Production of Cellulosic Microfibers from Coffee Pulp via Alkaline Treatment, Bleaching and Acid Hydrolysis.

The present work deals with the production of cellulosic microfibers (CMFs) from coffee pulp. The experimental development corresponds to an experimental design of three variables (concentration, temperature and time) of alkaline treatment for delignification, finding that concentration, temperature and time were the most representative variables. Higher delignification was achieved by bleaching cellulosic fibers, followed by acid hydrolysis, thus producing cellulosic fibers with an average diameter of 5.2 µm, which was confirmed using scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS). An X-ray diffraction (XRD) analysis revealed, via the crystallinity index, the presence of Type I cellulose and removal of lignocellulosic compounds through chemical treatments. The proximate chemical analysis (PChA) of coffee pulp helped to identify 17% of the crude fiber corresponding to the plant cell wall consisting of lignocellulosic compounds. The initial cellulose content of 26.06% increased gradually to 48.74% with the alkaline treatment, to 57.5% with bleaching, and to 64.7% with acid hydrolysis. These results attested to the rich cellulosic content in the coffee pulp.

Obtaining of a rich-cellulose material from black wattle (Acacia mearnsii De Wild.) bark residues.

Black wattle (Acacia mearnsii De Wild.) barks are residues produced by tannin industries in huge quantities, which are normally discharged on environmental or used for energy production. Therefore, this study aimed to evaluate the use of black wattle bark residues as a raw material on obtaining of a rich-cellulose material by alkaline (MET1), acetosolv (MET2), and organosolv (MET3) procedures. The results obtained indicated that the alkaline methodology, followed by a bleaching step (MET1), promoted klason lignin and hemicellulose removals more efficiently. It was possible to observe that better results were achieved using NaOH concentration of 6% (wt%), at 65 °C for 2.5 h, presenting a yield of 63.24 ± 1.25%, and a reduction on klason lignin content of almost 90.45%. Regarding the bleaching step, it was possible to obtain a material free of non-cellulosic compounds with a yield of 78.28 ± 1.48%. Thermogravimetric analysis indicated the removal of lignin and hemicellulose as well as an increase in cellulose degradation temperature, due to changes in crystalline phases. According to X-ray diffraction (XRD), the procedures employed have led to an increase in crystallinity from 66.27 to 91.78% due to the removal of non-cellulosic compounds. Scanning electron microscopy (SEM) showed morphological alterations in accordance with the removal of non-cellulosic compounds.

Agro-industrial wastes revalorization as feedstock: production of lignin-modifying enzymes extracts by solid-state fermentation using white rot fungi.

The purpose of the study was to evaluate the production of lignin-modifying enzyme extracts and delignified biomass from agro-industrial wastes using white rot fungi (Inonotus sp. Sp2, Stereum hirsutum Ru-104, Bjerkandera sp. BOS55, Pleurotus eryngii IJFM 169 and Phanerochaete chrysosporium BKM-F-1767). These were screened based on their adaptability and colonization ability on different substrates, as well as by the Laccase, Manganese peroxidase, and Lignin peroxidase enzymatic production. Native strains (Inonotus sp. Sp2 and S. hirsutum Ru-104) showed the highest growth kinetics under the solid-substrate fermentation conditions and the growth rate parameters of the kinetic logistic model for the different substrates were between 0.39-0.81 (1/d) and 0.42-0.83 (1/d), respectively; the determination coefficients were ≥0.99. Inonotus sp. Sp2 was subsequently cultured in static flasks to produce crude enzyme extracts, obtaining manganese peroxidase activity levels of 18.5 and 31.3 (U/g) when growing in corn cob husk and spent tea leaves, respectively. Besides, it was to establish that the best conditions for lignin-modifying enzymes production using corn cob husk are 70% of initial moisture and 2.12 mm of particle size; reaching after 30 incubation days a manganese peroxidase activity of 21 ± 6 (U/g) under these conditions; enzyme that showed a suitable thermostability.

Enhancement of alkaline-oxidative delignification of wheat straw by semi-batch operation in a stirred tank reactor.

This paper compares a semi-batch operation and a conventional one of an alkaline oxidative pretreatment of wheat straw carried out in a stirred tank reactor. For the pretreatment, different concentrations of biomass (6% up to 12% w/v) and two different particle sizes (mesh #40-60 and #>60) were experimented. The performance of processes was evaluated through the analysis of lignocellulosic composition of the biomass, and the enzymatic hydrolysis of pretreated biomass using the Cellic® CTec2 enzyme complex by Novozymes®. The process time of semi-batch operation is significantly lower than the batch one and enables a higher load of biomass, showing a delignification yield between 55 and 60%. In the first 5 h of reaction time, the enzymatic hydrolysis experiments reached their maximum yields of 72 and 66% according to reducing sugars conversion when using the mesh #>60 mesh and #40-60, respectively.

