carcinogenic aromatic amines. Recently, yeasts have become a promising alternative, combining unicellular growth with oxidative mechanisms. This work reports the characterization of the first methylotrophic yeast with dye decolorizing ability, Candida boidinii MM 4035 and some insights into its decoloration mechanism. The analysis of two selected media revealed a possible two stages mechanism of Reactive Black 5 decoloration. In glucose poor media, decoloration is incomplete and only the first stage proceeds, leading to the accumulation of a purple compound. In media with higher glucose concentrations, the yeast is able to decolorize totally an initial concentration of 200mg/L. The entire process is co-metabolic, being largely dependent on glucose concentration but being able to proceed with several nitrogen sources. Manganese dependent peroxidase but not laccase activity could be detected during decoloration. Aromatic amines do not accumulate in culture media, supporting an oxidative decoloration mechanism of unknown ecophysiological relevance.

Disentangling metabolic pathways involved in copper resistance in Candida fukuyamaensis RCL-3 indigenous yeast.

Candida fukuyamaensis RCL-3 yeast strain isolated from a copper filter plant is able to lower copper concentration in culture medium. In the present study, effect of copper in proteins expression and mechanisms involved in copper resistance were explored using comparative proteomics. Mono-dimensional gel electrophoresis revealed differential band expressions between cells grown with or without copper. 2-DE analysis of C. fukuyamaensis RCL-3 revealed that copper exposure produced at least an over-expression of 40 proteins. Sixteen proteins were identified and grouped in four categories according to their functions: glycolysis and ATP production, synthesis of proteins, oxidative stress response, and processing and transport of proteins. Integral membrane proteins and membrane-associated proteins were analyzed, showing nine protein bands over-expressed in Cu-supplemented medium. Four proteins were identified, namely nucleoporin pom152, elongation factor 2, copper chaperone Sod1 Ccs1, and eiosome component Lsp1. The proteomic analysis performed allowed the identification of different metabolic pathways and certain proteins involved in metal input and storage related to cell ability to bioremediate copper. These proteins and mechanisms could be used for future applications of C. fukuyamaensis RCL-3 in biotechnological processes such as remediation of heavy metals.

Oleaginous yeasts from Antarctica: Screening and preliminary approach on lipid accumulation.

The capability of 17 Rhodotorula spp. isolated from Antarctica to accumulate intracellular lipids in nitrogen-limited medium was investigated. As results, 10 isolates were selected by Nile red staining, while 12 isolates were selected as oleaginous by analysis of total lipid content (20.4-73%, w/w of dry biomass). The higher lipid production and accumulation was exhibited for six strains belonging to three species of Rhodotorula (Rhodotorula glutinis, Rhodotorula glacialis, and Rhodotorula laryngis). This is the first report where R. laryngis have been identified within oleaginous specie. Lipid accumulation was evaluated comparatively in two nitrogen-limited glucose-based media (MI and MII). MI (low C/N ratio) was more suitable for biomass and lipid production while in MII (high C/N ratio) total lipid content was improved. R. glutinis R4, R. glacialis R15, and R. glutinis R48 showed high lipid concentrations (4.65-6.93 g L-1 ) and they were able to accumulate large amounts of lipids per gram of biomass (47-77%, w/w). A similar profile in fatty acids composition and content of neutral lipids to vegetable oils was observed, indicating that lipids produced by oleaginous Antarctic yeasts can be considered an alternative feedstock for biodiesel production. Antarctica represents an important source of oleaginous yeasts with adaptive capabilities to accumulate considerable amounts of lipids with biotechnological interest at 15 °C and 25 °C.

[Biomineralization of copper in Candida fukuyamaensis RCL-3]. / Biomineralización de cobre en Candida fukuyamaensis RCL-3.

Candida fukuyamaensis RCL-3 yeast has the ability to decrease copper concentration in a culture medium. High copper concentrations change the cell color from white/cream to brown. The effect of color change ceases with the addition of KCN or when cells are grown in a culture medium without sulfate ions. These results could be associated with CuS bioaccumulation in the cell surface. This report revealed that mineralization would be a mechanism used by this yeast for copper bioremediation.

Gluconacetobacter diazotrophicus PAL5 possesses an active quorum sensing regulatory system.

