Cryptococcus laurentii: a wild yeast for xylanase production from agricultural by-products.

The development of technologies that allow the production of enzymes at a competitive cost is of great importance for several biotechnological applications, and the use of agro-industrial by-products is an excellent alternative to minimize costs and reduce environmental impacts. This study aimed to produce endo-xylanases using agro-industrial substrates rich in hemicellulose as sources of xylan in culture media. For this purpose, the yeast Cryptococcus laurentti and five lignocellulosic materials (defatted rice bran, rice husk, corn cob, oat husks, and soybean tegument), with and without pretreatment, were used as a source of xylan for enzyme production. To insert the by-products in the culture medium, they were dried and treated (if applicable) with 4% (w.v-1) NaOH and then added in a concentration of 2% (w.v-1). The cultures were agitated for 96 h, and the aliquots were removed to determine the enzymatic activities. Among the by-products studied, the maximum activity (8.7 U. mL-1 at pH 7.3) was obtained where rice bran was used. In contrast, corn cob was the by-product that resulted in lower enzyme production (1.6 U.mL-1). Thus, the defatted rice bran deserves special attention in front of the other by-products used since it provides the necessary substrate for producing endo-xylanases by yeast.

Trichoderma koningiopsis fermentation in airlift bioreactor for bioherbicide production.

During scaling of fermentations, choosing a bioreactor is fundamental to ensure the product's quality. This study aims to produce bioherbicides using Trichoderma koningiopsis fermentation, evaluating process parameters in an Airlift bioreactor. As a response, we quantified the production of enzymes involved in the bioherbicide activity (amylase, cellulase, laccase, lipase, and peroxidase). In addition, it evaluated the agronomic efficiency of the fermented extract optimized through tests that promoted soybean growth and nodulation, soybean seed germination, and in vitro phytopathogen control. As a result of optimizing the scaling bioprocess, it was possible to obtain an adequate fermentation condition, which, when applied to soybean seeds, had beneficial effects on their growth. It allowed the production of an enzyme cocktail. These results add a crucial biotechnological potential factor for the success of the optimized formulation in the Airlift bioreactor, in addition to presenting relevant results for the scientific community.

Study and characterization of a product based on a vegetable extract of quinoa fermented with water kefir grains.

This work aimed to study and characterize a product based on vegetable extract of quinoa (WVEQ) fermented with water kefir grains. The effect of sucrose concentration (SC), inulin concentration (IC), and xanthan gum (XG) concentration were evaluated using a central composite design (CCD) 23. They were subsequently characterized regarding cellular growth of the grains, beverage yield, pH, soluble solids, carbon dioxide (CO2) production, lactic acid, and ethanol production. Therefore, for the final stage, two formulations (F1 and F8) of the CCD were chosen to be characterized in terms of proximate composition, microbiological composition of the kefir culture, analysis of organic compounds, sensory analysis, and enzymatic and microbiological characterization before and after simulation of in vitro gastrointestinal digestion. In the two chosen products, one can see that fermentation optimized the bioavailability of proteins due to the high proteolytic activity of the microorganisms in kefir and the increase in lipid content. In identifying microorganisms, there was a prevalence of Saccharomyces sp. yeasts. In the sensory analysis, the F8 formulation showed better results than the F1 formulation. In vitro, gastrointestinal digestion showed reduced lactic acid bacteria and yeast and increased acetic acid bacteria in the liquid phase for both formulations. In the enzymatic profile, there was a reduction in all enzymes analyzed for both formulations, except for amylase in F1, which went from 14.05 U/mL to 39.41 U/mL. Therefore, it is concluded that using WVEQ as a substrate for the product appears to be a viable alternative with nutritional and technological advantages for serving a specific market niche.

Prospecting and engineering yeasts for ethanol production under inhibitory conditions: an experimental design analysis.

The recently discovered wild yeast Wickerhamomyces sp. UFFS-CE-3.1.2 was analyzed through a high-throughput experimental design to improve ethanol yields in synthetic media with glucose, xylose, and cellobiose as carbon sources and acetic acid, furfural, formic acid, and NaCl as fermentation inhibitors. After Plackett-Burman (PB) and central composite design (CCD), the optimized condition was used in a fermentation kinetic analysis to compare this yeast's performance with an industrial Saccharomyces cerevisiae strain (JDY-01) genetically engineered to achieve a higher xylose fermentation capacity and fermentation inhibitors tolerance by overexpressing the genes XYL1, XYL2, XKS1, and TAL1. Our results show that furfural and NaCl had no significant effect on sugar consumption by UFFS-CE-3.1.2. Surprisingly, acetic acid negatively affected glucose but not xylose and cellobiose consumption. In contrast, the pH positively affected all the analyzed responses, indicating a cell's preference for alkaline environments. In the CCD, sugar concentration negatively affected the yields of ethanol, xylitol, and cellular biomass. Therefore, fermentation kinetics were carried out with the average concentrations of sugars and fermentation inhibitors and the highest tested pH value (8.0). Although UFFS-CE-3.1.2 fermented glucose efficiently, xylose and cellobiose were mainly used for cellular growth. Interestingly, the genetically engineered strain JDY-01 consumed ~ 30% more xylose and produced ~ 20% more ethanol. Also, while UFFS-CE-3.1.2 only consumed 32% of the acetic acid of the medium, JDY-01 consumed > 60% of it, reducing its toxic effects. Thus, the overexpressed genes played an essential role in the inhibitors' tolerance, and the applied engineering strategy may help improve 2G ethanol production.

The bioherbicidal potential of isolated fungi cultivated in microalgal biomass.

This study evaluated the bioherbicidal potential of wild fungi grown on microalgal biomass from the digestate treatment of biogas production. Four fungal isolates were used and the extracts were evaluated for the activity of different enzymes and characterized by gas chromatography coupled with mass spectrometry. The bioherbicidal activity was assessed by application on Cucumis sativus, and the leaf damage was visually estimated. The microorganisms showed potential as agents producing an enzyme pool. The obtained fungal extracts presented different organic compounds, most acids, and when applied to Cucumis sativus, showed high levels of leaf damage (80-100 ± 3.00%, deviation relative to the observed average damage). Therefore, the microbial strains are potential biological control agents of weeds, which, together with the microalgae biomass, offer the appropriate conditions to obtain an enzyme pool of biotechnological relevance and with favorable characteristics to be explored as bioherbicides, addressing aspects within the environmental sustainability.

Fungus-based bioherbicides on circular economy.

This review aimed to show that bioherbicides are possible in organic agriculture as natural compounds from fungi and metabolites produced by them. It is discussed that new formulations must be developed to improve field stability and enable the commercialization of microbial herbicides. Due to these bottlenecks, it is crucial to advance the bioprocesses behind the formulation and fermentation of bio-based herbicides, scaling up, strategies for field application, and the potential of bioherbicides in the global market. In this sense, it proposed insights for modern agriculture based on sustainable development and circular economy, precisely the formulation, scale-up, and field application of microbial bioherbicides.

Fruit residues as biomass for bioethanol production using enzymatic hydrolysis as pretreatment.

The commercialization of fruits in markets generates a large amount of waste because they are perishable and have a short shelf life, so, they are discarded. This study aimed to provide a noble end to discarded fruits that have fermentable sugars. Banana, apple, mango and papaya residues were collected from supermarkets and underwent an enzymatic hydrolysis process. The ability of four pectinases, two amylases, one xylanase and one cellulase to release reducing sugars from fruit biomass before fermentation with two yeast strains (S. cerevisiae CAT-1 and S. cerevisiae Angel) for bioethanol production was investigated, obtaining a total of RS (Reducing sugar) of 268.08 mg/mL in banana residues. A fermentation with yeast S. cerevisiae CAT-1 resulted in 98% consumption of RS and the production of a total of 28.02 g/L of ethanol. Furthermore, fermentation with the yeast S. cerevisiae Angel, resulted in 97% RS consumption and 31.87 g/L ethanol production, which was the best result obtained throughout all the tests of hydrolysis, highlighting the banana residue as a promising biomass for the production of bioethanol.

Influence of different PEG/salt aqueous two-phase system on the extraction of 2,3-butanediol.

