An unexpected guest: a green microalga associated with the arsenic-tolerant shrub Acacia farnesiana.

The best-known plant endophytes include mainly fungi and bacteria, but there are also a few records of microalgae growing endophytically in vascular land plants, some of which belong to the genus Coccomyxa. In this study, we isolated a single-celled photosynthetic microorganism from the arsenic-tolerant shrub Acacia farnesiana, thus we hypothesized that it is an endophytic arsenic-tolerant microalga. The microorganism was identified as belonging to the genus Coccomyxa, and the observation of algal cells within the root tissues strongly suggests its endophytic nature. The alga's tolerance to arsenate (AsV) and its influence on the fitness of A. farnesiana in the presence of AsV were evaluated. Coccomyxa sp. can tolerate up to 2000 µM of AsV for periods shorter than 10 days, however, AsV-tolerance decreased significantly in longer exposure periods. The association with the microalga increased the pigment content in aboveground tissues of A. farnesiana seedlings exposed to AsV for 50 days, without changes in plant growth or arsenic accumulation. This work describes the association, probably endophytic, between an angiosperm and a microalga, confirming the ability of the genus Coccomyxa to form associations with land plants and broadening the known variety of plant endophytes.

Enhanced esterase activity during the degradation of dibutyl phthalate by Fusarium species in liquid fermentation.

Dibutyl phthalate (DBP) is one of the most abundantly produced and used plasticizers and is incorporated into plastic to make it more flexible and malleable. DBP has been found to be an environmental contaminant and reported as an endocrine disruptor. Therefore, it is crucial to develop ecofriendly alternatives to eliminate phthalate pollution. In the present research, the growth of F. culmorum and F. oxysporum in the presence of DBP was studied in liquid fermentation. The esterase activity, specific growth rate, and growth and enzymatic yield parameters were determined in DBP-supplemented media (1,500 or 2,000 mg/L) and in control medium (lacking DBP). These results show that in general, for both Fusarium species, the highest esterase activities, specific growth rates, and yield parameters were observed in media supplemented with DBP. It was observed that 1,500 and 2,000 mg of DBP/L did not inhibit F. culmorum or F. oxysporum growth and that DBP induced esterase production in both fungi. These organisms have much to offer in the mitigation of environmental pollution caused by the endocrine disruptor DBP. This study reports, for the first time, esterase production during the degradation of high concentrations (i.e., 1,500 and 2,000 mg/L) of DBP by F. culmorum F. oxysporum.

Solid-state fermentation increases secretome complexity in Aspergillus brasiliensis.

Aspergillus is used for the industrial production of enzymes and organic acids, mainly by submerged fermentation (SmF). However, solid-state fermentation (SSF) offers several advantages over SmF. Although differences related to lower catabolite repression and substrate inhibition, as well as higher extracellular enzyme production in SSF compared to SmF have been shown, the mechanisms undelaying such differences are still unknown. To explain some differences among SSF and SmF, the secretome of Aspergillus brasiliensis obtained from cultures in a homogeneous physiological state with high glucose concentrations was analyzed. Of the regulated proteins produced by SmF, 74% were downregulated by increasing the glucose concentration, whereas all those produced by SSF were upregulated. The most abundant and upregulated protein found in SSF was the transaldolase, which could perform a moonlighting function in fungal adhesion to the solid support. This study evidenced that SSF: (i) improves the kinetic parameters in relation to SmF, (ii) prevents the catabolite repression, (iii) increases the branching level of hyphae and oxidative metabolism, as well as the concentration and diversity of secreted proteins, and (iv) favors the secretion of typically intracellular proteins that could be involved in fungal adhesion. All these differences can be related to the fact that molds are more specialized to growth in solid materials because they mimic their natural habitat.

Secretomic Insight into Glucose Metabolism of Aspergillus brasiliensis in Solid-State Fermentation.

The genus Aspergillus is ubiquitous in nature and includes various species extensively exploited industrially due to their ability to produce and secrete a variety of enzymes and metabolites. Most processes are performed in submerged fermentation (SmF); however, solid-state fermentation (SSF) offers several advantages, including lower catabolite repression and substrate inhibition and higher productivity and stability of the enzymes produced. This study aimed to explain the improved metabolic behavior of A. brasiliensis ATCC9642 in SSF at high glucose concentrations through a proteomic approach. Online respirometric analysis provided reproducible samples for secretomic studies when the maximum CO2 production rate occurred, ensuring consistent physiological states. Extracellular extracts from SSF cultures were treated by SDS-PAGE, digested with trypsin, and analyzed by LC-MS/MS. Of 531 sequences identified, 207 proteins were analyzed. Twenty-five were identified as the most abundant unregulated proteins; 87 were found to be up-regulated and 95 were down-regulated with increasing glucose concentration. Of the regulated proteins, 120 were enzymes, most involved in the metabolism of carbohydrates (51), amino acids (23), and nucleotides (9). This study shows the high protein secretory activity of A. brasiliensis under SSF conditions. High glucose concentration favors catabolic activities, while some stress-related proteins and those involved in proteolysis are down-regulated.

Tolerance, arsenic uptake, and oxidative stress in Acacia farnesiana under arsenate-stress.

