Efficient integrated production of bioethanol and antiviral glycerolysis lignin from sugarcane trash.

BACKGROUND: Sugarcane trash (SCT) represents up to 18% of the aboveground biomass of sugarcane, surpassing 28 million tons globally per year. The majority of SCT is burning in the fields. Hence, efficient use of SCT is necessary to reduce carbon dioxide emissions and global warming and establish agro-industrial biorefineries. Apart from its low costs, conversion of whole biomass with high production efficiency and titer yield is mandatory for effective biorefinery systems. Therefore, in this study, we developed a simple, integrated method involving a single step of glycerolysis pretreatment to produce antiviral glycerolysis lignin (AGL). Subsequently, we co-fermented glycerol with hydrolyzed glucose and xylose to yield high titers of bioethanol. RESULTS: SCT was subjected to pretreatment with microwave acidic glycerolysis with 50% aqueous (aq.) glycerol (MAG50); this pretreatment was optimized across different temperature ranges, acid concentrations, and reaction times. The optimized MAG50 (opMAG50) of SCT at 1:15 (w/v) in 1% H2SO4, 360 µM AlK(SO4)2 at 140 °C for 30 min (opMAG50) recovered the highest amount of total sugars and the lowest amount of furfural byproducts. Following opMAG50, the soluble fraction, i.e., glycerol xylose-rich solution (GXRS), was separated by filtration. A residual pulp was then washed with acetone, recovering 7.9% of the dry weight (27% of lignin) as an AGL. AGL strongly inhibited the replication of encephalomyocarditis virus (EMCV) in L929 cells without cytotoxicity. The pulp was then saccharified in yeast peptone medium by cellulase to produce a glucose concentration similar to the theoretical yield. The total xylose and arabinose recoveries were 69% and 93%, respectively. GXRS and saccharified sugars were combined and co-fermented through mixed cultures of two metabolically engineered Saccharomyces cerevisiae strains: glycerol-fermenting yeast (SK-FGG4) and xylose-fermenting yeast (SK-N2). By co-fermenting glycerol and xylose with glucose, the ethanol titer yield increased to 78.7 g/L (10% v/v ethanol), with a 96% conversion efficiency. CONCLUSION: The integration of AGL production with the co-fermentation of glycerol, hydrolyzed glucose, and xylose to produce a high titer of bioethanol paves an avenue for the use of surplus glycerol from the biodiesel industry for the efficient utilization of SCT and other lignocellulosic biomasses.

Adsorptive Inhibition of Enveloped Viruses and Nonenveloped Cardioviruses by Antiviral Lignin Produced from Sugarcane Bagasse via Microwave Glycerolysis.

Antiviral lignin was produced by acidic microwave glycerolysis of sugarcane bagasse. The lignin exhibited antiviral activity against nonenveloped (encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelitis virus (TMEV)) and enveloped (vesicular stomatitis virus (VSV), Sindbis virus (SINV), and Newcastle disease virus (NDV)) viruses. A series of lignins with different antiviral activities were prepared by reacting bagasse at 140, 160, 180, and 200 °C to analyze the antiviral mechanism. No difference in ζ-potential was observed among the lignin preparations; however, the lignin prepared at 200 °C (FR200) showed the strongest anti-EMCV activity, smallest hydrodynamic diameter, highest hydrophilicity, and highest affinity for EMCV. FR200 inhibited viral propagation through contact with the virion at the attachment stage to host cells, and the EMCV RNA was intact after treatment. Therefore, the lignin inhibits viral entry to host cells through interactions with the capsid surface. The nonvolatile antiviral substance is potentially useful for preventing the spread of viruses in human living and livestock breeding environments.

Production of Antiviral Substance from Sugarcane Bagasse by Chemical Alteration of its Native Lignin Structure through Microwave Solvolysis.

The production of bioactive agents from lignocelluloses has received limited attention because plant cell walls are essentially non-bioactive. In this study, a chemical reaction is reported, which produces a lignin-derived antiviral substance from sugarcane bagasse by microwave heating at 200 °C in aqueous glycerol containing 0.5 % H2 SO4 . The purified fraction, designated as FR200 , strongly inhibited the replication of encephalomyocarditis virus (EMCV) in L929 cells without cytotoxicity. HSQC NMR spectra demonstrated that the principal interunit linkages in the native lignin were cleaved by the reaction. Gel permeation chromatography (GPC) and pyrolysis-GCMS revealed that FR200 is composed of oligomeric lignin with a weight average molecular weight of approximately 2000. When the bagasse was reacted at lower temperatures, 140 °C and 160 °C, the native lignin substructures were partially retained and the antiviral activity significantly decreased. The results thus indicate that the antiviral activity emerged through severe alteration of the native lignin structure. Furthermore, it was revealed that the antiviral lignin inactivated the EMCV virions through direct contact, as the innate immune system of L929 was not activated by FR200 treatment, and no antiviral activity was found when L929 was pre-treated with the lignin before viral infection.

Structure-dependent antiviral activity of catechol derivatives in pyroligneous acid against the encephalomycarditis virus.

The pyrolysis product, wood vinegar (WV), from Japanese larch exhibited strong antiviral activity against the encephalomycarditis virus (EMCV). Catechol, 3-methyl-, 4-methyl-, 4-ethyl-, and 3-methoxycatechol, and 2-methyl-1,4-benzenediol were identified as the major antiviral compounds. The viral inhibition ability of these compounds was affected by the structure and position of the substituent group attached to the aromatic skeleton. The IC50 of catechol was 0.67 mg mL-1 and those of its derivatives were <0.40 mg mL-1. Methyl and ethyl substitution in the para position relative to a hydroxyl group obviously increased the antiviral activities. The mode of antiviral action was investigated by adding catechol derivatives at different times of the viral life cycle. It was found that direct inactivations of EMCV by these compounds were the major pathway for the antiviral activity. The effect of catechol derivatives on the host immune system was studied by quantification of Il6 and Ifnb1 expression levels. Increased Il6 expression levels indicate NF-κB activation by reactive oxygen species from auto-oxidations of catechol derivatives, which is also a possible antiviral route. The present research provides indices for production of potent antiviral agents form lignocellulose biomass.

Novel Characteristics of Trypanosoma brucei Guanosine 5'-monophosphate Reductase Distinct from Host Animals.

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5'-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5'-monophosphate (GMP) to inosine 5'-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine ß-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K+ and NH4+ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR's was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis.