Kluyveromyces marxianus: a potential biocatalyst of renewable chemicals and lignocellulosic ethanol production.

Kluyveromyces marxianus is an ascomycetous yeast which has shown promising results in cellulosic ethanol and renewable chemicals production. It can survive on a variety of carbon sources under industrially favorable conditions due to its fast growth rate, thermotolerance, and acid tolerance. K. marxianus, is generally regarded as a safe (GRAS) microorganism, is widely recognized as a powerhouse for the production of heterologous proteins and is accepted by the US Food and Drug Administration (USFDA) for its pharmaceutical and food applications. Since lignocellulosic hydrolysates are comprised of diverse monomeric sugars, oligosaccharides and potential metabolism inhibiting compounds, this microorganism can play a pivotal role as it can grow on lignocellulosic hydrolysates coping with vegetal cell wall derived inhibitors. Furthermore, advancements in synthetic biology, for example CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, will enable development of an engineered yeast for the production of biochemicals and biopharmaceuticals having a myriad of industrial applications. Genetic engineering companies such as Cargill, Ginkgo Bioworks, DuPont, Global Yeast, Genomatica, and several others are actively working to develop designer yeasts. Given the important traits and properties of K. marxianus, these companies may find it to be a suitable biocatalyst for renewable chemicals and fuel production on the large scale. This paper reviews the recent progress made with K. marxianus biotechnology for sustainable production of ethanol, and other products utilizing lignocellulosic sugars.

Impact of long-term high-fat diet intake gestational protein-restricted offspring on kidney morphology and function.

Emerging evidence highlights the far-reaching consequences of high-fat diet (HFD) and obesity on kidney morphological and functional disorders. In the present study, we aim to evaluate the effects of early HFD intake on renal function and morphology in maternal protein-restricted offspring (LP). LP and normal protein-intake offspring (NP) were fed HFD (LPH and NPH, respectively) or standard rodent (LPN and NPN) diet from the 8th to 13th week of age. Blood pressure, kidney function, immunohistochemistry and scanning electron microscopy were analyzed. Increased total cholesterol and low-density lipoprotein serum levels were observed in LPH offspring. The adiposity index was reduced in the (LPN) group and, conversely, increased in the NPH and LPH groups. Blood pressure was higher beyond the 10th week of age in the LPH group compared with the other groups. Decreased urinary sodium excretion was observed in LP offspring, whereas the HFD-treated groups presented a decreased urine pH in a time-dependent fashion. The LPN, NPH and LPH groups showed increased expression of type 1 angiotensin II (AngII) receptor (AT1R), TGF-ß1, collagen and fibronectin in the kidneys. Moreover, the adult fetal-programmed offspring showed pronounced effacement of the podocyte foot process associated with the rupture of cell membranes and striking urinary protein excretion, exacerbated by HFD treatment. To the best of our knowledge, this is the first study demonstrating that young fetal-programmed offspring submitted to long-term HFD intake have increased susceptibility to renal structural and functional disorders associated with an accentuated stage of fibrosis and tubular dysfunction.

Evaluation of sorghum straw hemicellulosic hydrolysate for biotechnological production of xylitol by Candida guilliermondii

A preliminary study on xylitol production by Candida guilliermondii in sorghum straw hemicellulosic hydrolysate was performed. Hydrolysate had high xylose content and inhibitors concentrations did not exceed the commonly found values in other hemicellulosic hydrolysates. The highest xylitol yield (0.44 g/g) and productivity (0.19 g/Lh) were verified after 72 hours.

Evaluation of sorghum straw hemicellulosic hydrolysate for biotechnological production of xylitol by Candida guilliermondii.

A preliminary study on xylitol production by Candida guilliermondii in sorghum straw hemicellulosic hydrolysate was performed. Hydrolysate had high xylose content and inhibitors concentrations did not exceed the commonly found values in other hemicellulosic hydrolysates. The highest xylitol yield (0.44 g/g) and productivity (0.19 g/Lh) were verified after 72 hours.

Evaluation of sorghum straw hemicellulosic hydrolysate for biotechnological production of xylitol by Candida guilliermondii

A preliminary study on xylitol production by Candida guilliermondii in sorghum straw hemicellulosic hydrolysate was performed. Hydrolysate had high xylose content and inhibitors concentrations did not exceed the commonly found values in other hemicellulosic hydrolysates. The highest xylitol yield (0.44 g/g) and productivity (0.19 g/Lh) were verified after 72 hours.

