Simultaneous enzymatic saccharification and comminution for the valorization of lignocellulosic biomass toward natural products.

BACKGROUND: Large-scale processing of lignocellulosics for glucose production generally relies on high temperature and acidic or alkaline conditions. However, extreme conditions produce chemical contaminants that complicate downstream processing. A method that mainly rely on mechanical and enzymatic reaction completely averts such problem and generates unmodified lignin. Products from this process could find novel applications in the chemicals, feed and food industry. But a large-scale system suitable for this purpose is yet to be developed. In this study we applied simultaneous enzymatic saccharification and communition (SESC) for the pre-treatment of a representative lignocellulosic biomass, cedar softwood, under both laboratory and large-scale conditions. RESULTS: Laboratory-scale comminution achieved a maximum saccharification efficiency of 80% at the optimum pH of 6. It was possible to recycle the supernatant to concentrate the glucose without affecting the efficiency. During the direct alcohol fermentation of SESC slurry, a high yield of ethanol was attained. The mild reaction conditions prevented the generation of undesired chemical inhibitors. Large-scale SESC treatment using a commercial beads mill system achieved a saccharification efficiency of 60% at an energy consumption of 50 MJ/kg biomass. CONCLUSION: SESC is very promising for the mild and clean processing of lignocellulose to generate glucose and unmodified lignin in a large scale. Economic feasibility is highly dependent on its potential to generate high value natural products for energy, specialty chemicals, feed and food application.

Functional maintenance and structural flexibility of microbial communities perturbed by simulated intense rainfall in a pilot-scale membrane bioreactor.

Intense rainfall is one of the most serious and common natural events, causing the excessive inflow of rainwater into wastewater treatment plants. However, little is known about the impacts of rainwater dilution on the structure and function of the sludge microorganisms. Here, high-throughput sequencing of 16S ribosomal RNA (rRNA) genes was implemented to describe the microbial community dynamics during the simulated intense rainfall situation (event i) in which approximately 45 % of the sludge biomass was artificially overflowed by massive water supply in a pilot-scale membrane bioreactor. Thereafter, we investigated the functional and structural responses of the perturbed microbial communities to subsequent conditional changes, i.e., an increase in organic loading rate from 225 to 450 mg chemical oxygen demand (COD) l(-1) day(-1) (event ii) and an addition of a microbiota activator (event iii). Due to the event i, the COD removal declined to 78.2 %. This deterioration coincided with the decreased microbial diversity and the proliferation of the oligotrophic Aquabacterium sp. During the succeeding events ii and iii, the sludge biomass increased and the COD removal became higher (86.5-97.4 %). With the apparent recovery of the reactor performance, microbial communities became diversified and the compositions dynamically changed. Notably, various bacterial micropredators were highly enriched under the successive conditions, most likely being involved in the flexible reorganization of microbial communities. These results indicate that the activated sludge harbored functionally redundant microorganisms that were able to thrive and proliferate along with the conditional changes, thereby contributing to the functional maintenance of the membrane bioreactor.

Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (-)-epicatechin to (+)-taxifolin.

This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (-)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H2O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (-)-epicatechin, which are major structural units in plants.

High-Resolution Dynamics of Microbial Communities during Dissimilatory Selenate Reduction in Anoxic Soil.

Selenate is one of the most common toxic metal compounds in contaminated soils. Its redox status can be changed by microbial activity, thus affecting its water solubility and soil mobility. However, current knowledge of microbial dynamics has been limited by the low sensitivity of past isolation and identification protocols. Here, high-throughput Illumina sequencing of 16S rRNA genes was applied to monitor the shift of the microorganisms in an anoxic contaminated soil after Se(VI) and acetate amendment. An autoclaved soil with both chemicals and a live soil with acetate alone were used as controls. Preliminary chemical analysis clearly showed the occurrence of biological selenate reduction coupled with acetate oxidation. Principal coordinate analysis and diversity indices of Illumina-derived sequence data showed dynamic succession and diversification of the microbial community in response to selenate reduction. High-resolution phylogenetic analysis revealed that the relative frequency of an operational taxonomic unit (OTU) from the genus Dechloromonas increased remarkably from 0.2% to 36% as a result of Se(VI) addition. Multiple OTUs representing less abundant microorganisms from the Rhodocyclaceae and Comamonadaceae families had significant increases as well. This study demonstrated that these microorganisms are concertedly involved in selenate reduction of the employed contaminated soil under anoxic conditions.

