Bioconversion of ferulic acid into aroma compounds by newly isolated yeast strains of the Latin American biodiversity.

Nine yeast strains isolated from Latin American biodiversity were screened for ferulic acid (FA) consumption and conversion into aroma compounds such as vanillin, vanillic acid (VA), and 4-vinylguaiacol (VG). Selected strains (Rhodotorula mucilaginosa UFMG-CM-Y3647, UFMG-CM-Y2190, UFMG-CM-Y665) were evaluated in flask experiments to investigate the influence of the pH media on bioconversion and a two-step process was conducted to maximize the metabolites production. The effect of pH was found to be significantly important for FA bioconversion, as acidic conditions (pH < 6.0) improved VA accumulation, with highest production of 1.14 ± 0.02 and 1.25 ± 0.03 g/L shown by UFMG-CM-Y3647 and UFMG-CM-Y2190, respectively. The two-step process favored 4-VG production for most strains, being UFMG-CM-Y2190 the best producer, its cultures reaching 1.63 ± 0.09 g/L after 55 hr, showing a productivity of 29.59 ± 1.55 mg/(L·hr), as glucose affected the metabolites pool and redirected yeast metabolism. R mucilaginosa UFMG-CM-Y3647 was selected for scaled-up cultivations in a 2-L bioreactor, where pH-controlled pH 5.5 and aeration of 2.5 vvm was found to be the best condition to improve VA productivity, attaining final concentrations of 1.20 ± 0.02 g/L-1 (78% molar yield) and a productivity of 40.82 ± 0.57 mg/(L·hr).

Biosynthesis of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 using liquid acid protein residue of soybean as culture medium.

Vitamin B12 deficiency still persists, mainly caused by low intake of animal food products affecting vegetarians, vegans, and populations of underdeveloped countries. In this study, we investigate the biosynthesis of vitamin B12 by potential probiotic bacterium using an agroindustry residue, the liquid acid protein residue of soybean (LAPRS), as a low-cost, animal derivate-free alternative culture medium. Cultures of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 growing in LAPRS for vitamin B12 biosynthesis were studied using the Plackett-Burman experimental approach, followed by a central composite design 22 to optimize the concentration of significant variables. We also performed a proteolytic treatment of LAPRS and evaluated the optimized-hydrolyzed medium influence on the microbial growth and metabolism in shaker flask and bioreactor experiments. In this all-plant source medium, P. freudenreichii subsp. shermanii produced high concentrations of cells and high amounts of vitamin B12 (0.6 mg/g cells) after process optimization. These results suggest the possibility of producing vitamin B12 by a potential probiotic bacterium in a very cheap, animal derivate-free medium to address the needs of specific population groups, at the same time reducing the production costs of this essential vitamin.

Oleaginous yeast Meyerozyma guilliermondii shows fermentative metabolism of sugars in the biosynthesis of ethanol and converts raw glycerol and cheese whey permeate into polyunsaturated fatty acids.

We studied the biotechnological potential of the recently isolated yeast Meyerozyma guilliermondii BI281A to produce polyunsaturated fatty acids and ethanol, comparing products yields using glucose, raw glycerol from biodiesel synthesis, or whey permeate as substrates. The yeast metabolism was evaluated for different C/N ratios (100:1 and 50:1). Results found that M. guilliermondii BI281A was able to assimilate all tested substrates, and the most efficient conversion obtained was observed using raw glycerol as carbon source (C/N ratio 50:1), concerning biomass formation (5.67 g·L-1 ) and lipid production (1.04 g·L-1 ), representing 18% of dry cell weight. Bioreactors experiments under pH and aeration-controlled conditions were conducted. Obtained fatty acids were composed of ~67% of unsaturated fatty acids, distributed as palmitoleic acid (C16:1 , 9.4%), oleic acid (C18:1 , 47.2%), linoleic acid (C18:2 n-6 , 9.6%), and linolenic acid (C18:3 n-3 , 1.3%). Showing fermentative metabolism, which is unusual for oleaginous yeasts, M. guilliermondii produced 13.7 g·L-1 of ethanol (yields of 0.27) when growing on glucose medium. These results suggest the promising use of this uncommonly studied yeast to produce unsaturated fatty acids and ethanol using cheap agro-industrial residues as substrates in bioprocess.

Enzymatic synthesis of ethyl esters from waste oil using mixtures of lipases in a plug-flow packed-bed continuous reactor.