The production of 2,3-butanediol (2,3-BDO), a dialcohol of great interest for the food, chemical, and pharmaceutical industry, through the fermentation of biomass, is a sustainable process strategic position for countries with abundant biomass generated by the agribusiness. However, the downstream process of 2,3-BDO is onerous due to the complexity of fermentation broth and the physical-chemical characteristics of the 2,3-BDO. This study investigated the feasibility of 2,3-BDO extraction from model aqueous solutions using aqueous two-phase systems (ATPS). A central composite rotational design (CCRD) was employed to evaluate different ATPS compositions and the influences on the 2,3-BDO recovery and partition coefficient. The polyethylene glycol (PEG) and different concentrations of sodium citrate, ammonium sulfate, and potassium phosphate were investigated. The concentration of salt and PEG in the ATPS was identified as the most significant factors influencing the recovery and partition coefficient of 2,3-BDO. The recovery of 2,3-BDO reached 94.5% and was obtained when the system was composed of 36.22% (w/w) of PEG 4000 and 4.47% (w/w) of potassium phosphate. The results indicate that ATPS based on PEG-salt has a high potential for industrial application, using mild conditions and a simple process for recovering and purifying the 2,3-BDO produced from microbiological synthesis.

Analysis of glucose and xylose metabolism in new indigenous Meyerozyma caribbica strains isolated from corn residues.

Aiming to broaden the base of knowledge about wild yeasts, four new indigenous strains were isolated from corn residues, and phylogenetic-tree assemblings on ITS and LSU regions indicated they belong to Meyerozyma caribbica. Yeasts were cultivated under full- and micro-aerobiosis, starting with low or high cell-density inoculum, in synthetic medium or corn hydrolysate containing glucose and/or xylose. Cells were able to assimilate both monosaccharides, albeit by different metabolic routes (fermentative or respiratory). They grew faster in glucose, with lag phases ~ 10 h shorter than in xylose. The hexose exhaustion occurred between 24 and 34 h, while xylose was entirely consumed in the last few hours of cultivation (44-48 h). In batch fermentation in synthetic medium with high cell density, under full-aerobiosis, 18-20 g glucose l-1 were exhausted in 4-6 h, with a production of 6.5-7.0 g ethanol l-1. In the xylose medium, cells needed > 12 h to consume the carbohydrate, and instead of ethanol, cells released 4.4-6.4 g l-1 xylitol. Under micro-aerobiosis, yeasts were unable to assimilate xylose, and glucose was more slowly consumed, although the ethanol yield was the theoretical maximum. When inoculated into the hydrolysate, cells needed 4-6 h to deplete glucose, and xylose had a maximum consumption of 57%. Considering that the hydrolysate contained ~ 3 g l-1 acetic acid, it probably has impaired sugar metabolism. Thus, this study increases the fund of knowledge regarding indigenous yeasts and reveals the biotechnological potential of these strains.

Biochemical analysis of cellobiose catabolism in Candida pseudointermedia strains isolated from rotten wood.

We isolated two Candida pseudointermedia strains from the Atlantic rain forest in Brazil, and analyzed cellobiose metabolization in their cells. After growth in cellobiose medium, both strains had high intracellular ß-glucosidase activity [~ 200 U (g cells)-1 for 200 mM cellobiose and ~ 100 U (g cells)-1 for 2 mM pNPßG] and negligible periplasmic cellobiase activity. During batch fermentation, the strain with the best performance consumed all the available cellobiose in the first 18 h of the assay, producing 2.7 g L-1 of ethanol. Kinetics of its cellobiase activity demonstrated a high-affinity hydrolytic system inside cells, with Km of 12.4 mM. Our data suggest that, unlike other fungal species that hydrolyze cellobiose extracellularly, both analyzed strains transport it to the cytoplasm, where it is then hydrolyzed by high-affinity intracellular ß-glucosidases. We believe this study increases the fund of knowledge regarding yeasts from Brazilian microbiomes.

Co-contamination of food products from family farms in an environmental disaster area in Southeast Brazil with pathogenic bacteria and enteric viruses.