Application of aqueous saponin on the remediation of polycyclic aromatic hydrocarbons-contaminated soil.

The aim of this research was to evaluate the feasibility of aqueous saponin for the removal and biodegradation of polycyclic aromatic hydrocarbons (PAHs) from contaminated soil. Dissolution test confirmed the ability of saponin to increase the apparent solubility of the tested 3-5 rings PAH above the critical micelle concentration (approximately 1000 mg/L). Microbial test with pure culture of Sphingomonas sp. showed that saponin significantly enhanced the degradation of pyrene. For example, the percent degradation was 2.1 times higher in the presence of 2500 mg/L saponin than that of control without saponin after 60 hours incubation at around 10(8) CFU/mL initial cell loading. These results suggest that the binding of pyrene with saponin does not pose a serious constraint to bacterial uptake. Contrary to pyrene, saponin was chemically stable against the PAHs degrader. It is also not toxic to the cell at least up to 2500 mg/L. Finally, using a spiked soil sample, extraction tests with 10,000 mg/L of saponin showed that around 52.7% and 0.3% of pyrene was removed from low and high organic spiked soils, respectively. The results from this study indicate that aqueous saponin is appropriate as a washing agent as well as biodegradation enhancer for the detoxification of PAHs-contaminated low organic carbon soil.

Activated sludge microbiome in a membrane bioreactor for treating Ramen noodle-soup wastewater.

Population shifts in the activated sludge microbiome of a membrane bioreactor (MBR) during the treatment of Ramen noodle-soup wastewater were analyzed by high-throughput sequencing. An MBR underwent stable treatment of wastewater containing increasing oil concentrations (from 135 to 1,350 mg/L) for 26 days; however, after feeding with wastewater containing 2,700 mg/L of oil, the mixed liquor suspended solids and transmembrane pressure exhibited gradual and rapid increases, respectively, leading to clogging of the membrane. Phylogenetic analysis revealed an oil supply-dependent increase in the abundance of Cupriavidus gilardii (relative abundance of 26.2% at Day 30) in the sludge together with Parasegetibacter terrae (9.9%) and Ferruginibacter yonginensis (9.4%). These dominant species may play important roles in noodle-soup wastewater treatment.

Production of flavorful alcohols from woods and possible applications for wood brews and liquors.

This work explores the utilization of wood for high-value production of novel alcoholic brews and liquors with natural flavors. The process capitalizes on our original wet-type bead milling (WBM) technology that enables direct enzymatic saccharification and alcohol fermentation of wood without chemical and heat treatment, resulting in the absence of toxic compounds. When alcohol-based products from various wood species, including Cryptomeria japonica (cedar), Cerasus × yedoensis (cherry), and Betula platyphylla (birch), were analyzed by SPME-GC-MS, different natural flavor components were found in each. Correlation analysis using Heracles NEO and ASTREE V5 showed that the alcohols from wood have different flavor and taste characteristics when compared with those of existing commercial liquors. From pilot-scale experiments, the yield of alcoholic brew per biomass amount was determined. Pilot-scale runs established the importance of optimum wood particle size during WBM for efficient alcohol production. Although the alcohol produced from wood must first be established as safe for human consumption, this is the first description of drinking alcohols produced from wood. This work may open up important avenues for the exploitation of wood resources toward food production to further advance the current state of forestry.

Combined simultaneous enzymatic saccharification and comminution (SESC) and anaerobic digestion for sustainable biomethane generation from wood lignocellulose and the biochemical characterization of residual sludge solid.