This work describes the continuous synthesis of ethyl esters via enzymatic catalysis on a packed-bed continuous reactor, using mixtures of immobilized lipases (combi-lipases) of Candida antarctica (CALB), Thermomyces lanuginosus (TLL), and Rhizomucor miehei (RML). The influence of the addition of glass beads to the reactor bed, evaluation of the use of different solvents, and flow rate on reaction conditions was studied. All experiments were conducted using the best combination of lipases according to the fatty acid composition of the waste oil (combi-lipase composition: 40% of TLL, 35% of CALB, and 25% of RML) and soybean oil (combi-lipase composition: 22.5% of TLL, 50% of CALB, and 27.5% of RML). The best general reaction conditions were found to be using tert-butanol as solvent, and the flow rate of 0.08 mL min-1 . The combi-lipase reactors operating at steady state for over 30 days (720 h), kept conversion yields of ∼50%, with average productivity of 1.94 gethyl estersgsubstrate-1 h-1 , regardless of the type of oil in use. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:952-959, 2018.

Liberation of fermentable sugars from soybean hull biomass using ionic liquid 1-butyl-3-methylimidazolium acetate and their bioconversion to ethanol.

Optimized hydrolysis of lignocellulosic waste biomass is essential to achieve the liberation of sugars to be used in fermentation process. Ionic liquids (ILs), a new class of solvents, have been tested in the pretreatment of cellulosic materials to improve the subsequent enzymatic hydrolysis of the biomass. Optimized application of ILs on biomass is important to advance the use of this technology. In this research, we investigated the effects of using 1-butyl-3-methylimidazolium acetate ([bmim][Ac]) on the decomposition of soybean hull, an abundant cellulosic industrial waste. Reaction aspects of temperature, incubation time, IL concentration, and solid load were optimized before carrying out the enzymatic hydrolysis of this residue to liberate fermentable glucose. Optimal conditions were found to be 75°C, 165 min incubation time, 57% (mass fraction) of [bmim][Ac], and 12.5% solid loading. Pretreated soybean hull lost its crystallinity, which eased enzymatic hydrolysis, confirmed by Fourier Transform Infrared analysis. The enzymatic hydrolysis of the biomass using an enzyme complex from Penicillium echinulatum liberated 92% of glucose from the cellulose matrix. The hydrolysate was free of any toxic compounds, such as hydroxymethylfurfural and furfural. The obtained hydrolysate was tested for fermentation using Candida shehatae HM 52.2, which was able to convert glucose to ethanol at yields of 0.31. These results suggest the possible use of ILs for the pretreatment of some lignocellulosic waste materials, avoiding the formation of toxic compounds, to be used in second-generation ethanol production and other fermentation processes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:312-320, 2016.

Improved enzyme stability in lipase-catalyzed synthesis of fatty acid ethyl ester from soybean oil.

In this work, we describe the optimization of the ethanolysis of soybean oil by the enzyme Lipozyme TL-IM in the lipase-catalyzed biodiesel synthesis and the improvement of the enzyme stability over repeated batches. The studied process variables were: reaction temperature, substrate molar ratio, enzyme content, and volume of added water. Fractional factorial design was used to analyze the variables so as to select those with higher influence on the reaction and then perform a central composite design to find the optimal reaction conditions. The optimal conditions found were: temperature, 26 degrees C; substrate molar ratio, 7.5:1 (ethanol/oil); enzyme content, 25% in relation to oil weight; and added water, 4% in relation to oil weight. Under these conditions, the yield conversion obtained was 69% in 12 h. The enzyme stability assessment in repeated batches was carried out by washing the immobilized enzyme with different solvents (n-hexane, water, ethanol, and propanol) after each batch. In the treatment with n-hexane, around 80% of the enzyme activity still remains after seven cycles of synthesis, suggesting its economical application on biodiesel production.

Purification and properties of a transglutaminase produced by a Bacillus circulans strain isolated from the Amazon environment.

A new microbial transglutaminase (EC 2.3.2.13) from a Bacillus circulans strain isolated from the aquatic Amazonian environment was purified and characterized. Enzyme purification started with (NH(4))(2)SO(4) 'salting out' and proceeded with liquid chromatography on Q-Sepharose FF and octyl-Sepharose 4 FF. The purification factor was approx. 150-fold with a yield of 32%. The enzyme's molecular mass was estimated as 45000 Da on SDS/PAGE. The purified transglutaminase had an optimum temperature of 47 degrees C, the optimum pH of the reaction was 7 and it presented no calcium-dependent activity.