Produção de conídios de Metarhizium anisopliae em meio sólido à base de resíduos agroindustriais / Production of conidia of Metarhizium anisopliae in solid media based on agroindustrial residues

As crescentes despesas com o custo do meio para o cultivo de Metarhizium anisopliae levantam a necessidade de averiguar a eficiência de alguns resíduos agroindustriais que, além de propriedades nutricionais importantes, possuem grande disponibilidade e baixo custo. Os experimentos foram conduzidos no Laboratório de Tecnologia das Fermentações, UNIOESTE, Campus de Cascavel, PR. Neste trabalho, foram avaliados como substratos arroz polido (meio padrão), arroz vermelho, arroz polido suplementado com melaço (2,5%, 5% e 10%), resíduo de cervejaria, resíduo de fecularia, arroz polido + resíduo de cervejaria (1:1) e arroz polido + resíduo de fecularia (1:1). Os experimentos foram realizados em triplicata, em frascos Erlernmeyer de 250 mL contendo 30 g do substrato (ou 15 g de cada, quando em mistura) e umidade de 70 ± 10%. Os frascos foram inoculados com 0,5 mL da suspensão de conídios (1 × 108 conídios/mL) e incubados em BOD (25 ± 1o C, fotofase 12h, 7 dias). Dentre os meios testados, verificou-se uma elevada produção de conídios no meio composto por arroz + resíduo de cervejaria (11,3 × 108 conídios/g de substrato) e no arroz (9,6 × 108 conídios/g). Os conídios produzidos no meio de arroz + resíduo de cervejaria apresentaram atividade inseticida frente a Spodoptera frugiperda semelhante a dos conídios produzidos no arroz. Este novo meio de cultivo poderá proporcionar uma redução de custo de aproximadamente 50%, representando uma alternativa viável para a produção do fungo.

Increasing costs of substrate for production of Metarizhiuma nisopliae raise the necessity of investigating the efficiency of some agro-industrial residues that, in addition to important nutritional properties, are widely available at low cost. The assays were conducted at the Fermentation Technology Laboratory, UNIOESTE, Cascavel, PR, Brazil. In this work, substrates such as polished rice (standard media), red rice, polished rice + sugar molasses (2.5%, 5% and 10%), brewery residue, cassava residue, polished rice + brewery residue (1:1) and polished rice + cassava residue (1:1) were evaluated. The experiments were carried out in triplicate, in Erlernmeyer flasks of 250 mL, containing 30 g of the substrate (or 15 g of each one, when as mixture) and moisture content of 70 ± 10%. The flasks were inoculated with 0.5 mL of conidia suspension (1 × 108 conidia/mL) and then incubated in controlled temperature and light (25 ± 1o C, 12 hours photophase, 7 days). High conidial production was verified in rice + brewery residue media (11.30 × 108 conidia/g of substrate and in the polished rice (9.59 × 108 conidia/g of substrate). The conidia produced in rice + brewery residue media showed an insecticidal activity against Spodoptera frugiperda similar to that attained with the conidia produced in rice. This new culture media may provide a reduction of approximately 50% in the production cost, thus presenting a viable alternative for the fungus production.