In the present study, we evaluated the degree of contamination of fresh vegetables, cheeses and jellies from disaster area in Brazil with bacteria and enteric viruses. Food samples (n = 350) were tested for Escherichia coli, Salmonella spp., Listeria monocytogenes, Staphylococcus spp., and enteric viruses (rotavirus A (RVA), human adenovirus (HAdV), hepatitis A virus (HAV), and human norovirus (HNoV). E. coli was present in 56% of the samples, Salmonella spp. was present in 14% of the samples, L. monocytogenes and Staphylococcus spp. (coagulase-positive) were present in 36% of the samples. The enteric viruses RVA and HAdV were detected in cheeses and vegetables.

Extremophile Microbial Communities and Enzymes for Bioenergetic Application Based on Multi-Omics Tools.

Genomic and proteomic advances in extremophile microorganism studies are increasingly demonstrating their ability to produce a variety of enzymes capable of converting biomass into bioenergy. Such microorganisms are found in environments with nutritional restrictions, anaerobic environments, high salinity, varying pH conditions and extreme natural environments such as hydrothermal vents, soda lakes, and Antarctic sediments. As extremophile microorganisms and their enzymes are found in widely disparate locations, they generate new possibilities and opportunities to explore biotechnological prospecting, including biofuels (biogas, hydrogen and ethanol) with an aim toward using multi-omics tools that shed light on biotechnological breakthroughs.

Advanced oxidation processes applied for color removal of textile effluent using a home-made peroxidase from rice bran.

Enzymes are becoming tools in industrial processes because of several advantages, including activity in mild environmental conditions, and high specificity. Peroxidase, for one, stably oxidizes several substrates. The present study aimed to develop advanced oxidation processes (AOP), using non-commercial rice bran peroxidase to remove color and toxicity of synthetic textile wastewater. Using a microwave and shaker system, we obtained 38.9% and 100% of effluent color removal after peroxidase treatment, respectively. In addition, the shaker system decants residual dye particles through filtration, providing the textile industry with an economical and environmentally viable alternative to effluent treatment. In toxicity tests results, both treatment systems damaged the used genetic material. This damage occurs because of industrial discharge of wastewater into water bodies; effluent dilution reduced this damage. The data suggest that peroxidase as a textile effluent treatment has potential uses in industrial processes, because rice bran peroxidase has demonstrated affinity with dyes.

A review on alternative bioprocesses for removal of emerging contaminants.

Emerging contaminants (ECs) include endocrine-disrupting compounds, pharmaceuticals (lipid regulators, antibiotics, diuretics, non-steroid anti-inflammatory drugs, stimulant drugs, antiseptics, analgesic, beta blockers), detergents, disinfectants, and personal care products. The residues from these compounds have become a concerning because of their bioactive presence on environmental matrices, especially water bodies. The development of technologies, aiming the secure and efficient removal of these compounds from the environment or event to remove them before they achieve the environment, is necessary. In these context, the present review is about promising eco-friendly, low-cost and specially applied, including biological processes using microalgae, bacteria, enzymes produced by fungi, and adsorbent materials such as those recycled from other processes waste. The processes where revised considering the removal mechanism and the efficiency rate.

Cellulolytic enzyme production from agricultural residues for biofuel purpose on circular economy approach.

This study evaluated the production of cellulolytic enzymes from different agricultural residues. The crude enzyme extract produced was characterized and applied for saccharification of some agricultural residues. Maximum cellulolytic activities were obtained using soybean hulls. All enzymatic activities were highly stable at 40 °C at a pH range of 4.5-5.5. For stability at low temperatures, the enzyme extract was stored at freezing temperature and cooling for about 290 days without major loss of activity. The Km values found for total cellulase (FPase), endoglucanase (CMCase), and xylanase were 19.73 mg ml-1, 0.65 mg ml-1, and 22.64 mg ml-1, respectively, and Vmax values were 0.82 mol min-1 mg-1, 0.62 mol min-1 mg-1, and 104.17 mol min-1 mg-1 to cellulose, carboxymethyl cellulose, and xylan, respectively. In the saccharification tests, the total amount of total reducing sugars (TRS) released from 1 g of soybean hulls catalyzed by the enzymes present in the crude enzyme extract was 0.16 g g-1 dry substrate.

Evaluation of deammonification reactor performance and microrganisms community during treatment of digestate from swine sludge CSTR biodigester.