Study of supercritical carbon dioxide pretreatment processes on green coconut fiber to enhance enzymatic hydrolysis of cellulose.

Green coconut fiber was treated by supercritical CO2 with the aim to enhance hydrolysis of its enzymatic cellulose. To this end, different static conditions of CO2 contact times (3 and 5 h) and polarity modifiers (NaOH, NaHSO4, ethanol) were evaluated at 20 MPa, 70 °C and 1 h of dynamic extraction followed by fast depressurization. After supercritical CO2 exposition, SEM images showed fiber damage and FTIR spectra showed decreases of phenolic and wax contents, including a reduction in the degree of the hydrogen bond established between lignin and cellulose. Despite the apparent delignification, supercritical CO2 did not enhance cellulose enzymatic hydrolyses. Fiber exposed to supercritical CO2 (5 h) demonstrated that the highest sugar content (540.9 µmol glucose likely limited supercritical CO2 delignification; however, green coconut in natura can be an innovative substrate for fermentation in alcohol production.

Comparative data on effects of alkaline pretreatments and enzymatic hydrolysis on bioemulsifier production from sugarcane straw by Cutaneotrichosporon mucoides.

Bioemulsifiers are surface active compounds which could be potentially used in food processing, cosmetic sector and oil recovery. Sugarcane straw (SS), was used as the raw substrate for the production of bio-emulsifiers (BE) by Cutaneotrichosporon mucoides. Three different delignification strategies using dilute sodium hydroxide, sodium sulfite and ammonium hydroxide followed by enzymatic hydrolysis (Cellic CTec 2, 7.5% total solids, 15 FPU/g, 72 h) were studied. Enzyme hydrolysis of ammonium hydroxide pretreated SS showed a maximum of 62.19 ± 0.74 g/l total reducing sugars with 88.35% hydrolytic efficiency (HE) followed by sodium hydroxide (60.06 ± 0.33 g/l; 85.40% HE) and sodium sulfite pretreated SS (57.22 ± 0.52 g/l; 84.71% HE), respectively. The ultrastructure of SS (native and delignified) by fourier transform-infrared and near infrared spectroscopy, revealed notable structural differences. The fermentation of hydrolysates by C. mucoides into bioemulsifiers showing emulsification index (EI) of 54.33%, 48.66% and 32.66% from sodium sulfite, sodium hydroxide, and ammonium hydroxide pretreated SS, respectively.

A thermostable laccase from Thermus sp. 2.9 and its potential for delignification of Eucalyptus biomass.

Laccases are multicopper oxidases that are being studied for their potential application in pretreatment strategies of lignocellulosic feedstocks for bioethanol production. Here, we report the expression and characterization of a predicted laccase (LAC_2.9) from the thermophilic bacterial strain Thermus sp. 2.9 and investigate its capacity to delignify lignocellulosic biomass. The purified enzyme displayed a blue color typical of laccases, showed strict copper dependence and retained 80% of its activity after 16 h at 70 °C. At 60 °C, the enzyme oxidized 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP) at optimal pH of 5 and 6, respectively. LAC_2.9 had higher substrate specificity (kcat/KM) for DMP with a calculated value that accounts for one of the highest reported for laccases. Further, the enzyme oxidized a phenolic lignin model dimer. The incubation of steam-exploded eucalyptus biomass with LAC_2.9 and 1-hydroxybenzotriazole (HBT) as mediator changed the structural properties of the lignocellulose as evidenced by Fourier transform infrared (FTIR) spectroscopy and thermo-gravimetric analysis (TGA). However, this did not increase the yield of sugars released by enzymatic saccharification. In conclusion, LAC_2.9 is a thermostable laccase with potential application in the delignification of lignocellulosic biomass.

Influence of the alkaline delignification on the simultaneous saccharification and fermentation (SSF) of sugar cane bagasse.

Ethanol production from steam explosion alkaline delignified bagasse was investigated by saccharification and simultaneous fermentation. Non delignified bagasse (ND) contained 25% lignin, and after alkaline delignification, materials with 6% (D1 - NaOH 1% w/v) and 12% (D05 - NaOH 0.5% w/v) lignin, respectively, were obtained. Ethanol production increased 450% and 733% in relation to ND, when D05 and D1 material, respectively, were used. Higher productivity and EtOH/bagasse were observed for D1. However, higher enzymatic convertibility of cellulose was obtained with 0.5% w/v NaOH. Alkaline delignification increased the ethanol production despite decreased cellulose.

