Biomass Sorghum: Effect of Acid, Basic and Alkaline Peroxide Pretreatments on the Enzymatic Hydrolysis and Ethanol Production

Abstract This study evaluated the effects of three chemical pretreatments of biomass sorghum (BS): dilute alkaline (PTA1 and PTA2), dilute acid (PTB1 and PTB2) and alkaline hydrogen peroxide (PTC1 and PTC2) in the enzymatic hydrolysis and ethanol production. Among the six investigated conditions, the pretreatment with 7.36% H2O2 (PTC2) was the most efficient in the lignin removal and preservation of the polysaccharide fraction. After the enzymatic hydrolysis, increases in the glucose and xylose concentrations were observed in the pretreated BS hydrolysates, mainly in PTB1 and PTC1. All the hydrolysates obtained low concentrations of inhibitors. In the alcoholic fermentations with Pichia stiptis, the greatest ethanol yield was obtained in PTB1 hydrolysate (3.84 g L-1), corresponding to 16.15% of yield. The highest ethanol yield in PTB1 hydrolysate can be justified by the maximum concentration of xylose obtained in this hydrolysate, demonstrating the potential of P. stiptis in the fermentation of pentose to ethanol. The results indicated that biomass sorghum is an alternative lignocellulose source with potential for the production of second generation ethanol, opening up prospects for additional studies.

Ethanol Production and Other Bioproducts by Galactomyces geotrichum from Sugarcane Bagasse Hydrolysate.

This study investigated the enzymatic saccharification of alkaline-pretreated sugarcane bagasse (PSB) and the bioconversion of simple sugars from hydrolysates to ethanol and other bioproducts by the yeast Galactomyces geotrichum. The effects of percentage of dry substrate (3 and 10% w/v) and time of hydrolysis (24 and 72 h) in the content of released sugars were evaluated. The concentrations of monosaccharides and total reducing sugars (TRS) were calculated by high-performance liquid chromatography (HPLC) and by 3.5-dinitrosalicylic acid (DNS) method, respectively. The highest concentrations of TRS, glucose and xylose (73.96, 31.78 and 10.85 g/L, respectively) were obtained after the saccharification of 10% of PSB with Cellic CTec3 multi-enzyme cocktail (10 FPU/g cellulose) during 72 h (hydrolysate IV). G. geotrichum UFVJM-R150 fermented both glucose and xylose from the hydrolysates. The most efficient ethanol production was obtained after the fermentation of hydrolysate IV (9.99 g/L of ethanol, volumetric productivity-QP of 0.42 g/L.h and yield of ethanol as a function of the substrate-YP/S of 0.27 gethanol/gsugar). Besides ethanol, G. geotrichum was also able to produce other high-value chemicals such as isoamyl alcohol and galacturonic acid. This is the first report of the potential of the yeast G. geotrichum to fermentate sugarcane bagasse hydrolysates with the production of important bioproducts to further use by biorefineries.

Use of an (Hemi) Cellulolytic Enzymatic Extract Produced by Aspergilli Species Consortium in the Saccharification of Biomass Sorghum.

This study evaluated the production of lignocellulose-degrading enzymes by solid-state fermentation (SSF) using a microbial consortium of Aspergillus fumigatus SCBM6 and A. niger SCBM1 (AFN extract). The fungal strains were cultivated in sugarcane bagasse (SCB) and wheat bran (WB) as lignocellulosic substrates for 7 days at 30 °C. After SSF, the highest peaks of enzyme production were 150 and 80 U g-1 for ß-xylosidase and ß-glucosidase at 48 h, 375 U g-1 for xylanase at 96 h, and 80 U g-1 for endoglucanase and 4 U g-1 for cellulase activity on filter paper (FPase) at 144 h. The efficiency of the produced AFN extract was investigated in the enzymatic hydrolysis of crude biomass sorghum (BS) and after the removal of extractives (ES). After saccharification, the glucose and xylose concentrations were 10× superior in ES than in BS hydrolysate (2.5 g L-1 after 12 h). The presence of inhibitors of alcoholic fermentation, such as formic acid, was also reduced in ES hydrolysates, indicating that the removal of extractives positively contributed to the effectiveness of enzymatic hydrolysis of biomass sorghum using AFN extract.