Simultaneous enzymatic saccharification and comminution (SESC) was used for large-scale anaerobic digestion of wood lignocellulose to generate methane and unmodified lignin. During SESC, 10% aqueous mixture of powdered debarked wood from various species was subjected to bead milling with hydrolytic enzymes to generate particles below 1 µm. This slurry was directly used as a cosubstrate for anaerobic digestion in a 500 L stirred-tank reactor. Temperature and hydraulic retention time (HRT) were maintained at 50 °C and 30 days, respectively. At stable operation periods, an average yield of 224 L of methane per kg of cedar was attained. Comparable yields were achieved with red pine, elm, oak, and cedar bark. High-throughput microbial analysis established the presence of a relevant community to support the elevated level of methane production. The stability of the unmodified lignin in anaerobic digestion was also confirmed, allowing for its recovery as an important by-product.

Transcriptome analysis of activated sludge microbiomes reveals an unexpected role of minority nitrifiers in carbon metabolism.

Although metagenomics researches have illuminated microbial diversity in numerous biospheres, understanding individual microbial functions is yet difficult due to the complexity of ecosystems. To address this issue, we applied a metagenome-independent, de novo assembly-based metatranscriptomics to a complex microbiome, activated sludge, which has been used for wastewater treatment for over a century. Even though two bioreactors were operated under the same conditions, their performances differed from each other with unknown causes. Metatranscriptome profiles in high- and low-performance reactors demonstrated that denitrifiers contributed to the anaerobic degradation of heavy oil; however, no marked difference in the gene expression was found. Instead, gene expression-based nitrification activities that fueled the denitrifiers by providing the respiratory substrate were notably high in the high-performance reactor only. Nitrifiers-small minorities with relative abundances of <0.25%-governed the heavy-oil degradation performances of the reactors, unveiling an unexpected linkage of carbon- and nitrogen-metabolisms of the complex microbiome.

Influence of compost amendment on pyrene availability from artificially spiked soil to two subspecies of Cucurbita pepo.

The dissolved organic matter (DOM) fraction of soil organic matter (SOM) may positively contribute to polycyclic aromatic hydrocarbons (PAHs) bioavailability. This work investigated the effects of DOM-rich and PAHs-free compost amendment on the plant uptake of pyrene. Two subspecies of Cucurbita pepo (ssp. pepo cv. Raven and ssp. texana cv. Sunray) were grown for three weeks in a spiked soil containing 83.9 mg kg(-1) pyrene under four different treatments; inorganic fertilizer (IF) alone, 15% (v/v) mixed gardening compost with IF (MX15%+IF), MX30% alone, and no fertilization (NF). Equilibrium pyrene desorptions from a spiked soil (104 mg kg(-1)) under different concentrations (35-590 mg-C L(-1)) of DOM extracts derived from two types of composts including MX and cow manure were also conducted. After harvest, the decrease in the pyrene concentration of the soil ranged from 46-65% for the different treatments. The total dry biomass for both plants was highest under MX15%+IF. The bioconcentration factors of pyrene for both also tended to decrease with increasing MX dose from 15% to 30%. However, the total uptakes of pyrene with IF and MX15%+IF were not statistically different (36.7 and 33.7 microg for Raven, and 5.20 and 7.90 microg for Sunray, respectively). These values were around 100% higher than that with NF (17.4 microg for Raven and 2.0 microg for Sunray). The pyrene desorption data confirmed the ability of DOM to associate with pyrene as indicated by its increase in apparent water solubility. On the basis of these results, MX application at 15% (v/v) does not significantly reduce the phytoextraction of pyrene due to the enhancement of plant growth as well as the possible contribution of DOM fractions to pyrene bioavailability. The application of compost may not pose serious concerns regarding the efficiency of phytoremediation of PAHs-polluted soil.

Plant-Based Antioxidant Nanoparticles without Biological Toxicity.

Here, we present a function to derive non-deteriorated nanoparticulated lignin as an antioxidant without biological toxicity that is supplied through the simultaneous enzymatic saccharification and comminution of plants. The lignin exhibits an oxygen radical absorption capacity, even in its macromolecular nature. The non-deteriorated lignin nanoparticles never inhibit the biological activity of living things, despite their antioxidant nature. The oxygen radical absorption capacity of lignin is dependent on its botanical origin and monomeric structure. A stable organic radical in lignin is responsible for the antioxidant nature of non-deteriorated lignin. The organic radical of non-deteriorated lignin, which yields a distinct signal on electron spin resonance spectra, serves as a spin trap reagent that detects the emergence of short lifespan radicals as the change of radical concentration of the lignin. The presented discovery of non-deteriorated lignin will induce not only the industrial utilization of plant biomass polymers in pharmaceuticals and reagents, but also advance our scientific understanding of the antioxidant function of native lignin.