The endophytic bacterium Gluconacetobacter diazotrophicus colonizes a broad range of host plants. Its plant growth-promoting capability is related to the capacity to perform biological nitrogen fixation, the biosynthesis of siderophores, antimicrobial substances and the solubilization of mineral nutrients. Colonization of and survival in these endophytic niche requires a complex regulatory network. Among these, quorum sensing systems (QS) are signaling mechanisms involved in the control of several genes related to microbial interactions, host colonization and stress survival. G. diazotrophicus PAL5 possesses a QS composed of a luxR and a luxI homolog, and produces eight molecules from the AHL family as QS signals. In this report data are provided showing that glucose concentration modifies the relative levels of these signal molecules. The activity of G. diazotrophicus PAL5 QS is also altered in presence of other carbon sources and under saline stress conditions. Inactivation of the QS system of G. diazotrophicus PAL5 by means of a quorum quenching strategy allowed the identification of extracellular and intracellular proteins under the control of this regulatory mechanism.

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media.

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.

Proteomic study of the yeast Rhodotorula mucilaginosa RCL-11 under copper stress.

In order to understand the mechanism involved in Rhodotorula mucilaginosa RCL-11 resistance to copper a proteomic study was conducted. Atomic absorption spectroscopy showed that the copper concentration in the medium decreased from 0.5 to 0.19 mM 48 h after inoculation of the yeast. Analysis of one-dimensional gel electrophoresis of crude cell extracts revealed expression of differential bands between cells with and without copper. In order to study this difference, two-dimensional electrophoresis of R. mucilaginosa RCL-11 exposed to Cu for 16, 24, and 48 h was carried out. Identification of differentially expressed proteins was performed by MALDI-TOF/TOF. Ten of the 16 spots identified belonged to heat shock proteins. Superoxide dismutase, methionine synthase and beta-glucosidase were also found over-expressed at high copper concentrations. The results obtained in the present work show that when R. mucilaginosa RCL-11 is exposed to 0.5 mM copper, differential proteins, involved in cell resistance mechanisms, are expressed.

Unraveling the decolourizing ability of yeast isolates from dye-polluted and virgin environments: an ecological and taxonomical overview.

Microcosm assays with dye-amended culture media under a shot-feeding strategy allowed us to obtain 100 yeast isolates from the wastewater outfall channel of a dyeing textile factory in Tucumán (Argentina). Meanwhile, 63 yeast isolates were obtained from Phoebe porphyria (Laurel del monte) samples collected from Las Yungas rainforest (Tucumán), via a classical isolation scheme. Isolated yeasts, both from dye-polluted and virgin environments, were compared for their textile dye decolourization ability when cultured on solid and liquid media. Nine isolates from wastewater and 17 from Las Yungas showed the highest decolourization potential on agar plates containing six different reactive dyes, either alone or as a mixture. Five yeasts from each environment were further selected on the basis of their high dye removal rate in Vilmafix(®) Red 7B-HE- or Vilmafix(®) Blue RR-BB-amended liquid cultures. Yeasts from wastewater showed slightly higher decolourization percentages after 36 h of culture than yeasts from Las Yungas (98-100% vs. 91-95%, respectively). However, isolates from Las Yungas exhibited higher specific decolourization rates than isolates from effluents (1.8-3.0 vs. 0.9-1.3 mg g(-1)h(-1), respectively). All selected isolates were first grouped according to microsatellite-PCR analysis and representative isolates from each group were subsequently identified based on the 26S rRNA gene sequence analysis. Yeasts from wastewater were identified as the ascomycetous Pichia kudriavzevii (100%) and closely related to Candida sorbophila (99.8%), whilst yeasts from Las Yungas were identified as the basidiomycetous Trichosporon akiyoshidainum and Trichosporon multisporum. It is suggested that findings concerning yeast selection during screening programs for dye-decolourizing yeasts may be explained in the light of the copiotroph-oligotroph microorganisms rationale.

Responses of Candida fukuyamaensis RCL-3 and Rhodotorula mucilaginosa RCL-11 to copper stress.