PRODUÇÃO DE CONÍDIOS DE METARHIZIUM ANISOPLIAE EM MEIO SÓLIDO À BASE DE RESÍDUOS AGROINDUSTRIAIS

ABSTRACT Increasing costs of substrate for production of Metarizhiuma nisopliae raise the necessity of investigating the efficiency of some agro-industrial residues that, in addition to important nutritional properties, are widely available at low cost. The assays were conducted at the Fermentation Technology Laboratory, UNIOESTE, Cascavel, PR, Brazil. In this work, substrates such as polished rice (standard media), red rice, polished rice + sugar molasses (2.5%, 5% and 10%), brewery residue, cassava residue, polished rice + brewery residue (1:1) and polished rice + cassava residue (1:1) were evaluated. The experiments were carried out in triplicate, in Erlernmeyer flasks of 250 mL, containing 30 g of the substrate (or 15 g of each one, when as mixture) and moisture content of 70 ± 10%. The flasks were inoculated with 0.5 mL of conidia suspension (1 × 108 conidia/mL) and then incubated in controlled temperature and light (25 ± 1o C, 12 hours photophase, 7 days). High conidial production was verified in rice + brewery residue media (11.30 × 108 conidia/g of substrate and in the polished rice (9.59 × 108 conidia/g of substrate). The conidia produced in rice + brewery residue media showed an insecticidal activity against Spodoptera frugiperda similar to that attained with the conidia produced in rice. This new culture media may provide a reduction of approximately 50% in the production cost, thus presenting a viable alternative for the fungus production.

RESUMO As crescentes despesas com o custo do meio para o cultivo de Metarhizium anisopliae levantam a necessidade de averiguar a eficiência de alguns resíduos agroindustriais que, além de propriedades nutricionais importantes, possuem grande disponibilidade e baixo custo. Os experimentos foram conduzidos no Laboratório de Tecnologia das Fermentações, UNIOESTE, Campus de Cascavel, PR. Neste trabalho, foram avaliados como substratos arroz polido (meio padrão), arroz vermelho, arroz polido suplementado com melaço (2,5%, 5% e 10%), resíduo de cervejaria, resíduo de fecularia, arroz polido + resíduo de cervejaria (1:1) e arroz polido + resíduo de fecularia (1:1). Os experimentos foram realizados em triplicata, em frascos Erlernmeyer de 250 mL contendo 30 g do substrato (ou 15 g de cada, quando em mistura) e umidade de 70 ± 10%. Os frascos foram inoculados com 0,5 mL da suspensão de conídios (1 × 108 conídios/mL) e incubados em BOD (25 ± 1o C, fotofase 12h, 7 dias). Dentre os meios testados, verificou-se uma elevada produção de conídios no meio composto por arroz + resíduo de cervejaria (11,3 × 108 conídios/g de substrato) e no arroz (9,6 × 108 conídios/g). Os conídios produzidos no meio de arroz + resíduo de cervejaria apresentaram atividade inseticida frente a Spodoptera frugiperda semelhante a dos conídios produzidos no arroz. Este novo meio de cultivo poderá proporcionar uma redução de custo de aproximadamente 50%, representando uma alternativa viável para a produção do fungo.

Técnicas para avaliação in vitro do efeito de herbicidas sobre bacillus thuringiensis berliner var. kurstaki

ABSTRACT The objective of this research was to compare techniques for the in-vitro evaluation of the effect of herbicides onBacillus thuringiensis Berliner var. kurstaki (Btk). The herbicides sulfentrazone, lactofen, imazethapyr, fenoxaprop and metribuzin were tested at two concentrations, on cells and spores, in nutrient agar (NA) solid medium (SM), and in nutrient broth (NB) liquid medium (LM), in addition to their metabolization (MT) by Btk. For cells, the fungicides in NA were tested incorporated into the medium and on filter-paper disks impregnated with the products. Evaluation was made, respectively, of the number of colony-unit formers per milliliter CFU.mL-1 and of the formation of an inhibition halo. For cells, the Btk + products + NB mixture was incubated in Erlenmeyer flasks (30 ± 2° C, 150rpm, 24h) and then the NB was diluted and inoculated onto NA, followed by evaluation of the number of CFU.mL-1. The MT and spore tests were performed in liquid medium (water for MT and NB for spores). The effects of the products varied with their concentration, the evaluation technique used and the developmental stage of the microorganism. The LM evaluation technique proved to be the best for evaluating the effect of herbicides on Btk cells, as it did not show any diffusion limitations and it also allowed for quantification. The herbicides, regardless of their concentration, were incompatible with the cells, although sulfetrazone and imazethapyr were metabolized by Btk. All herbicides were compatible with the spores, allowing for germination, but the concentration and time influenced the results.