Pretreatment strategies for delignification of sugarcane bagasse: a review

The valorization of agro-residues by biological routes is a key technology that contributes to the development of sustainable processes and the generation of value-added products. Sugarcane bagasse is an agro-residue generated by the sugar and alcohol industry in Brazil (186 million tons per year), composed essentially of cellulose (32-44%), hemicellulose (27-32%) and lignin (19-24%). The conversion of sugarcane bagasse into fermentable sugars requires essentially two steps: pretreatment and hydrolysis. The aim of the pretreatment is to separate the lignin and break the structure of lignocellulose, and it is one of the most critical steps in the process of converting biomass to fermentable sugars. The aim of this review is to describe different pretreatment strategies to promote the delignification of the sugarcane bagasse by thermo-chemical and biological processes.

Caracterización de actinobacterias raras, degradadoras de lignocelulosa: demostración de actividad lacasa en dos aislados de Tsukamurella sp y Cellulosimicrobium sp / Characterization of lignocellulose-degrading rare actinobacteria: demostration of laccase activity in two isolates of Tsukamurella sp and Cellulosimicrobium sp

Las características fisicoquímicas de la lignina y su compactación con la celulosa han dificultado la explotación biotecnológica de enormes cantidades de biomasa vegetal. Las lacasas constituyen una subfamilia de oxidasas multicobre que intervienen en la despolimerización de la lignina. Si bien han sido ampliamente caracterizadas en los hongos, los estudios de la diversidad y las funcionalidades de las lacasas en los procariotas se han centrado especialmente en isoformas enzimáticas de Streptomyces sp. En este trabajo se aislaron 20 cepas de actinobacterias del suelo. La actividad lacasa de 17 de ellas fue evidenciada en ensayos cualitativos con guayacol y dos cepas seleccionadas fueron caracterizadas en detalle. Las pruebas morfológicas y el análisis de las secuencias del gen 16S rRNA apuntan a que estos dos aislados pertenecen a los géneros Tsukamurella y Cellulosimicrobium. En cultivo sumergido con agitación, AC01 (Tsukamurella sp.) expresó una máxima actividad de oxidación de ABTS (2,2’-azino-bis-(3-etilbenzotiazolin-6-sulfonato) de 108 U/L. Por otra parte, AC18 (Cellulosimicrobium sp.) que había exhibido una actividad oxidativa de guayacol superior a las 16 cepas restantes y demostró ser resistente a niveles tóxicos de cobre, logró un valor máximo de oxidación del ABTS de 0,56 U/L. Estos resultados sugieren que en el aislado AC18 operaría un fenómeno de especificidad de sustrato o de inductor, regulador de la expresión y de la actividad lacasa cuantificable. La caracterización genómica y funcional de las lacasas de nuevas actinobacterias lignocelulósicas ampliará la gama de centros redox con aplicaciones biotecnológicas específicas, además de facilitar el establecimiento de sus relaciones evolutivas con las eucariotas.

The physicochemical characteristics of lignin and its compaction with cellulose have restricted the biotechnological exploitation of enormous amounts of plant biomass. Laccases are a subfamily of multicopper oxidases involved in lignin depolymerization. Although they have been extensively characterized in fungi, studies of the diversity and functions of laccases in prokaryotes are mainly on enzyme isoforms of Streptomyces sp. In this work we isolated 20 strains of soil actinomycetes. The laccase activity of 17 of them was evidenced in qualitative assays with guaiacol, and two selected strains were characterized in detail. The morphological evidence and the analysis of the 16S rRNA gene sequences suggest that these two isolates belong to the genera Tsukamurella and Cellulosimicrobium. In submerged cultures with shaking, AC01 (Tsukamurella sp.) exhibited a maximal oxidation activity of ABTS (2,2 '-azino-bis-(3-ethylbenzthiazoline-6-sulfonate) of 108 U/L. On the other hand, AC18 (Cellulosimicrobium sp.) that exhibited a higher oxidative activity of guaiacol than the other 16 isolated strains and showed resistance to toxic levels of copper, reached a maximum ABTS oxidation rate of 0.56 U/L. These results suggest that in AC18 operates a mechanism of substrate or inducer specificity, regulating the measurable laccase activity and laccase gene expression. Genomic and functional characterization of laccases of new ligninolytic actinomycetes may help to extend the range of redox centers with specific biotechnological applications, as well as establishing their evolutionary relationships with eukaryotes.