Acacia farnesiana is a shrub widely distributed in soils heavily polluted with arsenic in Mexico. However, the mechanisms by which this species tolerates the phytotoxic effects of arsenic are unknown. This study aimed to investigate the tolerance and bioaccumulation of As by A. farnesiana seedlings exposed to high doses of arsenate (AsV) and the role of peroxidases (POX) and glutathione S-transferases (GST) in alleviating As-stress. For that, long-period tests were performed in vitro under different AsV treatments. A. farnesiana showed a remarkable tolerance to AsV, achieving a half-inhibitory concentration (IC50) of about 2.8 mM. Bioaccumulation reached about 940 and 4380 mg As·kg(-1) of dry weight in shoots and roots, respectively, exposed for 60 days to 0.58 mM AsV. Seedlings exposed to such conditions registered a growth delay during the first 15 days, when the fastest As uptake rate (117 mg kg(-1) day(-1)) occurred, coinciding with both the highest rate of lipid peroxidation and the strongest up-regulation of enzyme activities. GST activity showed a strong correlation with the As bioaccumulated, suggesting its role in imparting AsV tolerance. This study demonstrated that besides tolerance to AsV, A. farnesiana bioaccumulates considerable amounts of As, suggesting that it may be useful for phytostabilization purposes.

Effect of a saprophytic fungus on the growth and the lead uptake, translocation and immobilization in Dodonaea viscosa.

Phytoremediation is a feasible alternative to remediate soils polluted with toxic elements, which can be enhanced by manipulating plant-microbe interactions. Regarding this, free-living saprophytic fungi that interact beneficially with roots have been scarcely studied. Thus, the aim of this study was to assess the effect of a saprophytic fungus, Lewia sp., on the plant growth and the ability of Dodonaea viscosa to phytoaccumulate or phytostabilize soluble and insoluble sources of lead in a solid support. The growth of D. viscosa was influenced by both Pb and Lewia sp. While seedlings exposed to Pb showed a decrease in biomass production, in seedlings grown without Pb the biomass was stimulated by Lewia sp. The fungus strongly stimulated the weight-to-length ratio in roots. Regardless of the treatment, D. viscosa accumulated 4.4-6.5 times more Pb in roots than in shoots, conducting to low translocation factors (< 0.2). The presence of Lewia sp. significantly improved Pb accumulation, achieving high bioconcentration factors (> 22), which was attributed to an increased bioavailability and uptake of Pb due to the fungus. This study demonstrated that Lewia sp. could improve Pb-phytostabilization by D. viscosa in soils polluted with soluble and insoluble forms of Pb.

Lipases production by solid-state fermentation: the case of Rhizopus homothallicus in perlite.

Lipases are widely used in the industry for different purposes. Although these enzymes are mainly produced by submerged fermentation, lipase production by solid-state fermentation (SSF) has been gaining interest due to the advantages of this type of culture. Major advantages are higher production titers and productivity, less catabolite repression, and use of the dried fermented material as biocatalyst. This chapter describes a traditional methodology to produce fungal (Rhizopus homothallicus) lipases by SSF using perlite as inert support. The use of different devices (glass columns or Erlenmeyer flasks) and type of inoculum (spores or growing mycelium) is considered so that lipase production by SSF could be easily performed in any laboratory.

Evaluation of feed COD/sulfate ratio as a control criterion for the biological hydrogen sulfide production and lead precipitation.

The ability of sulfate-reducing bacteria to produce hydrogen sulfide and the high affinity of sulfide to react with divalent metallic cations represent an excellent option to remove heavy metals from wastewater. Different parameters have been proposed to control the hydrogen sulfide production by anaerobic bacteria, such as the organic and sulfate loading rates and the feed COD/SO4(2-) ratio. This work relates the feed COD/SO4(2-) ratio with the hydrogen sulfide production and dissolved lead precipitation, using ethanol as carbon and energy source in an up-flow anaerobic sludge blanket reactor. A maximum dissolved sulfide concentration of 470+/-7 mg S/L was obtained at a feed COD/SO4(2-) ratio of 2.5, with sulfate and ethanol conversions of approximately 94 and 87%, respectively. The lowest dissolved sulfide concentration (145+/-10 mg S/L) was observed with a feed COD/SO4(2-) ratio of 0.67. Substantial amounts of acetate (510-1730 mg/L) were produced and accumulated in the bioreactor from ethanol oxidation. Although only incomplete oxidation of ethanol to acetate was observed, the consortium was able to remove 99% of the dissolved lead (200 mg/L) with a feed COD/SO4(2-) ratio of 1.5. It was found that the feed COD/SO4(2-) ratio could be an adequate parameter to control the hydrogen sulfide production and the consequent precipitation of dissolved lead.

Biodegradation of high concentrations of hexadecane by Aspergillus niger in a solid-state system: kinetic analysis.

Solid-state microcosms were used to assess the influence of constant and variable C/N ratios on the biodegradation efficiency by Aspergillus niger at high hexadecane (HXD) concentrations (180-717 mg g-1). With a constant C/N ratio, 100% biodegradation (33-44% mineralization) was achieved after 15 days, at rates increasing as the HXD concentration increased. Biomass yields (YX/S) remained almost independent (approximately 0.77) of the carbon-source amount, while the specific growth rates (mu) decreased with increasing concentrations of HXD. With C/N ratios ranging from 29 to 115, complete degradation was only attained at 180 mg g-1, corresponding to 46% mineralization. YX/S diminished (approximately 0.50 units) as the C/N ratio increased. The highest values of mu (1.08 day-1) were obtained at low C/N values. Our results demonstrate that, under balanced nutritional conditions, high HXD concentrations can be completely degraded in solid-state microcosms, with a negligible (<10%) formation of by-products.