RESUMO Objetivou-se comparar técnicas para a avaliação in vitro do efeito de herbicidas sobre Bacillus thuringiensis Berliner var. kurstaki (Btk). Foram testados os herbicidas sulfentrazone, lactofen, imazethapyr, fenoxaprop e metribuzin em duas concentrações, sobre células e esporos, em meio ágar nutriente sólido (MS) e caldo nutriente (CN) - líquido (ML), além da metabolização (MT) de tais produtos por Btk. Para células em MS, os herbicidas foram testados incorporados ao meio e em discos de papel-filtro impregnados nos produtos. Avaliou-se, respectivamente, as unidades formadoras de colônia por mililitro (UFC/mL) e a formação de halo de inibição. Para células, a mistura Btk + produtos + CN foi incubada em frascos erlemmeyer (30 ± 2° C, 150 rpm, 24h) e, em seguida, a mistura foi diluída e inoculada em MS, avaliando-se o número de UFC/mL. Os testes de MT e esporos foram realizados em líquido (água para MT e CN para esporos). O efeito dos produtos variou conforme a concentração utilizada, técnica de avaliação e a fase/estágio do microrganismo, sendo a técnica de avaliação em ML a mais indicada para a avaliação do efeito dos herbicidas sobre células de Btk, pois não apresenta limitações para difusão e permite quantificação. Os herbicidas, independentemente da concentração, foram incompatíveis para a célula, porém sulfetrazone e imazethapyr foram metabolizados por Btk. Todos herbicidas foram compatíveis com esporos, possibilitando a germinação, porém a concentração e tempo influenciaram nos resultados.

Técnicas para avaliação do efeito in vitro de fungicidas sobre bacillus thuringiensis var. kurstaki

ABSTRACT The aim of this study was to compare techniques for the invitro evaluation of the effect of fungicides on Bacillus thuringiensis var. kurstaki (Btk). The fungicides epoxiconazole, fentin hydroxide, azoxystrobin, tebuconazole, and carbendazim were tested at two concentrations, on cells and spores, in nutrient agar (NA) solid medium (SM), and in nutrient broth (NB) liquid medium (LM), with an evaluation also being made of their metabolization (MT) by Btk. For cells, the fungicides in NA were tested incorporated into the medium and on filterpaper disks. The evaluation consisted, respectively, of the number of CFU/mL and of the formation of an inhibition halo. For cells, the Bt+products + NB mixture was incubated in Erlenmeyer flasks (30 ± 2° C, 150rpm, 24h) and then was diluted and inoculated onto NA; the number of CFU/mL was evaluated. The MT and spore tests were performed in liquid medium (water). The effects of the products varied with their concentration, technique used, contact time, and developmental stage of the pathogen. The LM test is the most recommendable for evaluating the effect on cells. It was observed that most fungicides were compatible with cells, and only epoxiconazole and azoxystrobin were metabolized. Fentin hydroxide was the only fungicide incompatible with spores.

RESUMO Objetivou-se comparar técnicas para a avaliação in vitro do efeito de fungicidas sobre Bacillus thuringiensis var. kurstaki (Btk). Foram testados os fungicidas epoxiconazole, fentin hidróxido, azoxystrobin, tebuconazole e carbendazin em duas concentrações, sobre células e esporos, em ágar nutriente (AN)-sólido (MS) e caldo nutriente (CN)-líquido (ML), além da metabolização (MT) destes por Btk. Para células em AN, os fungicidas foram testados incorporados ao meio e em discos de papel-filtro. Avaliou-se, respectivamente, as UFC/mL e a formação de halo de inibição. Para células, a mistura Bt + produtos + CN foi incubada em erlemmeyers (30 ± 2° C, 150 rpm, 24h), e em seguida, diluida e inoculada em AN, avaliandose o número de UFC/mL. Os testes de MT e esporos foram realizados em líquido (água). O efeito dos produtos variou conforme a concentração, técnica, tempo de contato e fase/estágio do desenvolvimento do patógeno. O teste ML é o mais indicado para a avaliação do efeito em células, sendo observado que a maioria dos fungicidas mostrou-se compatível com as mesmas, e apenas epoxiconazole e azoxystrobin foram metabolizados. Fentin hidróxido foi o único incompatível com esporos.

Sugarcane bagasse as alternative packing material for biofiltration of benzene polluted gaseous streams: a preliminary study.

Removal of benzene vapor from gaseous streams was studied in two identically sized lab-scale biofiltration columns: one filled with a mixture of raw sugarcane bagasse and glass beads, and the other one packed with a mixture of ground sugarcane bagasse and glass beads, in the same volume ratio, as filter materials. Separate series of continuous tests were performed, in parallel, under the same operating conditions (inlet benzene concentration of 10.0, 20.0 or 50.0 mg m(-3), and superficial gas velocity of 30.6, 61.2 or 122.4 m h(-1)) in order to evaluate and compare the influence of the packing material characteristics upon the biofilter effectiveness. The maximum elimination capacities obtained, at an inlet load of 6.12 g m(-3) h(-1), were 3.50 and 3.80 g m(-3)packibng material h(-1) with raw and ground sugarcane bagasse, respectively. This was a preliminary study and the results obtained suggest only a limited application with more work needed.

Metabolic study of the adaptation of the yeast Candida guilliermondii to sugarcane bagasse hydrolysate.

Batch xylitol production from concentrated sugarcane bagasse hydrolysate by Candida guilliermondii was performed by progressively adapting the cells to the medium. Samples were analyzed to monitor sugar and acetic acid consumption, xylitol, arabitol, ethanol, and carbon dioxide production, as well as cell growth. Both xylitol yield and volumetric productivity remarkably increased with the number of adaptations, demonstrating that the more adapted the cells, the better the capacity of the yeast to reduce xylose to xylitol in hemicellulose hydrolysates. Substrate and product concentrations were used in carbon material balances to study in which way the different carbon sources were utilized by this yeast under microaerobic conditions, as well as to shed light on the effect of the progressive adaptation to the medium on its fermentative activity. Such a theoretical means allowed estimation for the first time of the relative contribution of each medium component to the formation of the main products of this fermentation system.

Preliminary kinetic characterization of xylose reductase and xylitol dehydrogenase extracted from Candida guilliermondii FTI 20037 cultivated in sugarcane bagasse hydrolysate for xylitol production.

Candida guilliermondii FTI 20037 was cultured in sugarcane bagasse hydrolysate supplemented with 2.0 g/L of (NH4)2SO4, 0.1 g/L of CaCl2 x 2H2O, and 20.0 g/L of rice bran at 35 degrees C; pH 4.0; agitation of 300 rpm; and aeration of 0.4, 0.6, or 0.8 vvm. The high xylitol production (20.0 g/L) and xylose reductase (XR) activity (658.8 U/mg of protein) occurred at an aeration of 0.4 vvm. Under this condition, the xylitol dehydrogenase (XD) activity was low. The apparent K(M) for XR and XD against substrates and cofactors were as follows: for XR, 6.4 x 10(-2)M (xylose) and 9.5 x 10(-3) mM (NADPH); for XD, 1.6 x 10(-1)M (xylitol) and 9.9 x 10(-2) mM (NAD+). Because XR requires about 10-fold less xylose and cofactor than XD for the condition in which the reaction rate is half of the Vmax, some interference on the overall xylitol production by the yeast could be expected.

Effects of environmental conditions on xylose reductase and xylitol dehydrogenase production by Candida guilliermondii.

The effects of environmental conditions, namely initial pH (2.5-7.0) and temperature (25 and 35 degrees C), on xylose reductase and xylitol dehydrogenase levels, as well as on xylitol production, were evaluated. Although the fermentative parameter values increased with an increase in pH and temperature (the maximum Yp/s and Qp were 0.75 g/g and 0.95 g/[L.h], respectively, both attained at pH 6.0, 35 degrees C), the highest xylose reductase activities (nearly 900 IU/mg of protein) were observed at an initial pH varying from 4.0 to 6.0. Xylitol dehydrogenase was favored by an increase in both initial pH and temperature of the medium. The highest xylitol dehydrogenase specific activity was attained at pH 6.5 and 35 degrees C (577 IU/mg of protein).

Adaptation and reutilization of Candida guilliermondii cells for xylitol production in bagasse hydrolysate.

The xylitol productivity increased by about 15% with the use of cells of Candida guilliermondii FTI 20037 previously recycled through four consecutive batch cultures and adapted to the sugar cane bagasse hemicellulosic hydrolysate. Furthermore, the more concentrated the hydrolysate, the more necessary was the adaptation of the cells, owing to the presence of toxic substances at high concentration which inhibited the xylose-xylitol conversion by the yeast.