Cloning and sequencing of the gene encoding the enzyme for the reductive cleavage of diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Geobacillus thermodenitrificans UZO 3.

We have previously reported that a cell-free extract prepared from Geobacillus thermodenitrificans UZO 3 reductively cleaves diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), a dioxin with the highest toxicity, in a sequential fashion producing 3',4',4,5-tetrachloro-2-hydroxydiphenyl ether (TCDE) as the intermediate, and 3,4-dichlorophenol (DCP) as the final reaction product. The detection of TCDE implicated the discovery of an unprecedented dioxin-degrading enzyme that reductively cleaves the diaryl ether bonds. In this study, we report the cloning and sequencing of the dioxin reductive etherase gene dreE which codes for the 2,3,7,8-TCDD-degrading enzyme. We showed that dreE was expressed in Escherichia coli and that the product of the expression could reductively cleave diaryl ether bonds of 2,3,7,8-TCDD to produce TCDE. Furthermore, we established that the amino acid sequence encoded by dreE was homologous to an enzyme with yet unknown function that is encoded by a gene located in the riboflavin (vitamin B2) biosynthesis operon in Bacillus subtilis. We also showed that the amino acid sequence possesses a coenzyme A (CoA) binding site that is conserved in the N-acyltransferase superfamily. For the first time, the degradation of 2,3,7,8-TCDD at the molecular level using a enzyme of bacterial origin has been demonstrated. A novel mechanism model for the reductive cleavage of diaryl ether bond of 2,3,7,8-TCDD was also proposed.

Removal of PCDD/Fs and PCBS from sediment by oxygen free pyrolysis.

Abstract: Few studies have dealt on the evaluation of volatilization and decomposition reactions of dioxins from sediment by oxygen free pyrolysis. In this study, the performance of pyrolysis on the removal of dioxins from sediment was investigated. Dioxin concentrations of the raw sediment and the solid residues after pyrolysis were analyzed at different conditions. Results showed a removal efficiency of 99.9999% for total dioxins at 800 degrees C and retention time of 30 min. All the polychlorinated dibenzo-furans (PCDFs) have been removed and were not formed in the solid residues at the retention time range of 30-90 min at 800 degrees C. Close to 100% removal of polychlorinated dibenzo-p-dioxins (PCDDs) was also achieved. Only trace PCDDs were detected in the solid yields at a retention time of 60 min. The highest removal efficiency of polychlorinated biphenyls (PCBs) was more than 99.9994% at a retention time of 30 min. During cooling period following pyrolysis, however, the concentration of total dioxins in solid residues increased 130 times as compared to that of the raw sediment under air atmosphere. This confirmed that some complex reactions do occur to form PCDD/Fs and PCBs from 800 to 400 degrees C in the presence of oxygen. Oxygen-free atmosphere therefore can prevent formation of dioxin during thermal process thus generating clean solid residues.

High-resolution phylogenetic analysis of residual bacterial species of fouled membranes after NaOCl cleaning.

Biofouling is one of the major problems during wastewater treatment using membrane bioreactors (MBRs). In this regard, sodium hypochlorite (NaOCl) has been widely used to wash fouled membranes for maintenance and recovery purposes. Advanced chemical and biological characterization was conducted in this work to evaluate the performance of aqueous NaOCl solutions during washing of polyacrylonitrile membranes. Fouled membranes from MBR operations supplemented with artificial wastewater were washed with 0.1% and 0.5% aqueous NaOCl solutions for 5, 10 and 30 min. The changes in organics composition on the membrane surface were directly monitored by an attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectrometer. In addition, high-throughput Illumina sequencing of 16S rRNA genes was applied to detect any residual microorganisms. Results from ATR-FT-IR analysis indicated the complete disappearance of functional groups representing different fouling compounds after at least 30 min of treatment with 0.1% NaOCl. However, the biomolecular survey revealed the presence of residual bacteria even after 30 min of treatment with 0.5% NaOCl solution. Evaluation of microbial diversity of treated samples using Chao1, Shannon and Simpson reciprocal indices showed an increase in evenness while no significant decline in richness was observed. These implied that only the population of dominant species was mainly affected. The high-resolution phylogenetic analysis revealed the presence of numerous operational taxonomic units (OTUs) whose close relatives exhibit halotolerance. Some OTUs related to thermophilic and acid-resistant strains were also identified. Finally, the taxonomic analysis of recycled membranes that were previously washed with NaOCl also showed the presence of numerous halotolerant-related OTUs in the early stage of fouling. This further suggested the possible contribution of such chemical tolerance on their survival against NaOCl washing, which in turn affected their re-fouling potential.

Fine-scale monitoring of shifts in microbial community composition after high organic loading in a pilot-scale membrane bioreactor.

In biological wastewater treatment, municipal wastewater sometimes undergoes unexpected changes in physicochemical parameters, such as organic carbon concentration. The aim of this study was to understand how microbial communities in activated sludge in a membrane bioreactor (MBR) adapt to high organic loading and maintain their degradation ability during reactor operation. A pilot-scale MBR was operated for 19 days. On day 8, the concentration of organic matter in the synthetic wastewater increased from 450 to 900 mg chemical oxygen demand (COD)/L. Even under conditions of high organic loading, COD removal rates were high, ranging from 85.3 to 91.4%. High-throughput sequencing of 16S rRNA genes revealed that microbial communities changed drastically with increased organic loading. After day 8, Aquabacterium- and Azospira-related operational taxonomic units (OTUs) belonging to the class ß-proteobacteria became dominant; this potentially enhanced the degradation of organic substances and decreased activated sludge microbial diversity. Due to the use of dissolved oxygen (DO) for degradation of organic substances, DO levels in the reactor decreased. This led to an increase in a subset of OTUs related to not only aerobic but also anaerobic bacteria, e.g., those in the class Clostridia. During this period, anaerobic microorganisms may have contributed to the degradation of organic substances to maintain MBR performance. On the other hand, high-throughput sequencing also made it possible to identify yet-to-be cultured or minor microorganisms affiliated with the candidate phylogenetic division SR1 and ammonia-oxidizing archaea in activated sludge.

Effects of Organic-Loading-Rate Reduction on Sludge Biomass and Microbial Community in a Deteriorated Pilot-Scale Membrane Bioreactor.

The effects of a precipitous decrease in the inlet organic loading rate on sludge reductions and the microbial community in a membrane bioreactor were investigated. The sludge biomass was markedly reduced to 47.4% of the initial concentration (approximately 15,000 mg L(-1)) within 7 d after the organic loading rate was decreased by half (450 to 225 mg chemical oxygen demand L(-1) d(-1)). An analysis of the microbial community structure using high-throughput sequencing revealed an increase in the abundance of facultative predatory bacteria-related operational taxonomic units as well as microorganisms tolerant to environmental stress belonging to the classes Deinococci and Betaproteobacteria.

Heavy metal precipitation by polycation-polyanion complex of PEI and its phosphonomethylated derivative.

The removal of various heavy metal ions such as Cu2+, Co2+, Zn2+, Ni2+ and Pb2+ from aqueous solutions by precipitation with polyelectrolyte complex of PPEI and PEI was conducted. Heavy metal binding with PPEI was initially allowed to occur and then upon equilibration, PEI was added to initiate precipitation of the polyelectrolyte complex together with the heavy metal ion. The PPEI-PEI system was found effective for heavy metal scavenging purposes even in the presence of high concentrations of non-transition metal ions like Na+. Heavy metal concentration may be reduced beyond emission standards for industrial wastewaters. The PPEI-PEI polyelectrolyte complex was found to be more effective than traditional precipitation methods for the treatment of a representative electroless Ni plating waste solution.