The effect of high Cu(II) concentrations on superoxide dismutase (SOD) and catalase (CAT) activity in Candida fukuyamaensis RCL-3 and Rhodotorula mucilaginosa RCL-11, previously isolated from a copper filter at a mine plant in Argentina, was studied. Addition of 0.1, 0.2 and 0.5 mM Cu(II) to the culture medium increased total SOD and CAT activity in both strains. Native polyacrylamide gel electrophoresis revealed two bands with SOD activity for C. fukuyamaensis RCL-3 and only one for R. mucilaginosa RCL-11; the three bands corresponded to MnSOD.Intracellular accumulation of copper and morphological changes was observed using electron microscopy. Dark bodies examined with transmission electron microscopy (TEM) after 48 h of incubation probably corresponded to copper deposits. The number of dark bodies in R. mucilaginosa RCL-11 grew with increasing incubation time, whereas in C. fukuyamaensis RCL-3 the amount decreased. Scanning electron micrographs (SEM) of C. fukuyamaensis RCL-3 did not reveal any differences compared with the control, but R. mucilaginosa RCL-11 cells were bigger than control ones. TEM confirmed absence of compartmentalization mechanisms in Cu(II) detoxification since electron-dense bodies were mainly found in the cytoplasm.

Cu(II) removal by Rhodotorula mucilaginosa RCL-11 in sequential batch cultures.

The present study explored the ability of the yeast Rhodotorula mucilaginosa RCL-11 to adapt to increasing Cu(II) concentrations, measuring oxidative stress through superoxide dismutase and catalase activity in two parallel sequential batch assays. One assay was performed in Erlenmeyer flasks without aeration and a second in a fermentor in which the dissolved oxygen was maintained at 30% saturation. Both assays were carried out by increasing Cu(II) concentrations in five sequential steps: 0; 0.1; 0.2; 0.5 and 1 mM. Each assay was incubated at 30 degrees C, 250 rpm and pH 5.5. While growth parameters of R. mucilaginosa RCL-11 decreased 90-95% with increasing Cu(II) concentration in the culture medium, the oxidative stress level increased from 30 to 55% in both assays. Cells grown under controlled oxygen conditions showed 30% more copper bioaccumulation and 10% glucose consumption when compared with cells grown without aeration. SOD activity was higher under controlled than without aeration, whereas CAT activity was similar under both test conditions. Cu(II) bioaccumulation by R. mucilaginosa RCL-11 and a possible increase in this capacity by adaptation of the strain under controlled aeration represent a potential valuable tool for treatment of effluents or water bioremediation with high copper contents.

Optimization and mechanisms for biodecoloration of a mixture of dyes by Trichosporon akiyoshidainum HP 2023.

Trichosporon akiyoshidainum HP2023 is a basidiomycetous yeast isolated from Las Yungas rainforest (Tucumán, Argentina) and selected based on its outstanding textile-dye-decolorizing ability. In this work, the decolorization process was optimized using Reactive Black 5 as dye model. Lactose and urea were chosen as carbon and nitrogen sources through a one-at-time approach. Afterwards, factorial designs were employed for medium optimization, leading to the formulation of a simpler optimized medium which contains in g L-1: lactose 10, yeast extract 1, urea 0.5, KH2PO4 1 and MgSO4 1. Temperature and agitation conditions were also optimized. The optimized medium and incubation conditions for dye removal were extrapolated to other dyes individually and a mixture of them. Dye removal process happened through both biosorption and biodegradation mechanisms, depending primarily on the dye structure. A positive relation between initial inoculum and dye removal rate and a negative relation between initial dye concentration and final dye removal percentages were found. Under optimized conditions, T. akiyoshidainum HP2023 was able to completely remove a mixture of dyes up to a concentration of 300 mg L-1, a concentration much higher than those expected in real effluents.

Flavonoids from Argentine Tagetes (Asteraceae) with antimicrobial activity.

The flavonoids, constituting one of the most numerous and widespread groups of natural plant constituents, are important to humans not only because they contribute to plant colors but also because many members are physiologically active. These low-molecular-weight substances, found in all vascular plants, are phenylbenzopyrones. Over 4000 structures have been identified in plant sources, and they are categorized into several groups. Primarily recognized as pigments responsible for the autumnal burst of hues and the many shades of yellow, orange, and red in flowers and food, the flavonoids are found in fruits, vegetables, nuts, seeds, stems, flowers, and leaves as well as tea and wine and are important constituents of the human diet. They are prominent components of citrus fruits and other food sources. Flavonols (quercetin, myricetin, and kaempferol) and flavones (apigenin and luteolin) are the most common phenolics in plant-based foods. Quercetin is also a predominant component of onions, apples, and berries. Such flavanones as naringin are typically present in citrus fruit, and flavanols, particularly catechin, are present as catechin gallate in such beverages as green or black tea and wine. Some major sources of flavonoids are outlined in Table 1. The daily intake of flavonoids in humans has been estimated to be approx 25 mg/d, a quantity that could provide pharmacologically significant concentrations in body fluids and tissues, assuming good absorption from the gastrointestinal tract. Biological activity of flavonoids was first suggested by Szent-Gÿorgyi 1938, who reported that citrus peel flavonoids were effective in preventing the capillary bleeding and fragility associated with scurvy. The broad spectrum of biological activity within the group and the multiplicity of actions displayed by a certain individual members make the flavonoids one of the most promising classes of biologically active compounds.

