María Amelia Cubitto

Effects of Bacillus subtilis O9 biosurfactant on the bioremediation of crude oil-polluted soils

The application of a surfactant from Bacillus subtilis O9 (Bs) on the bioremediation of soils polluted with crude oil was assayed in soil microcosms under laboratory conditions. Three concentrations of biosurfactant were assayed (1.9, 19.5, and 39 mg kg−1 soil). Microcosms without biosurfactant were prepared as controls. During the experiment, the crude oil-degrading bacterial population, the aliphatic and aromatic hydrocarbons were monitored in each microcosm. The results indicated that applying Bs did not negatively affect the hydrocarbon-degrading microbial population Concentrations of 19 and 19.5 mg (Bs) per kilogram of soil stimulated the growth of the population involved in the crude oil degradation, and accelerated the biodegradation of the aliphatic hydrocarbons. However, none of the assayed Bs concentrations stimulated aromatic hydrocarbon degradation.

Biosynthesis of PHB from a new isolated Bacillus megaterium strain: Outlook on future developments with endospore forming bacteria

Diverse Bacillus strains are known as producers of polyhydroxyalkanoates (PHAs) under nutrient-limiting conditions. However, these limiting conditions have the same nutritional characteristics that stimulate spore generation in Gram-positive microorganisms. In the present work, a new isolated Bacillus megaterium strain was characterized based on 16S rRNA gene sequences (1,411 bp) and studied in terms of its ability for producing polyhydroxybutyrate (PHB) by implementing different fermentation configurations on formulated media. The isolated strain was able to produce PHB up to 59 and 60% of its dry cell weight during bioreactor experiments employing glucose and glycerol as carbon source, respectively. The produced biopolymer was characterized and identified by using carbon-13 nuclear magnetic resonance (13C-NMR) and Fourier transform infrared (FTIR) techniques. In spite of the sporulation phenomenon existing in Bacillus strains, obtained results demonstrate that the new isolated strain has the potential of accumulating high levels of intracellular PHB. Supported by these experimental results and by those reported by other authors, the last section of this paper gives an outlook of future research topics on PHB and polyhydroxyalkanoate (PHA) copolymers production by Gram-positive bacteria. The importance of combining bioprocessing/biorefinering concepts with bioreactor optimization approaches is stressed and analyzed based on current PHAs research trends.

Isolation, identification and antimicrobial activity of lactic acid bacteria from the Bahía Blanca Estuary

This study analyzed the biodiversity of lactic acid bacteria present in the Bahia Blanca Estuary and their antimicrobial activity against pathogens associate...

Pyrolysis of sunflower seed hulls for obtaining bio-oils

Bio-oils from pyrolysis of as received sunflower seed hulls (SSH), hulls previously washed with acid (SSHA) and hulls submitted to a mushroom enzymatic attack (BSSH) were analyzed. The concentration of lignin, hemicellulose and cellulose varied with the pre-treatment. The liquid corresponding to SSH presented a relatively high concentration of acetic acid and a high instability to storage. The bio-oil from SSHA showed a high concentration of furfural and an appreciable amount of levoglucosenone. Lignin was degraded upon enzymatic activity, for this reason BSSH led to the highest yield of bio-oil, with relative high concentration of acetic acid and stability to storage.

Pilot-scale bioconversion of rice and sunflower agro-residues into medicinal mushrooms and laccase enzymes through solid-state fermentation with Ganoderma lucidum

Solid-state fermentation was evaluated at the pilot-scale for the bioconversion and valorization of rice husks and straw (RSH), or sunflower seed hulls (SSH), into medicinal mushrooms and crude extracts, with laccase activity. The average mushroom yield was 56kg dry weight per ton of agro-residues. Laccase activity in crude aqueous extracts showed its maximum value of 10,927Ukg-1 in RSH (day 10, Exudate phase) and 16,442Ukg-1 in SSH (day 5, Full colonization phase), the activity at the Residual substrate phase being 511Ukg-1 in RSH and 803Ukg-1 in SSH, respectively. Crude extracts obtained with various protocols revealed differences in the extraction yields. Lyophilization followed by storage at 4°C allowed the preservation of laccase activity for more than one month. It is proposed that standard mushroom farms could increase their profits by obtaining laccase as a byproduct during the gaps in mycelium running.

DNA damaging potential of Ganoderma lucidum extracts

ETHNOPHARMACOLOGICAL RELEVANCE Ganoderma lucidum (Lingzhi or Reishi) is a medicinal mushroom historically used in Asian countries to treat a wide variety of diseases and prolong life. In the last years, G. lucidum has been internationally recognized as an effective adjuvant in cancer treatment. Among active components, the most recent research indicates that polysaccharides modulate the immune response favoring the recovery from toxicity of chemo and radiotherapy while triterpenes are cytotoxic to tumoral cells mainly by altering gene expression. Beyond this body of evidence on the efficacy of G. lucidum in cancer treatment, it is not yet understood whether these extracts exert the same mechanisms of action than current antitumoral drugs. AIM OF THE STUDY In this study, we tested the DNA damaging potential of G. lucidum extracts by the β-galactosidase biochemical prophage induction assay (BIA) using doxorubicin, a DNA intercalating agent, as a positive control. This assay was traditionally used to screen microbial metabolites towards antitumoral agents. Here, we used this bacterial assay for the first time to assess DNA damage of herbal drugs. RESULTS After a bioguided assay, only a purified fraction of G. lucidum containing a mixture of C16 and C18:1 fatty acids exerted weak activity which could not be attributed to direct interaction with DNA. At the same concentrations, the induction observed for doxorubicin was clearly contrasting. CONCLUSIONS The micro BIA assay could be successfully used to demonstrate differences in cellular effects between G. lucidum extracts and doxorubicin. These results showed that G. lucidum extracts display weak DNA damaging potential. Since DNA injury promotes aging and cancer, our results substantiate the traditional use of this mushroom to prolong life.

Modeling the bioconversion of starch to P(HB-co-HV) optimized by experimental design using Bacillus megaterium BBST4 strain

ABSTRACT Poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) is a prominent biopolymer as a potential candidate for use in the biomedical area. Several Bacillus spp. strains show promising characteristics in the use of several carbon sources and are an interesting alternative for the production of P(HB-co-HV). Sewage from the agricultural and food processing industries can be used to obtain abundantly starch as a carbon source for PHA production. The aim of the present study was to optimize by response surface methodology and desirability, the production of PHA by a Bacillus megaterium strain using starch as the sole carbon source. Two optimal conditions were determined without sporulation and were used to perform new experiments to calibrate and validate a mechanistic model, developed to simulate the dynamics of PHA and biomass production. The developed model successfully represents the kinetics of the microorganism. Employing different characterization techniques, it was determined that the PHA produced by the strain is a copolymer composed of different HB:HV proportions. Using starch as the sole carbon source in a minimal salt medium, this work shows the first reports in the literature of: 1) a mathematical model for predicting growth kinetic and PHA production for B. megaterium strain and 2) a Bacillus spp. producing P(HB-co-HV) copolymer.

Novel spectrophotometric technique for rapid determination of extractable PHA using Sudan black dye

Classical techniques employed to determine the amount of extractable poly(hydroxyalkanoate)s (PHAs) from cells, are laborious and destructive. Sudan black staining is commonly used in the laboratory to investigate the presence of intracellular PHA. The aim of the present study was to develop a low-cost alternative technique to achieve a quick determination of extractable intracellular PHA. This methodology employs a basic laboratory spectroscopy equipment and Sudan black dye for spectra determination. The correlation between the content of PHA in cell samples taken directly from the culture flask and its spectra was determined using partial least square regression analysis and simple linear regression analysis. The best fit obtained for calibration correlation analysis (R2=0.944, RSE: 1.24%), together with the good extractable PHA predictions (RSE=0.51%) demonstrate that the proposed methodology constitutes a fast way with high potential for the determination of extractable PHA. Based on its simplicity and flexibility, its application would be suitable in routine monitoring and rapid quantification in large-scale processes involving PHA metabolism.

Improved intracellular PHA determinations with novel spectrophotometric quantification methodologies based on Sudan black dye

The presence of intracellular polyhydroxyalkanoates (PHAs) is usually studied using Sudan black dye solution (SB). In a previous work it was shown that the PHA could be directly quantified using the absorbance of SB fixed by PHA granules in wet cell samples. In the present paper, the optimum SB amount and the optimum conditions to be used for SB assays were determined following an experimental design by hybrid response surface methodology and desirability-function. In addition, a new methodology was developed in which it is shown that the amount of SB fixed by PHA granules can also be determined indirectly through the absorbance of the supernatant obtained from the stained cell samples. This alternative methodology allows a faster determination of the PHA content (involving 23 and 42 min for indirect and direct determinations, respectively), and can be undertaken by means of basic laboratory equipment and reagents. The correlation between PHA content in wet cell samples and the spectra of the SB stained supernatant was determined by means of multivariate and linear regression analysis. The best calibration adjustment (R2 = 0.91, RSE: 1.56%), and the good PHA prediction obtained (RSE = 1.81%), shows that the proposed methodology constitutes a reasonably precise way for PHA content determination. Thus, this methodology could anticipate the probable results of the above mentioned direct PHA determination. Compared with the most used techniques described in the scientific literature, the combined implementation of these two methodologies seems to be one of the most economical and environmentally friendly, suitable for rapid monitoring of the intracellular PHA content.