Highlight on the problems generated by p-coumaric acid analysis in fermentations

p-Coumaric acid is a natural hydroxycinnamic acid existing in grapes and wine. It is the precursor of the 4-ethylphenol molecule through the bioconversion reaction by Brettanomyces yeast. Chromatographic methods are the most common techniques to detect p-coumaric acid. It is known that this acid is highly unstable in analysis and fermentation experiments. This paper highlights the problems occurring in p-coumaric acid analysis in wine fermentation conditions when studying its bioconversion. First, it was shown that p-coumaric acid was unstable at elevated temperature. On the other hand, it was found that in our experimental conditions p-coumaric acid reacted with ethanol. This work revealed also that the p-coumaric acid is partially adsorbed on Brettanomyces yeast, certainly on cell walls. Because of these phenomena the quantity of p-coumaric acid which can participate to the bioconversion into ethylphenol decreases

In-situ physical analysis of cellulose fibre suspensions during enzymatic hydrolysis

In-situ physical analysis of cellulose fibre suspensions during enzymatic hydrolysi

Fungi isolated from Miscanthus and sugarcane: biomass conversion, fungal enzymes, and hydrolysis of plant cell wall polymers.

BackgroundBiofuel use is one of many means of addressing global change caused by anthropogenic release of fossil fuel carbon dioxide into Earth's atmosphere. To make a meaningful reduction in fossil fuel use, bioethanol must be produced from the entire plant rather than only its starch or sugars. Enzymes produced by fungi constitute a significant percentage of the cost of bioethanol production from non-starch (i.e., lignocellulosic) components of energy crops and agricultural residues. We, and others, have reasoned that fungi that naturally deconstruct plant walls may provide the best enzymes for bioconversion of energy crops.ResultsPreviously, we have reported on the isolation of 106 fungi from decaying leaves of Miscanthus and sugarcane (Appl Environ Microbiol 77:5490-504, 2011). Here, we thoroughly analyze 30 of these fungi including those most often found on decaying leaves and stems of these plants, as well as four fungi chosen because they are well-studied for their plant cell wall deconstructing enzymes, for wood decay, or for genetic regulation of plant cell wall deconstruction. We extend our analysis to assess not only their ability over an 8-week period to bioconvert Miscanthus cell walls but also their ability to secrete total protein, to secrete enzymes with the activities of xylanases, exocellulases, endocellulases, and beta-glucosidases, and to remove specific parts of Miscanthus cell walls, that is, glucan, xylan, arabinan, and lignin.ConclusionThis study of fungi that bioconvert energy crops is significant because 30 fungi were studied, because the fungi were isolated from decaying energy grasses, because enzyme activity and removal of plant cell wall components were recorded in addition to biomass conversion, and because the study period was 2 months. Each of these factors make our study the most thorough to date, and we discovered fungi that are significantly superior on all counts to the most widely used, industrial bioconversion fungus, Trichoderma reesei. Many of the best fungi that we found are in taxonomic groups that have not been exploited for industrial bioconversion and the cultures are available from the Centraalbureau voor Schimmelcultures in Utrecht, Netherlands, for all to use

Assembly, growth and conductive properties of tellurium nanorods produced by Rhodococcus aetherivorans BCP1

Tellurite (TeO32-) is a hazardous and toxic oxyanion for living organisms. However, several microorganisms can bioconvert TeO32- into the less toxic form of elemental tellurium (Te0). Here, Rhodococcus aetherivorans BCP1 resting (non-growing) cells showed the proficiency to produce tellurium-based nanoparticles (NPs) and nanorods (NRs) through the bioconversion of TeO32-, depending on the oxyanion initial concentration and time of cellular incubation. Te-nanostructures initially appeared in the cytoplasm of BCP1 cells as spherical NPs, which, as the exposure time increased, were converted into NRs. This observation suggested the existence of an intracellular mechanism of TeNRs assembly and growth that resembled the chemical surfactant-assisted process for NRs synthesis. The TeNRs produced by the BCP1 strain showed an average length (>700 nm) almost doubled compared to those observed in other studies. Further, the biogenic TeNRs displayed a regular single-crystalline structure typically obtained for those chemically synthesized. The chemical-physical characterization of the biogenic TeNRs reflected their thermodynamic stability that is likely derived from amphiphilic biomolecules present in the organic layer surrounding the NRs. Finally, the biogenic TeNRs extract showed good electrical conductivity. Thus, these findings support the suitability of this strain as eco-friendly biocatalyst to produce high quality tellurium-based nanomaterials exploitable for technological purposes