Digestate from anaerobic processes still contains relatively high amount of total organic carbon (TOC) that can inhibit deammonification. In this sense, the present study investigated the interference of TOC in a lab-scale expanded granular sludge bed (EGSB) deammonification reactor treating digestate from a continuous stirred tank reactor (CSTR) swine sludge biodigester. Additionally, the microorganisms community was analyzed when the process was submitted to different operational conditions. The study was divided into three phases according to the C/N ratio (0, 0.5 and 1 for phase I, phase II and phase III, respectively). At phase I the average nitrogen removal efficiency (NRE) was 65 ±â€¯1.6%. With the increase of TOC in phase II (156 ±â€¯8.15 mg L-1) the average NRE was 61 ±â€¯9.8% which is statically equivalent to phase I (p < 0.05). On the other hand, at phase III (TOC was increased to 255 ±â€¯3.50 mg L-1) the NRE decreased to 50 ±â€¯3.9% which was 22% lower than in phase II. Stoichiometric coefficients of N2 was close to theoretical values during all experimental phases, while stoichiometric coefficient of N-NO3- was lower than theoretical values specially during phase III. Ca. Jettenia was favored when the reactor was fed with digestate although its proportion decreased in phase III. Thus, at the conditions employed in the present study it is recommended to use a C/N ratio of 0.5 (TOC concentration around 156 mg L-1) to treat digestate by deammonification process, in order to not diminish anammox microorganisms abundance. Thereby, the microorganisms community can be modulated based on carbon and nitrogen loading rates of a deammonification reactor for swine manure treatment purpose.

Efficient and low-cost alternative of lipase concentration aiming at the application in the treatment of waste cooking oils.

In this study, we evaluated the concentration of lipases from Aspergillus niger using efficient and low-cost methods aiming at application in the treatment of waste cooking oils. The change in ionic strength of the medium by the addition of salt and precipitation with ethanol increased the specific activity from 2.90 to 28.50 U/mg, resulting in a purification factor of 9.82-fold. The use of acetone resulted in a specific activity of 33.63 U/mg, resulting in a purification factor of 11.60-fold. After that, the concentrated lipase was used in the hydrolysis of waste cooking oil and 753.07 and 421.60 µmol/mL of free fatty acids were obtained for the enzyme precipitated with ethanol and acetone, respectively. The hydrolysis of waste cooking oil catalyzed by homemade purified lipase in ultrasonic media can be considered a pretreatment of oil by converting a significant amount of triglycerides into free fatty acids.

Biotechnological potential of microbial inulinases: Recent perspective.

Among microbial enzymes, inulinases or fructo-furanosylhydrolases have received considerable attention in the past decade, and as a result, a variety of applications based on enzymatic hydrolysis of inulin have been documented. Inulinases are employed for generation of fructose and inulo-oligosaccharides (IOS) in a single-step reaction with specificity. The high fructose syrup can be biotransformed into value-added products such as ethanol, single cell protein, while IOS are indicated in nutraceutical industry as prebiotic. Myriad microorganisms produce inulinases, and a number of exo- and endo-inulinases have been characterized and expressed in heterologous hosts. Initially, predominated by Aspergilli, Penicillia, and some yeasts (Kluyveromyces spp.), the list of prominent inulinase producers has gradually expanded and now includes extremophilic prokaryotes and marine-derived microorganisms producing robust inulinases. The present paper summarizes important developments about microbial inulinases and their applications made in the last decade.

Purification of inulinases by changing the ionic strength of the medium and precipitation with alcohols.

The present study evaluated the purification of inulinase by changing the ionic strength of the medium by addition of NaCl and CaCl2 followed by precipitation with n-propyl alcohol or iso-propyl alcohol. The effects of the concentration of alcohols and the rate of addition of alcohols in the crude extract on the purification yield and purification factor were evaluated. Precipitation caused an activation of enzyme and allowed purification factors up to 2.4-fold for both alcohols. The purification factor was affected positively by the modification of the ionic strength of the medium to 0.5 mol.L-1 NaCl before precipitation with the alcohol (n-propyl or iso-propyl). A purification factor of 4.8-fold and an enzyme yield of 78.1 % could be achieved by the addition of 0.5 mol.L-1 of NaCl to the crude extract, followed by the precipitation with 50 % (v/v) of n-propyl alcohol, added at a flow rate of 19.9 mL/min.