Gluconic acid produced by Gluconacetobacter diazotrophicus Pal5 possesses antimicrobial properties.

Gluconic acid is produced in large quantities by the endophytic and diazotrophic bacterium Gluconacetobacter diazotrophicus Pal5. This organic acid derives from direct oxidation of glucose by a pyrroloquinoline-quinone-linked glucose dehydrogenase in this plant growth-promoting bacterium. In the present article, evidence is presented showing that gluconic acid is also responsible for the antimicrobial activity of G. diazotrophicus Pal5. The broad antagonistic spectrum includes Gram-positive and -negative bacteria. Eukaryotic microorganisms are more resistant to growth inhibition by this acid. Inhibition by gluconic acid can be modified through the presence of other organic acids. In contrast to other microorganisms, the Quorum Sensing system of G. diazotrophicus Pal5, a regulatory mechanism that plays a key role in several microbe-microbe interactions, is not related to gluconic acid production and the concomitant antagonistic activity.

Chromate removal by yeasts isolated from sediments of a tanning factory and a mine site in Argentina.

Twenty-one yeast-like microorganisms were isolated from tannery effluents and from a nickel-copper mine in Argentina. They were tested for their Cu(II), Ni(II), Cd(II) and Cr(VI) tolerance in qualitative assays on solid medium. Three isolates were selected for their multiple tolerance to the different heavy metals and highest tolerance to Cr(VI). According to morphological and physiological analysis and 26S rDNA D1/D2 domain sequences the isolates were characterized as: Lecythophora sp. NGV-1, Candida sp. NGV-9 and Aureobasidium pullulans VR-8. Resistance of the three strains to high Cr(VI) concentrations and their ability to remove Cr(VI) were assessed using YNB-glucose medium supplemented with 0.5 and 1 mM Cr(VI). Chromate removal activity was estimated by measuring remaining Cr(VI) concentration in the supernatant using the colorimetric 1,5-diphenylcarbazide method and total chromium was determined by flame atomic absorption spectroscopy. The results indicate that the initial Cr(VI) concentration negatively influenced growth and the specific growth rate but stimulated the metabolic activity of the three strains; resistance to Cr(VI) by these strains was mainly due to reduction of Cr(VI) rather than chromium bioaccumulation. This study showed the potential ability of these strains as tools for bioremediation of Cr(VI) from contaminated sites.

Copper tolerant yeasts isolated from polluted area of Argentina.

Eleven yeasts were isolated from wastewater sediment samples collected from a copper filter mine plant, located in the province of Tucumán, Argentina, and tested for their heavy metal tolerance. Two isolates were selected based on their multiple tolerance to different heavy metals and their copper biosorption capacity was studied. Analysis of the 26S rDNA D1/D2 domain sequences indicates that isolate RCL-3 showed similarity with Candida sp. and RCL-11 with Rhodotorula mucilaginosa . Growth performance and copper toxicity of both yeasts were evaluated using YNB-glucose medium supplemented with 0.1, 0.2, 0.5 and 1 mM of Cu2+ solutions. Candida sp. RCL-3 was able to grow up to 7 mg ml(-1) biomass in the presence of either 0.1 or 0.2 mM Cu2+, and at 0.5 mM Cu2+ growth reached 5.5 mg ml(-1). R. mucilaginosa RCL-11 reached 8 mg ml(-1) in the presence of 0.1 mM Cu2+, and values of 6.5 and 5.5 mg ml(-1) biomass were obtained at 0.2 and 0.5 mM Cu2+, respectively. Copper accumulation profiles were different: the metal was librated from the intact cells by Candida sp. whereas R. mucilaginosa did not show release from the cells indicating intracellular storage. Specific biosorption of copper by both isolated yeasts showed increase with the initial copper supplied with the medium (up to 11.5 and 8.0 mg Cu g(-1) biomass for Candida sp. and R. mucilaginosa , respectively). However, specific biosorption decreased with time.