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47{Delta}B) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts

Liquid state bioconversion of palm oil mill effluent for cellulase production: statistical optimization of process conditions

The filamentous fungus Trichoderma harzianum was used for liquid state bioconversion of POME for cellulase production. Statistical optimization was carried out to evaluate the physico-chemical parameters (factors) for maximum cellulase production by 2-level fractional factorial design with six central points. The polynomial regression model was developed using the experimental data including the effects of linear, quadratic and interaction of the factors. The factors involved were substrate (POME) and co-substrate (wheat flour) concentrations, temperature, pH, inoculum and agitation. Statistical analysis showed that the optimum conditions were: temperature of 300C, substrate concentration of 2%, wheat flour concentration of 3%, pH of 4, inoculum of 3% and agitation of 200 rpm. Under these conditions, the model predicted the enzyme production to be about 14 FPU/ml. Analysis of variance (ANOVA) of the design showed a high coefficient of determination (R2) value of 0.99, thus ensuring a high satisfactory adjustment of the quadratic model with the experimental data

Biocomposting process for utilization agro-industrial wastes

Biological treatment has played prominent roles in bioremediation of wastes and contaminants. Composting is one of the biological process that has been considered to be one of the most suitable ways of converting organic wastes into products that are beneficial for plant growth. The study of the bio-composting process by solid-state bioconversion utilizing palm oil mill effluent (POME) and empty fruit bunch (EFB) were studied in lab scale. From the study it is found that horizontal rotary drum bioreactor was the most suitable to run this study. In this project, four filamentous fungi were used; (i) Phanerochaete chrysosporium. (ii) Trichoderma harzianum. (iii) Aspergillus niger. (iv) Penicillium sp. The bioconversion of lignocellulosic materials by evaluating the C/N ratio and other parameters using horizontal rotary drum bioreactor were studied. The good and mature of the compost is reflected by C/N ratio, germination index and glucosamine assay. The result showed that the C/N ratio drop from days 10 onwards and in the range of 25 to 30. The germination indexes of 50 to 70% indicate that the compost produce was a phyto toxic-free product and merely achieved as mature compost. The composting period required to complete this process was two months. Thus, this study developed an effective and feasible composting technique of POME and EFB using horizontal rotary drum bioreactor by solid state bioconversion process

Nutritional value of Pleurotus (Flabellatus) Djamor (R-22) cultivated on sawdusts of different woods

The sawdust of different woods were investigat ed for the cultivation of exotic strain of Pleurotus (flabellatus) djamor (R-22) to find out the efficiency of different nutrients including protein, fat, crude fiber, ash, dry matter and moisture. Among all type of nutrients, protein, fat, cr ude fiber, ash, dry matter and moisture of Pleurotus ostreatus on sawdust of different woods were observed. Protein was observed on cont rol treatment (cotton waste, kikar, mango, mixed sawdust, simbal and kail (21.89), (21.64), (21.34), (21.16), (21.03) and (20.75) % respectively. Fat was observed on control treatment (cotton waste, kikar, mango, mi xed sawdust, simbal and kail (0.80), (0.53), (0 .41), (0.33), (0.24) and (0.11)% respectively. Crude fiber was observed on control treatment (cotton waste, kikar, mango, mixed sawdust, simbal and kail (8.92), (8.45), (8.17), (7.96), (7.70) and (7.32) % respectively. Ash was observ ed on control treatment (cotton waste, kikar, mango, mixed sawdust, simbal and kail (7.65), (6.75), (6 .47), (6.39), (6.33) and (6.23%) respectively. Dry matter was observed on control treatment (cotton waste, kikar, mango, mixed sawdust, simbal and kail (6.47), (6.27), (6.13), (6.01), (5.87) and (5.67) % respectively. Moisture was observed on control treatment (c otton waste, kikar, mango, mixed sawdust, simbal and kail (84.55), (81.20), (79.85), (76.26), (74.35) and (71.14) % respectively. Oyster mushroom showed relatively more contents on control treatment cotton waste as compared to other substrates. The maximum protein, fat, crude fiber, ash, dry matter and moisture contents in Pleurotus (flabellatus) djamor (R-22) was obtained on Kikar sawdust .The lowest contents was obtained on kail sawdust