Substract and temperature effect on xylanase production by aspergillus fumigatus using low cost agricultural wastes / Efeito da temperatura e do substrato na produção de xilanase por aspergillus fumigatus utilizando resíduos agroindustriais de baixo custo

This study reports the optimization of xylanase production under solid state fermentation (SSF) by a thermotolerant Aspergillus fumigatus strain (SCB4) isolated from sugarcane bagasse piles of Brazilian Cerrado. Different combinations of low-cost agricultural byproducts in SSF were evaluated: sugarcane bagasse and wheat bran (1:1), sugarcane bagasse and corn straw (1:1) and only sugarcane bagasse. The enzyme biosynthesis by SSF was carried out at different temperatures (40, 45, 50 and 55 oC). The maximum levels of xylanase activity were obtained after 24 h at 45 °C using a culture medium containing sugarcane bagasse and wheat bran (1:1). Under optimal conditions, the fungal culture produced 574 U g-1 of xylanase (units/g of dry substrate). The crude enzyme showed optimal activity at 60 °C and pH 4.5. It exhibited thermostability up to 55 °C, wide range of pH stability and tolerance to ethanol, xylose and glucose. The physicochemical properties shown by this enzyme are appropriate for its application in hydrolysis of lignocellulosic residues for ethanol production and other bioproducts.

Este estudo descreve a otimização da produção de xilanase por fermentação em estado sólido (FES) por uma linhagem termotolerante de Aspergillus fumigatus isolada de pilhas de bagaço de cana-de-açúcar do Cerrado Brasileiro (linhagem SCB4). Combinações de diferentes subprodutos agrícolas de baixo custo foram avaliadas como substratos na FES: bagaço de cana-de-açúcar e farelo de trigo (1:1), bagaço de cana-de-açúcar e palha de milho (1:1) e somente bagaço de cana-de-açúcar. A produção da enzima por FES foi realizada em diferentes temperaturas (40, 45, 50 e 55 oC). Níveis máximos de xilanase (574 U g-1 de substrato seco) foram obtidos após 24 h a 45 °C, utilizando bagaço de cana-de-açúcar e farelo de trigo (1:1) no meio de cultura. O extrato enzimático bruto apresentou atividades ótimas a 60 °C e pH 4,5. A enzima exibiu estabilidade térmica até 55 °C, ampla faixa de pH de estabilidade e tolerância ao etanol, xilose e glucose. Tais propriedades físico-químicas indicam que o extrato enzimático obtido é apropriado para aplicação na hidrólise de resíduos lignocelulósicos para a produção de etanol e outros bioprodutos.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production.

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best ß-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For ß-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0-8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Purification and characterization of an ethanol-tolerant ß-glucosidase from Sporidiobolus pararoseus and its potential for hydrolysis of wine aroma precursors.

An extracellular ethanol-tolerant ß-glucosidase from Sporidiobolus pararoseus was purified to homogeneity and characterized, and its potential use for the enhancement of wine aroma was investigated. The crude enzymatic extract was purified in four steps (concentration, dialysis, ultrafiltration, and chromatography) with a yield of around 40 % for total activity. The purified enzyme (designated Sp-ßgl-P) showed a specific activity of approximately 20.0 U/mg, an estimated molecular mass of 63 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric point of 5.0 by isoelectric focusing. Sp-ßgl-P has optimal activity at pH 4.0 and at 55 °C. It was stable in a broad pH range at low temperatures and it was tolerant to ethanol and glucose, indicating suitable properties for winemaking. The hydrolysis of glycosidic terpenes was analyzed by adding Sp-ßgl-P directly to the wines. The released terpene compounds were evaluated by gas chromatography/mass spectrometry. The enzymatic treatment significantly increased the amount of free terpenes, suggesting that this enzyme could potentially be applicable in wine aroma improvement.

