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.

Inactivation of bacterial quorum sensing signals N-acyl homoserine lactones is widespread in yeasts.

The inactivation of quorum sensing signals, a phenomenon known as quorum quenching, has been described in diverse microorganisms, though it remains almost unexplored in yeasts. Beyond the well-known properties of these microorganisms for the industry or as eukaryotic models, the role of yeasts in soil or in the inner tissues of a plant is largely unknown. In this report, the wider survey of quorum quenching activities in yeasts isolated from Antarctic soil and the inner tissues of sugarcane, a tropical crop, is presented. Results show that, independently of their niche, quorum quenching activities are broadly present in unicellular fungi. Although yeasts showing a broad range of quorum quenching activity are present in the two niches, at the same time specific AHL inactivation profiles can also be found. Furthermore, yeasts from both sampling sites show quorum quenching activities compatible with lactonase-like and acylase-like inactivations of AHLs. Interestingly, the characterization of Rhodotorula mucilaginosa 7Apo1 showed that the presence of a particular AHL does not interfere with the quenching of a second molecule. Evidence suggests that yeasts could play a role in the modulation of the quorum sensing activity of bacteria. The relationship among phylogeny, sampling sites and yeast quorum quenching activities of the isolates is analyzed.

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.

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.

The Ralstonia solanacearum pathogenicity regulator HrpB induces 3-hydroxy-oxindole synthesis.

The transcriptional activator HrpB of the bacterial wilt causing betaproteobacterium Ralstonia solanacearum represents a key regulator for pathogenicity. In particular, it drives expression of hrp genes encoding a type III secretion system (T3SS) as well as effector molecules for delivery into the host cytosol to promote disease. However, the HrpB regulon extends beyond this T3SS. We describe here an HrpB-activated operon of six genes that is responsible for the synthesis of a fluorescent isatin derivative of 149 Amu that we named HDF for HrpB-dependent factor and that we purified from culture supernatants. The structure of the labile molecule was solved by using NMR and CD spectroscopy to be (3S)-3-hydroxy-indolin-2-one and confirmed by its chemical synthesis and MS spectrometry. HDF was found to be present at 20 nM in wild-type cultures grown on minimal medium, and its synthesis increased 15-fold upon overproduction of HrpB, confirming that HrpB activates HDF synthesis. The addition of tryptophan significantly stimulated HDF biosynthesis and was shown to represent the precursor molecule for HDF synthesis. A search for the biological function of the molecule revealed that HDF induces acyl-homoserine lactone receptor-mediated reporter activity of the well studied LuxR transcriptional regulator of Vibrio fischeri. Thus, our results provide evidence that the specificity of acyl-homoserine lactone (acyl-HSL) receptors is clearly broader than previously considered. The failure to detect induction by HDF of the described endogenous quorum-sensing circuits of the pathogen points to a role in interfering with cell-cell signaling of rivalling bacteria.

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions.

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.