Effect of a microbiota activator on accumulated ammonium and microbial community structure in a pilot-scale membrane bioreactor.

Microbiota activators (MAs) have been used to improve the reactor performances of biological wastewater treatment processes. In this study, to remove ammonium (NH4(+)) accumulated during the pre-operation of a pilot-scale membrane bioreactor (MBR) under high-organic-loading conditions, an MA was added to the MBR system and the resulting changes in reactor performances and microbial communities were monitored for 12 days. The NH4(+) concentrations in the sludge and effluent decreased (from 427 to 246 mg/L in the sludge (days 1-9)), and mixed liquor suspended solid increased (from 6,793 to 11,283 mg/L (days 1-12)) after the addition of MA. High-throughput Illumina sequencing of 16S rRNA genes revealed that the microbial community structure changed along with the NH4(+) removal resulting from the MA addition. In particular, the relative abundance of an Acidovorax-related operational taxonomic unit (OTU) increased significantly, accounting for approximately 50% of the total microbial population at day 11. In contrast, the ammonia-oxidizing bacteria and archaea showed low abundances (<0.05%), and no anaerobic ammonia oxidizers were detected. These results suggested that the Acidovorax-related OTU was mainly involved in the NH4(+) removal in the MBR, probably due to its ammonia-assimilating metabolism.

Dynamic transition of chemolithotrophic sulfur-oxidizing bacteria in response to amendment with nitrate in deposited marine sediments.

Although environmental stimuli are known to affect the structure and function of microbial communities, their impact on the metabolic network of microorganisms has not been well investigated. Here, geochemical analyses, high-throughput sequencing of 16S rRNA genes and transcripts, and isolation of potentially relevant bacteria were carried out to elucidate the anaerobic respiration processes stimulated by nitrate (20 mM) amendment of marine sediments. Marine sediments deposited by the Great East Japan Earthquake in 2011 were incubated anaerobically in the dark at 25∘C for 5 days. Nitrate in slurry water decreased gradually for 2 days, then more rapidly until its complete depletion at day 5; production of N2O followed the same pattern. From day 2 to 5, the sulfate concentration significantly increased and the sulfur content in solid-phase sediments significantly decreased. These results indicated that denitrification and sulfur oxidation occurred simultaneously. Illumina sequencing revealed the proliferation of known sulfur oxidizers, i.e., Sulfurimonas sp. and Chromatiales bacteria, which accounted for approximately 43.5% and 14.8% of the total population at day 5, respectively. These oxidizers also expressed 16S rRNA to a considerable extent, whereas the other microorganisms, e.g., iron(III) reducers and methanogens, became metabolically active at the end of the incubation. Extinction dilution culture in a basal-salts medium supplemented with sulfur compounds and nitrate successfully isolated the predominant sulfur oxidizers: Sulfurimonas sp. strain HDS01 and Thioalkalispira sp. strain HDS22. Their 16S rRNA genes showed 95.2-96.7% sequence similarity to the closest cultured relatives and they grew chemolithotrophically on nitrate and sulfur. Novel sulfur-oxidizing bacteria were thus directly involved in carbon fixation under nitrate-reducing conditions, activating anaerobic respiration processes and the reorganization of microbial communities in the deposited marine sediments.

Nature of bioavailability of DNA-intercalated polycyclic aromatic hydrocarbons to Sphingomonas sp.

The nature of bioavailability of DNA-intercalated PAHs in aqueous solution was investigated. The degradability of different PAHs including anthracene, phenanthrene and pyrene by Sphingomonas sp. was not inhibited even at a high DNA concentration of 2%. The DNA was stable against the PAH-degrader as indicated by the unchanged electrophoresis gel chromatograms after treatment. This shows that a structural change in the polymer is not necessary for the release of PAHs. Partitioning experiments using phenanthrene as a model PAH illustrated the presence of an initial passive uptake by autoclaved cells. Subsequent intracellular degradation became apparent from parallel data with live cells. Phenanthrene transfer from the DNA was diffusion-controlled and the exit of this molecule from their intercalation sites is favored in lieu of the presence of stronger hydrophobic binding sites in the cell membrane.