Yeast diversity isolated from grape musts during spontaneous fermentation from a Brazilian winery.

Saccharomyces and non-Saccharomyces yeast species from a winery located in Brazil were identified by ribosomal gene-sequencing analysis. A total of 130 yeast strains were isolated from grape surfaces and musts during alcoholic fermentation from Isabel, Bordeaux, and Cabernet Sauvignon varieties. Samples were submitted to PCR-RFLP analysis and genomic sequencing. Thirteen species were identified: Candida quercitrusa, Candida stellata, Cryptococcus flavescens, Cryptococcus laurentii, Hanseniaspora uvarum, Issatchenkia occidentalis, Issatchenkia orientalis, Issatchenkia terricola, Pichia kluyveri, Pichia guilliermondii, Pichia sp., Saccharomyces cerevisiae, and Sporidiobolus pararoseus. A sequential substitution of species during the different stages of fermentation, with a dominance of non-Saccharomyces yeasts at the beginning, and a successive replacement of species by S. cerevisiae strains at the final steps were observed. This is the first report about the yeast distribution present throughout the alcoholic fermentation in a Brazilian winery, providing supportive information for future studies on their contribution to wine quality.

Fungi isolated from olive ecosystems and screening of their potential biotechnological use.

This study investigated the fungi diversity of fresh olive (Olea europaea L.) fruits, olive paste (crushed olives) and olive pomace (solid waste) and screened and quantified enzymatic activities with biotechnological applications. Fungi were randomly isolated from olive cultivars from Castilla La Mancha region (Spain). Identification included comparison of their polymerase chain reaction (PCR) amplicons of the ITS1-5.8S-ITS2 ribosomal DNA region, followed by nucleotide sequence analysis. Fourteen different species with DNA sequences of different similarities were identified, belonging to seven different genera (Aspergillus, Penicillium, Rhizomucor, Mucor, Rhizopus, Lichtheimia and Galactomyces). Aspergillus fumigatus, followed by Galactomyces geotrichum, Penicillium commune and Rhizomucor variabilis var. regularior were the most frequent species. Specific enzyme screening was assayed on agar plates, using cellobiose, carboxymethylcellulose (CMC), polygalacturonic acid and CaCl(2)/Tween 80 as substrates for ß-glucosidase, carboxymethylcellulase (CMCase), polygalacturonase and lipase, respectively. Species exhibiting the best activities were: Aspergillus fumigatus (for ß-glucosidase, CMCase and lipase); Rhizopus oryzae (for ß-glucosidase and lipase); Rhizomucor variabilis (for ß-glucosidase, CMCase and polygalacturonase); Mucor fragilis (ß-glucosidase, CMCase and lipase); Galactomyces geotrichum (for ß-glucosidase, polygalacturonase and lipase) and Penicillium commune and Penicillium crustosum (for lipase). The species that had shown the best enzymatic activities were grown on hemicellulose, cellulose and pectin and some activities were quantified (xylanase, cellulase, ß-glucosidase and pectinase). An isolate of A. fumigatus and one of A. niger showed the best cellulase and xylanase activities, while no species presented good pectinase and ß-glucosidase activities. The selected species with potential enzymatic activities could be used for future applications of industrial interest.

Esterase enzymes involved in pyrethroid and organophosphate resistance in a Brazilian population of Riphicephallus (Boophilus) microplus (Acari, Ixodidae).

Esterases are a group of enzymes that are reportedly associated with acaricide resistance in Riphicephallus (Boophilus) microplus. A comparative analysis was made of the esterase patterns in malathion and deltamethrin-sensitive, tolerant and resistant tick groups, using non-denaturing polyacrylamide gel electrophoresis. Electrophoretical profiles revealed four bands of esterase activity against alpha-naphthyl acetate; which were dubbed EST-1 to EST-4. The EST-3 and EST-4 were detected in all strains and were classified as carboxylesterases (CaEs). The EST-2, classified as an acetylcholinesterase (AChE), was detected in all groups, but its staining intensity increased from susceptible to resistant groups, indicating an altered production according to the degree of resistance. EST-1, which was also classified as an AChE, was detected exclusively in tolerant and resistant groups to both acaricides, but displayed greater activity in the malathion-resistant group. These data suggest that these AChEs may represent an important detoxification strategy developed to overcome the effects of acaricides.

Identification of point mutations in a putative carboxylesterase and their association with acaricide resistance in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Chemical control based on the use of pyrethroid and organophosphate compounds has selected resistant genotypes in populations of Rhipicephalus (Boophilus) microplus. Point mutations in esterase-encoding genes represent one of the main resistance mechanisms in this species. In this study, the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique was used to investigate the presence of mutations in a fragment of a putative carboxylesterase in a population of ticks with a history of resistance. The digestion of a fragment of 372 pb with EcoRI revealed three genotypes: W, H and M, observed in different frequencies. The homozygous wild-type genotype (W) was detected only in sensitive strains, with high frequency. The heterozygous genotype (H) was observed in all the strains, albeit with higher frequency in the strains with a moderate resistance, while the homozygous mutant genotype (M) was found only in the moderate resistant strain and resistant strains, with higher frequency in the resistant strains. A comparison of the sequences indicated the presence of other mutations, besides EcoRI polymorphism in the moderate resistant and resistant strains. Also found was the presence of stop codons generating truncated proteins in the sensitive and moderate resistant strains. A domain analysis revealed the presence of additional domains in the resistant strain. These findings suggest that different point mutations, as well as the influence of post-translational modification mechanisms, are altering the activity of the translated proteins and may be associated with resistance.

Esterase profile in a pyrethroid-resistant Brazilian strain of the cattle tick Boophilus microplus (Acari, Ixodidae)

The cattle tick Boophilus microplus causes great damage in livestock and is considered one of the most important tropical ectoparasites. The traditional method of control is based on the intensive use of pesticides, however the indiscriminate use of these compounds over the years has led to the selection of resistant ticks. Hydrolases, particularly esterases (EST), have been reported to be associated with acaricide resistance in B. microplus. We compared the esterase profile of susceptible and cypermethrin-resistant strains of adult B. microplus and a pyrethroid susceptible reference strain (the Mozzo strain) using polyacrylamide gel electrophoresis and specific staining. The electrophoretic profiles of protein extracts revealed the presence of four regions with esterase activity in the cypermethrin-resistant strain and three of these regions in the susceptible strains. The bands were numbered EST-1 to EST-4 in sequence (starting from the anode) according to their decrease in negative charge. The EST-1A and EST-1B enzymes were detected only in the resistant strain. The inhibition studies with eserine sulfate, copper sulfate, p- p-chloromercuribenzoate (pCMB), malathion and phenylmethylsulfonyl fluoride (PMSF) indicated that the EST-1A and EST-1B enzymes belong to the acetylcholinesterase class and are probably associated with resistance to acaricides in this Brazilian resistant strain of B. microplus.

Molecular analysis of the rDNA ITS-1 intergenic spacer in Drosophila mulleri, D. arizonae, and their hybrids.

We analyzed the ITS-1 spacer region of the rDNA in Drosophila mulleri and D. arizonae, two sibling species belonging to the mulleri complex (repleta group) and in hybrids obtained in both cross directions. In spite of several previous studies showing the incompatibility of crosses involving D. arizonae females and D. mulleri males, we were able to obtain hybrids in this direction. Complete ITS-1 region was amplified using primers with homology at the 3'-end of the 18S rDNA and the 5'-end of the 5.8S rDNA genes. Our data demonstrated that D. mulleri and D. arizonae can be differentiated as they present a difference in length for the ITS-1 region. The amplified fragment for this region in D. mulleri has a length of 600 bp, whereas in D. arizonae this fragment is about 500 bp. It was also observed that male and female hybrids obtained in both cross directions present two amplified fragments, confirming the location of the ribosomal cistrons in the X chromosomes and microchromosomes of both parental species.