Ochrobactrum sp. MPV1 from a dump of roasted pyrites can be exploited as bacterial catalyst for the biogenesis of selenium and tellurium nanoparticles

Background: Bacteria have developed different mechanisms for the transformation of metalloid oxyanions to non-toxic chemical forms. A number of bacterial isolates so far obtained in axenic culture has shown the ability to bioreduce selenite and tellurite to the elemental state in different conditions along with the formation of nanoparticles-both inside and outside the cells-characterized by a variety of morphological features. This reductive process can be considered of major importance for two reasons: firstly, toxic and soluble (i.e. bioavailable) compounds such as selenite and tellurite are converted to a less toxic chemical forms (i.e. zero valent state); secondly, chalcogen nanoparticles have attracted great interest due to their photoelectric and semiconducting properties. In addition, their exploitation as antimicrobial agents is currently becoming an area of intensive research in medical sciences. Results: In the present study, the bacterial strain Ochrobactrum sp. MPV1, isolated from a dump of roasted arsenopyrites as residues of a formerly sulfuric acid production near Scarlino (Tuscany, Italy) was analyzed for its capability of efficaciously bioreducing the chalcogen oxyanions selenite (SeO32-) and tellurite (TeO32-) to their respective elemental forms (Se0 and Te0) in aerobic conditions, with generation of Se- and Te-nanoparticles (Se- and TeNPs). The isolate could bioconvert 2 mM SeO32- and 0.5 mM TeO32- to the corresponding Se0 and Te0 in 48 and 120 h, respectively. The intracellular accumulation of nanomaterials was demonstrated through electron microscopy. Moreover, several analyses were performed to shed light on the mechanisms involved in SeO32- and TeO32- bioreduction to their elemental states. Results obtained suggested that these oxyanions are bioconverted through two different mechanisms in Ochrobactrum sp. MPV1. Glutathione (GSH) seemed to play a key role in SeO32- bioreduction, while TeO32- bioconversion could be ascribed to the catalytic activity of intracellular NADH-dependent oxidoreductases. The organic coating surrounding biogenic Se- and TeNPs was also characterized through Fourier-transform infrared spectroscopy. This analysis revealed interesting differences among the NPs produced by Ochrobactrum sp. MPV1 and suggested a possible different role of phospholipids and proteins in both biosynthesis and stabilization of such chalcogen-NPs. Conclusions: In conclusion, Ochrobactrum sp. MPV1 has demonstrated to be an ideal candidate for the bioconversion of toxic oxyanions such as selenite and tellurite to their respective elemental forms, producing intracellular Se- and TeNPs possibly exploitable in biomedical and industrial applications.[Figure not available: see fulltext.

Introducing the concept of biocatalysis in the classroom: The conversion of cholesterol to provitamin D 3

Biocatalysis is a fundamental concept in biotechnology. The topic integrates knowledge of several disciplines; therefore, it was included in the course “design and optimization of biological systems” which is offered in the biochemistry curricula. We selected the ciliate tetrahymena as an example of a eukaryotic system with potential for the biotransformation of sterol metabolites of industrial interest; in particular, we focused on the conversion of cholesterol to provitamin D3. The students work with wild type and recombinant strains and learn how sterol pathways could be modified to obtain diverse sterol moieties. During the course the students identify and measure the concentration of sterols. They also search for related genes by bioinformatic analysis. Additionally, the students compare biotransformation rates, growing the ciliate in plate and in a bioreactor. Finally, they use fluorescence microscopy to localize an enzyme involved in biotransformation. The last day each team makes an oral presentation, explaining the results obtained and responds to a series of key questions posed by the teachers, which determine the final mark. In our experience, this course enables undergraduate students to become acquainted with the principles of biocatalysis as well as with standard and modern techniques, through a simple and robust laboratory exercise, using a biological system for the conversion of valuable pharmaceutical moieties.Fil: De Luca, Belén M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gonzalez, Rodrigo Horacio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Nusblat, Alejandro David. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin